EP1281085A1

EP1281085A1 - Dynamic superparamagnetic markers

Info

Publication number: EP1281085A1
Application number: EP01933876A
Authority: EP
Inventors: Martin Koch; Ernst Stetter; Bernhard Wolf
Original assignee: Individual
Current assignee: KOCH, MARTIN
Priority date: 2000-04-26
Filing date: 2001-04-24
Publication date: 2003-02-05
Also published as: WO2001081923A1; US20030201208A1; DE10020376A1; AU2001260238A1

Abstract

The invention relates to: the use of dynamic magnetic fields (DM fields) or DM field generators for identifying and/or sorting cells, cell components or pathogens; the use of said fields or field generators for eliminating pathogens contained in liquids; methods or procedures for treating infected cells or tumour cells; the use of superparamagnetically marked active substances for producing a preparation to be used in a method for treating infected cells or tumour cells, said method comprising treatment with a DM field or DM field generator; and the combination of superparamagnetically marked active substances or superparamagnetic beads with a DM field generator. Fig. 3 illustrates the example of a microscope (12), under which a superparamagnetically marked sample (15) is subjected to the DM alternating field that is created by the field generator (11), thus setting the displaced marked objects (e.g. cells) in motion. Said cells can then be identified in a specific manner.

Description

DYNAMIC SUPER PARAMAGNETIC MARKERS

The invention relates to the use of dynamic magnetic fields (DM fields) or DM field generators for recognizing and / or sorting cells, cell components or pathogens, the use of these fields or field generators for cleaning pathogen liquids, methods or methods for treatment of infected cells or tumor cells, the use of superparamagnetically labeled active ingredients for the production of a preparation for use in a method for the treatment of infected cells or tumor cells, which comprises treatment with a DM field or DM field generator, and the combination of Superparamagically labeled active ingredients or superparaqmagnetic beads with a generator of a DM field.

Background of the Invention

Systems for isolating cells marked with superparamagnetic beads or otherwise paramagnetically are known. These either use line sorting apparatuses (see e.g. US 5,837,200) which have a relatively low throughput, or they are based on the application of static DC magnetic fields in order to retain superparamagnetically marked cells by means of a column surrounded by a magnet which generates a non-homogeneous DC magnetic field and can only be made washable after removing unmarked cells by removing the magnet (MACS = Magnetically Activated Cell Sorter, commercially available from Miltenyi Biotec GmbH).

The use of alternating fields to suppress the chain formation of heme molecules in erythrocytes infected by the pathogen of malaria, a protozoan called Plasmodium, has also become known. One form of the pathogen normally resides in erythrocytes, where it releases the heme by absorbing the protein component from hemoglobin, which then stored in long chains. Alternating fields succeeded in inhibiting this chain formation or destroying existing chains. As a result, a 33 to 70% drop in the number of parasites was achieved. The weakly oscillating magnetic fields that were used in these experiments at Washington University are low-frequency magnetic fields. They are intended to help deprive the malaria pathogen of its livelihood by destroying the heme structures formed in the erythrocytes. The divalent iron ion in the heme molecule is magnetic only in the deoxygenated state, so that venous blood is of particular interest.

A method of a Danish company (MEDICO- CHEMICAL LAB, APS) is also known, in which magnetized medicine is injected directly into the bloodstream and at the location where the treatment is to take place, for example at the site of a tumor strong magnetic DC field is captured and enriched. The problem here was to build up a magnetic field that is strong enough to hold the active substance at the desired location.

Finally, it is also known to administer nanoparticles containing iron oxide to a tumor (e.g. by injection) and then to set them in vibration locally using alternating fields in such a way that temperatures of up to approx. 47 ° C. arise at the location of the nanoparticles. As a result, the degenerated tissue disintegrates. Breast tumors transplanted into mice disappeared within half an hour. This method, developed at Humbold University, uses high-frequency fields (kHz, MHz). However, the electromagnetic fields acting here are dependent on the material constants and may inhomogeneous. In contrast to that of the dynamic alternating fields, which can be described with the first Maxwell equation, their effect can be described with the second Maxwell equation (induction law).

1. Maxwell's equation: J H-ds = J (J + θD / 3t) -dA 2nd Maxwell 'see equation: XE "ds = - - / J .d / dt' dA

A = area

H = magnetic field

E = electric field

J = current density s = distance

B = magnetic flux density t = time dO / dt = displacement current density

All of the systems mentioned use either static magnetic

DC fields or simple (oscillating) alternating fields.

Magnetic fields generated by a permanent magnet are used in particular in the MACS technology mentioned at the outset. These inhomogeneous, static magnetic sliding fields are independent of material constants (J.C. Maxwell, On Faraday's Lines of Force, Scientific Papers 1 855, 1 856, reprinted by Dower, New York 1 952) and can therefore penetrate liquids and Reach the magnetic particles contained therein and act on them. However, the magnetic system cannot be changed after it has been installed in the system. There is no doubt that a large number of magnetically marked objects (e.g. cells) are attracted to a strong permanent magnet in solution, albeit to different extents.

The patent application WO 95/19217 (corresponds to EP 0 740 578) describes a device which makes it possible to generate literally moving electromagnetic fields. This is used in the stated patent application for the movement of ions in masonry with the aim of removing salts from masonry.

For the therapy of proliferative diseases, such as tumor diseases or cancer, chemotherapy, radiation, surgical treatment, cryotherie (freezing), hyperthermia (locally or throughout the body abnormally elevated temperature) and immunotherapy, or combinations thereof, have been used to treat infections, for example antibiotics or other chemotherapy, rapeutics, eg antiparasitic or antiviral agents. There is a need to have new devices and methods available for the therapy and diagnosis of such diseases.

In early 2000 it was reported in the Deutsche Ärztezeitung that cancer cultures in laboratory vessels can be impaired in their growth if they were exposed to loudspeakers. This simple experiment shows that the cell cultures (cells of a lung carcinoma) grow more slowly under mechanical stress than in an unexposed control group. It would be interesting to produce such mechanical effects in a different way.

Summary of the advantages of the invention

In contrast to the prior art, the dynamic marking technique according to the invention now makes it possible to work with magnetic movement fields (dynamic magnetic fields, hereinafter referred to as DM fields). These can be combined in such a way that even in standing (without Hall effect) or moving liquids with low to high (eg reliable) viscosity, dynamic effects can be generated on stationary or moving marked objects. The frequency of these magnetic fields can be selected so that they can also be regarded as independent of the material constants (the frequency-dependent summand of the first Maxwell's equation is negligible here, the first summand is completely independent of the material constants). This means that fields of this type can pass through corresponding liquids with almost no loss, in order to then be able to act on all magnetic objects, for example marked with magnetic beads, for example cells or, for example, liposomes or superpar magnetically marked active substances (for example by rotations) , Transportation or construction of (reversible after eliminating the fields dissolving) structural barriers). It is precisely this dynamic characteristic of the fields that can be used for dynamic marking (DM fields) that can also be used to generate electrical forces in liquids which can influence magnetic and non-magnetic ions (see FIG. 1 below).

The weakly oscillating fields that were used in the aforementioned experiments at the University of Washington against malaria can also be caused by a DM field generator used in the present invention, if the otherwise elongated DM field generator is made round, the stator comparable to a three-phase machine, and for example as a kind of cuff around an arm, a leg or the whole body. However, non-ring-shaped DM field generators are preferably used.

It is also possible to use the DM fields to cause mechanical stress on infected tissue or tumor tissue, e.g. by applying superparamagnetic beads _j.ii close to a tumor or an infected organ (e.g. liver, brain)

(For example, by injection at the tumor site or by transport using a DM field itself, or in particular by administration of antibodies directed against the tumor or infected cells, which are labeled superparamagnetically, so that they bind to tumor cells after, for example, injection or Enrich infusion on tumor cells) and then magnetically "shakes" or rotates using DM fields. The DM field generator can be adapted to the corresponding requirements in terms of shape, power and frequency.

When using ferrite materials instead of layered, mutually insulated sheets or solid components (for example suitable for frequencies below 15 Hz) as a component of the magnetic field generator, higher frequencies (for example in the kHz range) can also be generated by a DM device. According to reports by the Berlin researchers from the Charite / Humboldt University, the hyperthermic effects already described by iron oxide particles injected into tumors and external fields are said to have destroyed tumors in mice. Alternating electromagnetic fields, which are supposed to heat away magnetized tumors over large distances (human body), are problematic due to the long distances with different material constants (tissue, water, etc.). An additional problem is probably the "magnetization" of the tumor. The simple injection of superparamagnetic beads does not always guarantee an even, optimal distribution. However, this can be achieved with the DM technique in combination with beads to which antibodies against tumor cells or infected cells are conjugated, and, if necessary, forced dynamic pressing over the blood-supplied parts of the diseased tissue.

The fields of application of DM fields are therefore extremely diverse.

Detailed description of the invention

The invention relates to the use of dynamic magnetic fields (DM fields) that can be generated by AC-supplied multiphase systems, or of DM field generators for recognizing and / or sorting cells, cell components or pathogens, in particular those cells, cell components or pathogens, to the superparamagnetic Beads are bound.

The invention also relates to the use of DM fields or DM field generators for the purification of liquids from pathogens, in particular those to which superparamagnetic beads are bound.

The invention also relates to methods or methods for diagnosis

(Detection of sick, for example infected or tumor cells) and in particular treatment of infected cells or tumor cells which prevent the use of DM fields or DM field generators and special additional superparamagnetic beads which bind at the site of the infected cells or tumor cells (in particular after appropriately localized administration, for example injection, or, in particular if the superparamagnetic beads are bound to antibodies specific for the cells mentioned (directly, via a spacer or via liposomes) systemic administration) or are bound to it.

The invention also relates to the use of superparamagnetically labeled active ingredients for the manufacture of a preparation for use in a method for treating infected cells or tumor cells, which comprises treatment with a DM field or with a DM field generator and superparamagnetic beads, which (a) administered (eg injected) at the location of the cells, (b) maneuvered there with a DM field or DM field generator (especially through body cavities) and / or (c) to the cells mentioned, eg via antibodies bound to the beads, specific for antigens on the cells to be treated (which are bound, in particular, directly or via spacers or liposomes, which are bound to the beads), or are specifically bound and, if desired, an active ingredient which is active against infection or tumors (directly or via carry a spacer covalently attached to the beads or in liposomes attached to the beads); and the combination of superparamagically labeled active ingredients or superparamagnetic beads with a generator of a DM field (DM field generator).

Unless otherwise stated, the expressions mentioned above and below preferably have the following meanings within the scope of the present disclosure - one or more of the corresponding more specific definitions can be used instead of the above general definitions and then relate to preferred embodiments of the invention:

DM fields (dynamic magnetic fields) are those that can be generated by the DM field generators described in more detail below. be generated in particular. These are wandering (or pulsating) magnetic fields that arise from the superposition of 2 or more alternating fields that are staggered in time and location.

Using DM fields or DM field generators and superparamagnetic beads means in particular that DM fields or DM field generators are used to set the superparamagnetic beads, in particular those bound to cells, cell components and / or pathogens, in motion, for example in longitudinal motion or rotation, or (by generating static alternating fields using a DM field generator) to form structures such as barriers or saber-shaped structures from them.

DM-fields (magnetic motion fields) sinddadurchgekennzeich- net that they obey the first Maxwell equation and hence affect magnetic effects sufficiently low when selecting frequencies, since then the second term of the 1st Maxwell see 'equation is negligible (with änder _t en words , if there is hardly any measurable additional warming (warming due to eddy currents), show material-independent penetration

(In contrast to this, e.g. those used at the Charite are based

Fields on the second Maxwell equation and are based on the principle of induction).

DM field generators are described in WO 95/19217, their dimensions and shape can be adapted to the needs

(e.g. by dimensioning, such that they are on a

Microscope stage can be used up to whole-body coil systems). In principle, they correspond to "linear motors". For the generation of the DM fields of the present invention, AC-supplied multiphase systems, that is to say those with 2 or more coils which are spatially offset relative to one another and which are actuated at different times, are used as DM generators, for example in combination with a frequency converter. The multiphase system is preferably embedded in a magnetic material, whereby concentration and amplification are achieved. The windings can, for example, be introduced into a slotted, ferromagnetic, laminated core in which the laminations are insulated from one another (for example by insulating lacquers such as shellac, plastics or paper). In a further embodiment of the invention, resistors or coils are interposed between the supply network and the multiphase systems - this enables the DM alternating field amplitude to be varied. Concrete embodiments for such

Devices are found in WO 95/19217, which are incorporated herein by reference. In one embodiment, the DM field generators are fixed, so that the DM field is only generated by the alternating current, but they can preferably also be displaceable during the DM field generation. The generation of the magnetic motion field is therefore solely or at least mainly responsible for the AC supply of the multiphase system, but the location of the DM field can also be varied by shifting the DM field generator. This distinguishes the DM fields significantly from the relative movement of permanent magnets or (direct or alternating current controlled) single-phase electromagnets.

Detection of cells, cell components or pathogens means that after marking with superparamagnetic beads they can be set in rotation or moved in a directional manner by applying magnetic movement fields and can thus be observed and recognized (identified) in the presence of unmarked cells, preferably microscopically. This means that a method for diagnosis (for example detection of diseased cells from tissue cells or blood) is also the subject of the present invention.

Sorting cells, cell components or pathogens means that appropriately superparamagnetically labeled cells, cell components or pathogens from solutions by applying, for example, as from magnetic motion fields or by creating structures (Fig. 4, (18)) from mixtures with unmarked counterparts can be sorted out or enriched, for example from flowing solutions by directing them to one side and only branching off there, or from standing L solutions, in particular blood preserves, blood serum preserves or blood plasma preserves or nutrient media, for example for organ transplantation or cell cultures, which must be free of pathogens) by likewise concentrating them on one site, for example on one side, and removing them there (for example by suction). The advantage is that, for example, culture media for cell culture or canned blood can be cleaned in this way. By sequentially using different antibodies, several components can be obtained from one sample.

Cell components are, for example, organelles such as lysosomes, endoplasmic reticulum, vesicles of the cell membrane (e.g. micro soes, channelicular membrane vesicles from biliary tubules) and the like. Pathogens are also cancer cells, e.g. Tumor cells (for example capable of forming metastases), such as blood cancer cells, or abnormally proliferating cells from bone marrow.

Pathogens are, for example, bacteria including mycoplasma, viruses (e.g. HIV, hepatitis viruses such as HCV), fungi (such as yeasts) or parasites (such as protozoa, e.g. trypanosomes or plasmodia, worms or the like).

The superparamagnetic labeling is preferably achieved by superparamagnetically labeled antibodies or by superparamagnetically labeled liposomes, which have also bound antibodies which are specific for the corresponding cells or pathogens, ie specifically or intensively expressed there, and bind them. These antibodies are, for example, against (for example expressed on the cell surface) tumor antigens, on the cell surface (for example by antigen-presenting proteins such as those of the main compatibility complex (major Histocompatibility Complex) or the like) presented peptides (for example from pathogens such as viruses or mycoplasma) or directed directly to the antigens themselves and available by standard methods.

The sorting of cells, cell components or pathogens is also possible by means of correspondingly superparamagnetically labeled antibodies or liposomes, the labeled components also being able to be enriched at certain points from flowing or standing liquids and then selectively derived or suctioned off. The cleaning of liquids from cells, cell components or pathogens also works in an analogous manner.

The treatment of infected cells or tumor cells can be carried out, for example, extracorporeally (for example in cell or tissue cultures, for example for culturing liver cells from a virus-infected liver, or from isolated bone marrow of a tumor patient, in order to remove infected or tumor cells in each case and thus enable reimplantation ). In this way (by sorting out marked infected or tu oral cells, by mechanically destroying them, by vigorous movement and / or by administration of superparamagnetic, antibody-bound beads loaded with active substances, their active substances in the DM field due to the greater mechanical load on the bound Cells and the selective concentration on the cells have an increased effect), the unwanted cells are eliminated and purely "healthy" cell or tissue cultures or cells or tissue parts are obtained, which can be used, for example, for heterologous or preferably autologous transplantation.

However, the treatment can also be carried out in the body, preferably in the case of a warm-blooded animal, such as a person, in particular if the person requires appropriate treatment. Antibody and superparamagnetic labeled beads, preferably loaded with one or more active ingredients, can be administered to a warm-blooded animal and are put into motion by means of DM fields after "docking" on the diseased tissue, which on the one hand mechanically stresses the diseased cells and on the other hand exposes them to the active substance (for example, because this reaches the interior of the cell through the DM field).

Unlabelled beads can also be used (especially in body cavities such as the lungs, gastrointestinal tract, abdominal cavity, pleural space, brain cavities, spinal canal, cavities in the area of muscle fascia etc.). These can first be maneuvered to the desired location (e.g. tumor, infected organ, e.g. liver) using DM fields and then set in motion on site, thus making the diseased areas accessible to the body's defenses through mechanical stress.

The invention also relates to the use of 'superparamagnetically labeled active ingredients for the preparation of a preparation for use in a method for treating infected cells or tumor cells, which comprises treatment with a DM field, and to the combination of superparamagetically labeled active ingredients or superparamagnetic beads with a generator a DM field, in particular as described above or in WO 95/19217.

Superparamagnetic beads are known, can be produced by methods known per se or are commercially available. The term "bead" does not necessarily mean spherical, but is used in the sense of "particle".

For example, the international patent application WO 85/02772 describes particles based on a carbohydrate, polyamino acid or plastic matrix. Examples of corresponding carbohydrate matrices can be found in PCT / SE82 / 00381, PCT / SE83 / 00106 and PCT / SE83 / 00268, for corresponding polyamino acid matrices in US 4,247,406; Plastic matrices, for example based on polymers made of acrylates, polystyrene, etc., are also known. For example, iron oxide particles are in the matrix embedded. The patent US 4,219,411 describes polystyrene and in particular on polymers of acrylic acid and its derivatives

(such as acrylamide, methacrylamide, acrylic acid, methacrylic acid, dimethylaminomethacrylate, hydroxy-lower alkyl or amino-lower alkyl acrylates, such as 2-hydroxyethyl, 3-hydroxypropyl or 2-aminoethyl methacrylic acid) based matrices with free hydroxy groups that can be activated with cyanogen bromide or with free ones

Carboxy groups, or with free amino groups, all of which allow covalent binding to molecules such as antibodies, avidin or streptavidin. Further patents which describe suitable particle matrices are US 3,957,741 and US 4,035,316.

"Superparamagnetic" means in particular that the permeability U _j . lies between that of paramagnetic materials and that of ferromagnetic materials (otherwise,

Illtlllθ ⁱ L • r (paramagnet. Material) ^ r ~ r (superparamagnet. Material) r (f erromagnet. Material) '' for example in the range of μ. approximately equal to 2 to μ_. approximately equal to 100. All materials which meet the aforementioned conditions are suitable for the purposes of the present invention. The superparamagnetic properties are preferably achieved by embedding metal oxides, especially iron oxide (Fe ₃ 0 ₄ ), or other suitable metals or alloys. The metal particles are preferably fine and of relatively uniform size, so that the resulting particle diameter preferably has the sizes mentioned below. The metals are in particular iron, nickel or cobalt, or alloys, for example gadolinium, dysprosium or erbium

Vanadium, or other transition metals can contain. iron oxide

(especially magnetite) is preferred. Some ferrites, such as lithium ferrites, are also suitable.

Preferred beads have an average diameter of 2 μm or less, in particular 1 μm or less (in order not to get stuck in the capillary system, for example), preferably from 30 to 1000 nm, in particular from 30 to 300 nm. Beads with biodegradable are particularly preferred Matrix, e.g. from carbohydrates ten (especially polysaccharides) or polyamino acids. All of these beads, as well as analogs thereof, are suitable for the purposes of the present invention. The beads can also be additionally marked with low activity by gamma emitters (such as technetium-99m) in order to track the movement of globules loaded with active substances by means of a gamma camera, for example in the body.

In particular, the company Miltenyi Biotec GmbH, BergischGladbach, Germany, offers under the name "MicroBeads" free or antibody-conjugated beads with an average size of around 50 nm, which contain iron oxide in a polysaccharide matrix - these have the great advantage, also bioab- to be buildable, and are therefore very preferred because of their small size. From the company Polysciences, Inc., under the name "BioMag ^® " beads of approximately 1 μm in size are offered, which consist of an iron oxide core with a silane shell and are functionalized with amino or carboxy groups, which covalently bind to proteins (such as Antibodies, avidin, streptavidin), glycoproteins, polysaccharides, lectins and other ligands allowed. Sigma-Aldrich also offers superparamagnetic beads with a diameter of approx. 1 μm based on iron oxide, which carry either carboxy or amino groups on the surface as functional groups. Other superparamagnetic beads are offered by Deutsche Dynal GmbH, Hamburg, Germany, and by a number of other companies.

Examples of active substances which can be used according to the invention are, in particular, antitumor chemotherapeutic agents which (alone or as a combination of two or more of the substances mentioned) can be used as active substances according to the invention, in particular those contained in the following list:

(A) alkylating agents such as dacarbazines (DTIC domes); Mustard gas derivatives such as mechlorethamines (mustarges); Ethyleneimine derivatives, eg triethyleneethiophosphoramide (thio-tepa); Procarbazine (Matu lane); Alkyl sulfonates such as busulfan (myeleran); cyclophosphamide; 4-hydroxyperoxycyclophosphamide (4-HC); mafosfamide; ifosfamide; Melphalan (Alkeran); Chlorambucil (Leukeran); Nitrosoureas such as cyclohexylnitrosourea (meCCNU; Carmustin, BCNU, BiCNU) orLomustin (CCNU, CeeNU), cis-platinum (II) -diamine dichloride (platinol or cisplatin); Carboplatin (paraplatin); preferably other cross-linking Che otherapeutics, in particular bis-alkylating agents, especially mustard gas derivatives such as alkyl sulfonates, eg. Busulfan; or compounds that cause cross-links via ionic bonds, such as ethyleneimine derivatives, e.g. B. Triethylene thiophosporamide (Thio-tepa);

(B) antitumor antibiotics, preferably selected from the group consisting of bleomycin (blenoxanes); Anthracyclines such as daunomycin, dactinomycin (Cosmegen), daunorubicin (cerubidine), doxorubicin (Adriamycin, Rubex), epirubicin, esorubicin, idarubicin (idamycin), plicamycin (mithracin, formerly known as mithramycin end) and in particular cross-linking network Antitumor antibiotics, such as mitomycin C (mitomycin, mutamycin);

(C) antimetabolites, e.g. Folic acid analogues such as methotrexate (Folex, Mexate) or tri-etrexate; Purine nucleoside analogues such as

Cladribine (leustatin; 2-chloro-2'-deoxy-ß-D-adenosine), 6-

Mercaptopurine (Mercaptopurine, Purinethol, 6-MP), Pentostatin

(Nipent) or 6-thioguanine (6-TG, tabloid); Pyrimidine analogues such as

5-fluorouracil (fluorouracil, 5-FU), 5-fluorodesoxyuridine (floxuridine, FUDR), cytosine arabinoside (Ara-C, cytarabine, Cytosar-U or

Tarabin PFS), fludarabine phosphate (Fludara) or 5-azacytidine; Hydroxy urea (hydrea); or polyamine biosynthesis inhibitors, especially ornithine decarboxylase or S-adenosylmethionine decarboxylase inhibitors, e.g. those mentioned in EP 0 456 1 33, in particular 4-amidino-1-indanone-2 '-amininohydrazone;

(D) plant alkaloids, in particular vinca alkaloids, such as viriblastin (Velban), vincristine (Oncovin) or vindesine; Epipodophylloxoxins such as etoposide (VP-1 6, VePesid) or teniposide (VM-26, Vumon);

(E) agents and antagonists with hormone activity, in particular adrenocorticoids, such as prednisone (Deltason) or dexamethasone (Decadron); Progestins such as hydroxyprogesterone (Prodox), megestrolace tat (Megace) or medroxyprogesterone (Provera, Depo-Provera); Androgens such as testosterone or fluoxymesterone (halotestin); Estrogens such as diethylstilbestrol (DES), estradiol or chlorotriansien (Tace); synthetic analogs of LHRH, such as goserelin (Zoladex); Synthetic analogs of LH-releasing hormones, such as leuprolide (Lupron, Lupron Depot); Antiandrogens such as flutamide (Eulexin); Anti-estrogens such as tamoxifen; Aromatase inhibitors such as aminogluthetimide (cytadren), lentarone (formestane, 4-hydroxy-4-androsten-3, 17-dione) (see EP 0 162 510), fadrozole (5- (p-cyanophenyl) -5, 6, 7, 8 -tetra-hydroimidazo [1, 5-a] pyridine, see EP 0437415 and EP 0165904), letrozole (4,4'- (1H-1, 2, 4-triazol-1-yl-methylene) -bis-benzonitrile, see US 4,976,672), Arimidex, 4- (α- (4-cyanophenyl) -α-fluoro-1 - (1, 2,4-triazolyl) methyl) -benzonitrile (see EP 0 490 816) or 4- (α- (4-cyanophenyl) - (2-tetrazolyl) methyl) benzonitrile (see EP 0 408 509); adrenal-cytoxic agents, such as mitotane (lysodes); Somatostatin analogs such as octreotide (sandostatin); or 5α-reductase inhibitors, such as N- (1-cyano-1-methyl-ethyl) -4-aza-3-oxo-5-androst-1-en-17β-carboxamide (see EP 0 538 192);

(F) Modifiers for biological processes (biological response modulators), in particular lymphokines, such as aldesleukin (human recombinant IL-2, proleukin); or interferons, such as interferon-α (Intron-A, Roferon) or interferon "B ^ B _j D '(see EP 0 205 404);

(G) inhibitors of protein tyrosine kinases and / or serine / threonine kinases, ieN- {5- [4-methyl-piperazino-methyl) -benzoylamido] -

2-methyl-phenyl} -4- (3-pyridyl) -2-pyrimidine (see EP 0 546 409), N- (3-chlorophenyl) -4- (2- (3-hydroxy) propylamino-4 -pyridyl) -2-pyrimidineamine (see EP 0 606 046), N-benzoyl-staurosporine (see EP 0 296 110), 4,5-bis (anilino) phthalimide (see EP 0 516 588), N- (5-N-Benzoylamido-2-methylphenyl) -4- (3-pyridyl) -2-pyridinamine (see EP 0 564 409) or 4- (m-chloroanilino) -5, 6-dimethyl-7H-pyr rolo [2, 3-d] pyrimidine (see EP 0 682 027);

(H) antisense oligonucleotides or oligonucleotide derivatives, for example targeting raf (see WO 95/32987) or PKC, targeting SAMDC (PCT application WO 96/05298); or (I) mixed-acting agents or agents with other or unknown mechanisms of action, for example S-triazine derivatives, for example altrematin (hexals); Enzymes such as asparaginase (Elspar); Methylhydrazine derivatives such as dacarbazine and procarbazine; Matrix metalloproteinase inhibitors, hexamethylmelamine, pentamethylmelela; Anthraquinones such as Mitoxantrone (Novantrone); Mitotic spindle poisons such as paclitaxel (Taxol), epothilone A, epothilone B, epothilone derivatives or discodermolide; Streptozocin (Zanosar); Estracyt (Estramustine); amsacrine; Agents with cell differentiation action, such as all-trans retinoic acid (TRA); Immunomodulators such as levamisole (ergamisole); Vaccines, for example anti-melanoma vaccines (see EP 0 674 097); or antibodies which are effective against tumors, for example antibodies directed against melanoma antigens (see EP 0 640 131), antibodies for active immunotherapy of melanomas (see EP 0 428 485), antibodies against

Colon cancer (Panorex®), antibodies against ^" Non-Hodkin lymphoma

(Rituximab), antibodies to breast cancer (trastuzumab),

Antiidiotype antibodies such as TriaAb® or CeaVac® (Titan Pharmaceuticals, Inc.) and with the from eleven,. Amino acid-containing section of the TAT protein of the AIDS virus, which causes the penetration of cell membranes, conjugated (for example recombining) proteins, such as proteins influencing cell regulation or correspondingly modified antibodies, the degenerate proteins, such as degenerate tyrosine or within the cancer cell Can bind and thus inactivate serine / threonine kinases).

Antibiotics, antiviral agents, such as e.g. Inhibitors of reverse transcriptase or retroviral proteases, such as HIV protease, or active substances which are active against virus hepatitis (such as HCV), such as interferon (in particular interferon-alpha-2) and / or ribavirin, or antibodies are used.

The compounds mentioned can also be present as salts, in particular pharmaceutically usable salts, if they have suitable salt-forming groups. Salts of active ingredients with basic Groups can be, for example, acid addition salts, such as halides, methanesulfonates or sulfates, active substances with acidic groups can be salts with bases, such as metals or ammonium salts of ammonia or substituted amines. The active compounds can either be directly (preferably via spacers) covalently coupled (conjugated) to superparamagnetic beads, or incorporated into liposomes which are or can be labeled with superparamagnetic beads which are non-covalent (for example by antigens conjugated with lipids) the liposome surface are presented and allow the docking of antibodies marked by superparamagnetic beads, or are bound using the biotin / avidin or biotin / streptavidin interaction), or are covalently bound to the liposomes by direct binding or binding via spacers (ge - Bund.e.g. on components of the liposome envelope, such as amino groups of lecithins, or amino, hydroxyl or carboxy groups on acyl residues, which belong to the liposome-forming phospholipids, or the like). Alternatively, superparamagnetic material, for example directly corresponding iron oxide particles, can be built directly into the liposomes. Superparamagnetic beads labeled only with antibodies (which can also act as therapeutic agents at the same time) can also be used, since they also recognize corresponding diseased cells and make them accessible for treatment with DM fields.

Examples of liposome formulations are known; Thus, a liposome dispersion (phospholipid-stabilized dispersion) which can be used in the context of the invention comprises a) one or more phospholipids of the formula A,

1

(A), wherein R _A C ₁₀ . ₂₀ acyl, R _{B is} hydrogen or C ₁₀ . ₂₀ -acyl, R _a , R _b and R _{c are} hydrogen or C, _ ₄ -alkyl and n are an integer from two to four, if desired b) another phospholipid or more phospholipids; c) the active ingredient (s) and d) a pharmaceutically acceptable carrier liquid and, if desired, further auxiliaries and / or preservatives.

The production process for these dispersions is characterized in that a solution or suspension of components a) and c) or a), b) and c), preferably a) and b), in a weight ratio of 20: 1 to 1 : 5, in particular from 5: 1 to 1: 1, converted into a dispersion by dilution with water, then the organic solvent is removed, for example by centrifugation, gel filtration, ultrafiltration or in particular by dialysis, e.g. B. tangehtial dialysis, preferably against water, the dispersion obtained, preferably after addition of auxiliaries or preservatives, if necessary with adjustment to an acceptable pH by adding pharmaceutically acceptable buffers such as phosphate salts or organic acids (pure or dissolved in water), such as Acetic acid or citric acid, preferably between pH 3 and 6, e.g. B. pH 4 - 5, if it does not already have the correct active ingredient concentration, preferably concentrated to an active ingredient concentration of 0.2 to 30 mg / ml, in particular 1 to 20 mg / ml, the concentration preferably by the latter methods for removing an organic solvent, in particular by ultrafiltration, for. B. using an apparatus for performing tangential dialysis and ultrafiltration.

The dispersion which can be prepared by this process and stabilized by phospholipids is stable at room temperature for at least several hours, is reproducible with regard to the proportion of the components and is toxicologically harmless and is therefore particularly suitable for oral or intravenous administration to warm-blooded animals, in particular special people, suitable.

The order of magnitude of the particles obtained in the dispersion is variable and is preferably between about 1.0 x 10 ^" ⁸ to about 1.0 x 10 ^{" 5} m, in particular between about 10 ^"7 and about 2 x 10 ^{~ 6} m.

The nomenclature of the phospholipids of the formula A and the numbering of the C atoms is carried out using the method described in Eur. J. of Biochem. 79, 11-21

(1977) "Nomenclature of Lipids" recommendations given by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) (sn-nomenclature, stereospecific numbering).

In a phospholipid of the formula A R _A and R _B are ₁₀ _ ₂₀ acyl, preferably straight-chain C ₁₀ _ ₂₀ alkanoyl having an even number of carbon atoms (unsubstituted or substituted with the meanings of C, in particular by functional groups containing a coupling allow for antibodies, beads or the like, for example hydroxyl, amino or carboxyl) and straight-chain Cι ₀ _ ₂₀ alkenoyl with a double bond and an even number of carbon atoms (unsubstituted or substituted, in particular by functional groups that couple to antibodies , Beads or the like, for example hydroxyl, amino or carboxyl, where amino or hydroxyl should not be bound to C atoms from which the double bond originates for reasons of stability).

Straight chain C ₁₀ . ₂₀ -alkanoyl R _A and R _B with an even number of carbon atoms are, for example, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoyl. Straight-chain C ₁₀ _ ₂₀ alkenoyl R _A and R _B with a double bond and an even number of C atoms are, for example, 6-cis, 6-trans, 9-cis or 9-trans-dodecenoyl, -tetradecenoyl, - hexadecenoyl, octadecenoyl or icosenoyl, especially 9-cis-octadecenoyl (oleoyl).

In a phospholipid of formula A, n is an integer from two to four, preferably two. The group of the formula - (C _n H _2n ) - represents unbranched or branched alkylene, for example 1,1-ethylene, 1,1-, 1,2- or 1,3-propylene or 1,2-, 1,3- or 1,4-butylene. 1,2-Ethylene (n = 2) is preferred.

Phospholipids of the formula A are, for example, naturally occurring cephalins in which R _a , R _b and R _{c are} hydrogen or naturally occurring lecithins in which R _a , R _b and R _{c are} methyl, for example cephaline or lecithin from soybeans, bovine brain and bovine liver or hen's egg with different or identical acyl groups R _A and R _B or mixtures thereof. The term "naturally occurring" phospholipids of the formula A "defines phospholipids which have no uniform composition with respect to R _A and R _B. Such natural phospholipids are also lecithins and kephalins, the acyl groups R _A and R _{B of which are} structurally indefinable and derived from naturally occurring fatty acid mixtures are.

Synthetic, essentially pure phospholipids of the formula A with different or identical acyl groups R _A and R _B are preferred. The term “synthetic” phospholipid of the formula A defines phospholipids which have a uniform composition with respect to R _A and R _B. Such synthetic phospholipids are preferably the lecithins and kephalins defined below, the acyl groups R _A and R _{B of which have} a defined structure and are derived from a defined fatty acid with a degree of purity higher than approximately 95%. R _A and R _B can be the same or different and unsaturated or saturated. R _A is preferably saturated, for example n-hexadecanoyl, while R _{B is} unsaturated, for example 9-cis-octadecenoyl (= oleoyl).

The term "substantially pure" phospholipid defines a degree of purity of more than 70% (by weight) of the phospholipid of formula A, which can be determined using suitable determination methods, e.g. paper chromatographically, is detectable.

Particularly preferred are synthetic, substantially pure phospholipids of the formula A, wherein R _A is straight-chain C ₁₀ _ ₂₀ -

Alkanoyl with an even number of carbon atoms and R _B the meaning straight chain C ₁₀ . ₂₀ -Alkenoyl with a double bond and an even number of carbon atoms. R _a , R _b and R _c are methyl and n = 2.

In a particularly preferred phospholipid of the formula A, R _{A is} n-do decanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoyl and R _{B is} 9-cis-dodecenoyl, 9-cis-tetradecenoyl, 9-cis-hexad- cenoyl, 9-cis-octadecenoyl or 9-cis-icosenoyl. R _a , R _b and R _c are methyl and n is 2. A very particularly preferred phospholipid of the formula A is synthetic 1-n-hexadecanoyl-2- (9-cis-octadecenoyl) -3-sn-phosphatidylcholine with one Purity more than 95%. Preferred natural, essentially pure phospholipids of the formula A are in particular lecithin (L-α-phosphatidylcholine) from soybeans or chicken eggs.

For the acyl residues in the phospholipids of the formula A, the names given in brackets are also common: 9-cis-decenoyl (lauroleoyl), 9-cis-tetradecenoyl (myristoleoyl), 9-cis-hexa decenoyl (palmitoleoyl), 6- cis-octadecenoyl (petroseloyl), 6-trans-octadecenoyl (petroselaidoyl), 9-cis-octadecenoyl (oleoyl), 9-trans-octadecenoyl (elaidoyl), 11-cis-octadecenoyl (vaccenoyl), 9-cis-ioleoyl ), n-dodecanoyl (lauroyl), n-tetradecanoyl (myristoyl), n-hexadecanoyl (palmitoyl), n-octadecanoyl (stearoyl), n-icosanoyl (arachidoyl). Further phospholipids are preferably esters of phosphatidic acid (3-sn-phosphatidic acid) with the acyl radicals mentioned, such as phosphatidylserine and phosphatidylethanolamine.

Components a), b) and c) or a) and c) are contained in the carrier liquid d) as liposomes in such a way that no solids or solid aggregates such as micelles form for several days to weeks, and the liquid with the components mentioned, if appropriate after filtration, preferably orally or intravenously, can be applied.

Pharmaceutically acceptable, Contain non-toxic auxiliaries, for example water-soluble auxiliaries which are suitable for producing isotonic conditions, for example ionic additives such as table salt or nonionic additives (scaffolding agents) such as sorbitol, mannitol or glucose or water-soluble stabilizers for the liposome dispersion such as lactose, fructose or sucrose. In addition to the water-soluble auxiliaries, emulsifiers, wetting agents or surfactants which can be used for liquid pharmaceutical formulations can be present in the carrier liquid, in particular emulsifiers such as oleic acid, nonionic surfactants of the fatty acid polyhydroxy alcohol ester type such as sorbitan monolaurate, oleate, stearate or palmitate, sorbitan tristearate or trioleate, polyoxyethylene adducts of fatty acid, polyhydroxy alcohol esters such as polyoxyethylene sorbitan monolaurate, oleate, stearate, palmitate, tristearate or trioleate, polyethylene glycol fatty acid esters such as polyoxyethyl stearate, polyethylene glycol 400 stearate 2000, polyethylene glycol stearate, in particular ethylene oxide-propylene oxide block polymers of the Pluronic ^® type (Wyandotte Chem. Corp.) or Synperonic ^® (ICI).

The liposomes can be separated from free active substance, for example by gel filtration, so that little or no active substance is present outside the liposomes in the remaining dispersion.

Superparamagnetic beads can either be subsequently covalently bound (for example by adding bifunctional cross-linkers), or antigenic components that are incorporated into the membrane (for example recombinant membrane proteins such as CD4 or CD8, or low molecular weight haptens, such as dinitrophenol, which are present, for example, instead of the radicals R _a , R _b or R _c ), to which superparamagnetic beads conjugated with the corresponding antibodies can then be bound, or by binding biotin over one Spacers, for example instead of one of the radicals R _a , R _b and / or R _c , and binding of superparamagnetic conjugated with avidin or streptavidin Beads the supermparamagnetically labeled liposomes; the liposome (s) themselves are loaded with these particles with paramagnetic materials, such as the smallest iron oxide particles, which are added directly during the production of the liposomes, and are themselves a kind of superparamagnetic beads. Preferred preservatives are e.g. B. antioxidants, such as ascorbic acid, or microbicides, such as sorbic acid or benzoic acid.

Particularly preferred are conjugated superparamagnetic beads (especially against infected cells, such as HIV-infected lymphocytes or virus (eg HCV) -infected liver cells, or tumor cells), or active ingredient-containing liposomes coupled with superparamagnetic beads and corresponding antibodies, which are each after injection Enrich at the location of the tumor and which are set in motion directly by means of the DM fields and thus enable anti-tumor effects directly at the location of the infected cells or tumor site due to mechanical stress or, in the case of liposomes, additional drug release under the influence of the DM field. The use of antibodies directed against parasites is also possible.

The routes of administration include, inter alia, enteral, such as nasal, oral or rectal; or parenteral, such as intradermal, subcutaneous, intramuscular, but in particular intravascular (especially intravenous), intralumbar, intracranial or intracavitary (eg into the abdominal cavity or other body cavities, in muscular fasciae or the like) injection or intravascular infusion ,

The enteral (eg oral) administration is particularly suitable for the treatment of diseases which are accessible from the intestinal, lung, pharynx, mouth and / or nasal lumen. Within the corresponding rooms, the superparamagnetic beads, for example coupled with active substances or active substance-carrying liposomes, can be applied to the desired ones by means of DM fields Places to be maneuvered in the body.

Parenteral administration is particularly suitable for the treatment of diseases that can be reached via the bloodstream (in particular infusion, intravascular injection), behind the blood-brain barrier are protected from the access of the active substance or from body cavities (eg abdominal cavity, interpleural gap, interfascicular wall) , Spinal fluid or the like) are accessible, and in the case of the body cavities there is in turn the possibility of maneuvering superparamagnetically labeled active ingredient or appropriately labeled active ingredient-containing liposomes to the desired locations by means of DM fields. Administration can be local (at the site of the disease to be treated, e.g. by injection) or systemic (e.g. by intravascular injection or infusion).

The warm-blooded animals, e.g. B. People of about 70 kg body weight, doses to be administered, expressed as the amount of active substance, vary depending on the species, age, individual condition, mode of application and the particular clinical picture and are in particular between about non-polymeric active ingredients (other than proteins or antibodies) 0.1 mg and about 10 g, preferably between about 0.4 mg and about 4 g, e.g. B. at about 1 mg to 1.5 g per person per day, divided into preferably 1 to 3 individual doses, the z. B. can be the same size. In the case of polymeric active substances, the dose, expressed as the amount of the active substance, is preferably between 0.05 and 50 mg, in particular between 0.1 and 10 mg, per person and day. Children are usually given half the dose of adults. If necessary, the treatment can be carried out as necessary to treat tumors and / or to prevent the formation of metastases.

For coupling active substances or liposomes (with or without antibody marking, loaded with active substance) (hereinafter both referred to as reaction partner A) to superparamagnetic beads (hereinafter referred to as reaction partner B), or from (in particular Infection-, parasite- or tumor-specific) antibodies (reaction partner A) on liposomes or superparamagnetic beads (reaction partner B in each case) which are loaded with active substance are commonly used methods, for example the cyanogen bromide activation, for example, of the surface of the beads when OH— is present. Groups, or the more gentle treatment with heterobifunctional coupling reagents, which initially contain reactive groups or those containing functional groups, in particular hydroxyl, amino, carboxy, epoxy, thiol or diene groups, which are present on the molecule to be coupled or the surface of the beads or liposo Forms thereof can react and then subsequently or in the same batch at essentially the same time with groups on the molecules or antibodies to be bound. Noncovalent binding is possible by coupling, for example, avidin or streptavidin to reaction partner A, biotin to the reaction partner B to be bound, or vice versa.

The covalent coupling can be carried out, for example, on epoxy groups or carboxy groups functionalized as activated esters (reactive form). The reactive carboxy groups can also be prepared in situ (for example using reagents customary in peptide chemistry, for example for the preparation of 1-hydroxybenzotriazole, succinimide or N-hydroxysuccinimide esters, or in-situ derivatization, for example with carbodiimides, such as dicyclohexylcarbodiimide, with carbonylimidazole, with N- [(dimethylamino) -1H-1, 2, 3-triazo-lo [4, 5-b] pyridin-1-ylmethylene] -N-methylmethanaminiumhexa- fluorophosphate-N-oxide (HATU ); with 2- (1H-benzotriazol-1 -yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate (HBTU), with 2- (pyridon-1 -yl) - 1,1,3, 3-tetramethyluronium tetrafluoroborate (TPTU ); or benzotriazole-1 -yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), or similar reagents). The reaction then takes place primarily with amino, but also hydroxyl or mercapto groups on the counterpart to be coupled. (I) Azide- or (ii) diene-modified reactants A or B can also be reacted with their respective complementary reactants, which in the case of (i) contain diene groups, in the case of (ii) azide groups, which together are suitable for a Diels-Alder coupling.

Examples of heterobifunctional reagents which can be used are those which comprise a group which reacts with amino, hydroxyl or mercapto groups and which contain a further group which is a disulfide and subsequently with the liberation of a mercapto group (for example with dithiothreitol or similar reducing agents) ) can be implemented. Other possible heterobifunctional reagents e.g. an amino reactive group and a photoactivatable group, e.g. N-hydroxysuccinimidoyl-4-azido-salicylic acid. Still other heterobifunctional reagents include e.g. one amino and one mercapto-reactive group, or two different amino-reactive groups, e.g. Succini idoyl-maleimide derivatives such as succinimidoyl-butylphenyl-maleicide, N-ε-maleimidocaproic acid or N- (ε-maleimidocaproic acid), or the like. An example of a hydroxy- and sulfhydryl-reactive heterobifunctional reagent is N- (ρ-maleimidophenyl) isocyanate.

As mentioned, superparamagnetic beads or other reaction partners B can be activated by cyanogen bromide, but can also be activated by epoxy, nitrophenyl chloroformate, N-hydroxysuccinimide or chloroformate groups, by polyacrylic acid residues (photo-activatable), epoxide groups, bromoacetyl groups, epichlorohydrin groups activation, tresyl chloride activation, vinyl sulfone activation, or the like.

New combination products (e.g. of antibodies with superparamagnetic beads, or of active substances with liposomes and / or with active substances) are also the subject of the present invention.

The invention relates in particular to the embodiments of the invention mentioned in the examples.

Illustrations Fig. 1: The Lorentz force is described by Antoon Lorentz himself as an electrical force and is indeed formally similar the electrical distance effect (coulomb force). The Lorentz force acts on electrical charges in a magnetic field. The demonstration experiment in FIG. 1 demonstrates the effect of the Lorentz force on ions. For this purpose, a dynamic, homogeneous magnetic field is generated (see WO 95/19217), which penetrates a transparent plastic container (3) filled with a mixture of table salt, water and sand (1), which is hermetically sealed from the environment. The DM field is guided through layered plates (also field return path plates (2)) with little loss. Layered laminated cores are not necessary at very low frequencies of the dynamic fields (eg 15 Hz or less). (4) is a symbolic closed field line, (5) shows an example of a groove section with a turn part (5) of the DM field generator (6). See Example 3 for further details. An electrical force, the Lorentz force, is therefore able to transport both positive and negative ions - which can be magnetic or non-magnetic - in the same direction. This cannot be achieved with the Coulomb force.

Fig. 2: This time only shows the magnetic aspects. A transparent plastic container (8), which is filled with water, is located above a DM field generator (6). A magnetite ball (7) can e.g. be moved longitudinally in and against the direction of the arrow. This is not possible with a piece of aluminum instead of the magnetite ball (7). (9) symbolizes a closed field line, (10) a slot section with a winding section. It is not possible to describe a quantum mechanical multi-particle problem exactly, but the purely magnetic character of the effect of the DM field can be demonstrated.

Fig. 3: The figure shows one of the possible applications of the invention. The sample (15) lying under the microscope (12) in front of its objective (13) on the microscope table (14) contains, for example, cells, cell components or pathogens and a relatively very small number (in extreme cases only 1) with superparamagnetic beads (above corresponding antibodies bound) labeled cells, Cell components or pathogens. DM field generators (11) integrated into the microscope table for generating the field structures can be seen lying on both sides of the sample. These can be 2 active DM field generators, a DM field generator and a magnetic return path or just a DM field generator without a return path. The sample is penetrated by a dynamic magnetic field, for example variable in amplitude, frequency and direction. The observer can now independently change, for example, the frequency, amplitude or direction of the dynamic magnetic field passing through the sample. This is achieved in that electronic circuits, similar to classic frequency converters, are controlled by control devices, for example foot pedals, switches or the like, or are controlled and switched. In this way, the marked objects are correspondingly influenced electromagnetically. For example, constant / non-constant rotations of the marked objects (conservation of angular momentum) can be achieved in a mathematically positive or negative sense. As a result, only a few, even individually marked objects can be recognized among a large number of unmarked objects. However, the dynamic magnetic field can also be divided into several independent static alternating magnetic fields by observing the control circuits, which results in a magnetic "solidification" of the observed objects. All this enables the observation of individually marked objects between a large number of other unmarked objects with a high selectivity.

Fig. 4: This figure shows (16) disordered marked objects (e.g. cells). These are set into rotational movements in (17) (see description of this in FIG. 3). Opposing rotational movements can also be achieved, or permanent changes in the rotational movement. Under (18) one

Structure generation (rod-shaped) of the marked objects shown

(For a description of this, see FIG. 3). It can be targeted

Moving movements of the marked objects can be achieved, through which other, also unmarked objects can be marked and transported. The following examples serve to illustrate the invention without restricting its scope.

Example 1: Labeling of lymphocytes Extraction of lymphocytes from blood: 20 IU heparin per ml blood are placed in a 20 ml injection syringe and venous blood is drawn up therein. About 4 ml of Macrodex 6% (from Knoll, Ludwigshafen, Germany) are added to this heparin blood and the syringe is placed in a stand at room temperature for about 1 hour. After this time, the almost erythrocyte-free supernatant is taken up in a second syringe with a cannula. The blood is mixed 1: 1 with phosphate-buffered saline (PBS) (150 mM sodium chloride, 150 mM sodium phosphate, pH 7.2). To completely isolate the lymphocytes, 3 ml of lymphocyte separation medium (Ficoll solution with a density of 1.077 g / ml) are placed in a centrifuge tube and 4 ml of the blood / PBS solution are carefully layered on the separation medium (carefully pipette to mix the phases avoid). After sealing with a silicone stopper, the gradient is centrifuged at 400 g (based on the center of the tube) for 30 min at room temperature (during centrifugation, care must be taken to ensure that the centrifuge's electric brake remains switched off during the run); 4 phases are formed: top layer of plasma, including an opaque whitish band (peripheral monocytic blood cells), then the lymphocyte separation medium and, as a pellet, the remaining erythrocytes with the granulocytes. The plasma is aspirated using a Pasteur pipette.

If desired, existing monocytes can be removed by transferring the layer with the peripheral monocytic blood cells into a Petri dish. The B and T lymphocytes remain in the supernatant here, while other cell types adsorb to the surface of the petri dish.

The cells in the supernatant or (if the monocytes are no longer removed) the aspirated plasma are then in a Buffer solution - PBS (as free as possible of calcium and magnesium ions to prevent cell aggregation with one another or on surfaces) with 2 M EDTA and 0.5% bovine serum albumin (BSA), hereinafter referred to as PBS * - and taken up by a nylon Mesh or a nylon filter (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) to remove lumps. The cells are counted and washed in the last-mentioned buffer solution by centrifugation. The pellet with the cells is then labeled with MACS MultiSort Microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), which are labeled with CD4 (or CD-8) antibody (it is anti-CD4 antibody (or with anti-CD8 antibody) conjugated beads with iron oxide in a polysaccharide matrix, diameter approx. 50 nm), incubated: The cells (10 ⁷ cells) are taken up in 80 μl PBS *. After adding 20 μl of MACS CD4 (or alternatively CD8) microbeads suspension (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), the mixture is incubated at 4 ° C. for 10 min. After the incubation, the cells are taken up in a 10 to 20-fold amount of PBS *, centrifuged at 300 × g for 10 min and, after the supernatant has been completely removed, the cell pellet is taken up in 500 μl buffer of 10 ⁸ labeled cells each.

The magnetic enrichment is then carried out by means of columns which are filled with beads made of plastic-coated ferromagnetic material in the presence of a magnetic field: An MS ⁺ column (Miltenyi) is placed in the magnetic field of a permanent magnet (separator from Miltenyi). The column is prepared by washing with 500 μl PBS *; the cell suspension prepared above is then applied. The unlabeled cells are washed out with PBS * (3 x 500 μl). The column is then removed from the separator, placed over a suitable collecting tube and washed out with 1 ml PBS *. The CD4 (or CD8) cells with magnetically labeled cells are obtained.

Example 2: Detection of magnetically marked cells under the microscope The magnetically marked cells produced in Example 1 are then set microscopically in motion (rotation or migration) using a DM field according to the invention (FIG. 3), as a device for producing the magnetic field structures DM field generator (11), 2 active field generators being used , or alternatively 1 active DM field generator and a magnetic return path, or even just a DM field generator without a magnetic return path. It is possible to move the marked cells in this way. This shows that the principle of the invention is actually applicable.

The sample under the microscope contains a large number of unlabelled cells and a smaller (depending on the sample also very small) number of labeled cells, marked with the corresponding antibody and the magnetite particles conjugated to it. For example, by causing constant / non-constant rotations of the marked objects in the mathematically positive or negative sense, constant / non-constant rotations of the marked cells are achieved. Even a few or even individually marked objects can be recognized among a multitude of others. The dynamic magnetic field is divided into several independent static magnetic fields by observer wiring of the control loops. In this way, the marked cells can solidify. Even so, individual marked cells can be recognized between unmarked cells that remain mobile.

Example 3: Model for the transport of salts within a body

In a plastic container (Fig. 1, (3)) filled with a mixture of salt, water and sand (1) (as a model for a body), it is shown that a DM field generator (6) moves ions by generating traveling fields can be. Through the action of the Lorentz force it is achieved that when a dynamic, homogeneous magnetic field according to WO 95/19217, which penetrates the plastic container (3) and is guided through layered plates (field return plates (2)) with little loss, more of the non-magnetic sodium and chloride ions on one side than on the other after completion of the experiment Side of the box. In other words, the experiment also shows that it is possible to produce concentration gradients of salts and thus, for example, to investigate the effect of such gradients on cells (for example macrophages, protozoa) in corresponding test arrangements.

Example 4: Model for the transport of magnetic particles in a body or in solutions, in particular for sorting superparamagnetically labeled cells and separating them from unlabeled cells

FIG. 2 shows a further arrangement with which the movement of a magnetic particle (here a paramagnetic ball (7) - here as a magnetite ball - as a model) is shown. This experiment is carried out in a German university as a double-blind experiment. About a field producer

(6) there is a transparent plastic container (8) which is filled with water. The ball is moved longitudinally in the direction of the arrow, for example, by movement feeders. With a non-magnetic piece of aluminum, this movement does not succeed under the same conditions.

Example 5: Movement of marked superparamagnetic

Beads

4 shows disordered superparamagnetic beads (Muteyi). By means of a (not shown) DM field generator, the disordered beads (16) are either set in motion by magnetic movement fields (e.g. rotation (17) or targeted movement (19)) or they become compact, here linearly expanded structure by means of static alternating fields - Ren trained (this would, for example, in a body, the closing of blood vessels using superparamagnetic shear beads to prevent the blood supply to a tumor or infected tissue and thus kill them). Targeted migration, alternating with rotation, of cells marked with superparamagnetic beads is also possible (19). This shows the applicability, for example, for the detection of specific cells (for example detection of tumor cells by means of antibodies labeled accordingly with superparamagnetic beads) or in particular for cell sorting (pulling out the labeled cells from a mixture with non-labeled cells).

Example 6: Movement of rotifers

A handful of hay is slurried from a lake with sand and water and left to stand for a few days, whereupon rotifers develop. These are in one

Eppendorf cup held and 10 ul of a slurry of 100-250 nm beads (magnetic polyalkylcyanoacryla particles from micromod particle technology GmbH, Rostock, Germany) added. The omnivores take up the beads. After a few hours, the animals are then moved under a microscopic device, as shown in FIG. 3, by applying DM fields.

Claims

claims:

1. Use of dynamic magnetic fields (DM fields), which can be generated by AC-supplied multiphase systems, for the detection and / or sorting of cells, cell components or pathogens to which superparamagnetic beads are bound.

2. Use according to claim 1, characterized in that the detection is used for the diagnosis of diseases of cells.

3. Use according to claim 3, characterized in that tumor cells or infected cells are recognized, to which superparamagnetic beads are bound via antibodies specific for these cells.

4. Use according to one of claims 1 to 3, characterized in that it is used extracorporeally.

5. Use of a dynamic magnetic field, as defined in claim 1, for the purification of liquids from pathogens to which superparamagnetic beads are bound.

6. Use according to claim 5, wherein the liquid to be purified is a blood preserve, plasma preservation or a cell culture medium.

7. A method for diagnosis, in particular for recognizing sick, especially infected, cells or tumor cells, which involves the use of dynamic magnetic fields, as in

Claim 1 defined, and comprising superparamagnetically labeled beads binding to said cells.

8. The method according to claim 7, characterized in that it is applied extracorporeally.

9. A method for treating diseased cells, in particular infected cells or tumor cells, which comprises the use of dynamic magnetic fields as defined in claim 1 and superparamagnetic beads which are present at the site of the infected cells or tumor cells or are bound to these.

10. The method according to claim 9, wherein the diseased cells are selectively marked by superparamagnetic beads, which are bound to the diseased cells via antibodies, and are exposed to movement stress by the application of a dynamic magnetic field, as defined in claim 1.

11. The method according to claim 9, wherein the diseased cells by superparamagnetic beads which are directly attached to a

Active substance are coupled or are labeled on liposomes which carry such an active substance and are transported to the site of the action by applying a dynamic field as defined in claim 1, and / or are so excited after marking the diseased cells, that the cells are increasingly exposed to the active ingredient.

12. The method according to claim 9, characterized in that it is applied extracorporeally.

13. Use of superparamagnetically labeled active ingredients for the manufacture of a preparation for use in a method for the treatment of infected cells or tumor cells, which comprises treatment with a dynamic magnetic field as defined in claim 1, which (a) administers at the location of said cells , (b) maneuvered there with a DM field or DM field generator and / or (c) are or are bound to the cells mentioned, in particular via antibodies bound to the beads and specific for antigens on the cells to be treated.

1 . Combination of superparamagically labeled active ingredients or superparamagnetic beads with a generator of a dynamic magnetic field, suitable for the treatment of infected cells or tumor cells.