WO2002043708A2

WO2002043708A2 - Magnetic particles for the targeted regional therapy

Info

Publication number: WO2002043708A2
Application number: PCT/EP2001/013926
Authority: WO
Inventors: Christoph Alexiou; Christian Bergemann
Original assignee: Christoph Alexiou; Christian Bergemann
Priority date: 2000-11-29
Filing date: 2001-11-28
Publication date: 2002-06-06
Also published as: WO2002043708A3; EP1339396A2; DE10059151C2; DE10059151A1

Abstract

The invention relates to magnetic particles that comprise a magnetic substance combined with a therapeutic substance. The particles according to the invention are used for the regional magnetotherapy of diseases by intra-arterial application.

Description

description

The present invention relates to magnetic particles for targeted regional therapy of diseases, in particular for treatment in humans. The invention is particularly suitable for regional treatment against Tu ore, local infections and local inflammation and similar local disease states.

The treatment of diseases is usually a balancing act between the effectiveness and the toxicity of the active ingredient used. This applies in particular to chemotherapy with the generally toxic cytostatics. A number of strategies have therefore been developed to address this dilemma.

A strategy called "Magnetic Drug Targeting" is based on binding therapeutically active substances to magnetic particles as a carrier system in order to enrich the therapeutic substance regionally or locally with magnetic field support and thus a higher effectiveness at the desired therapy location with a simultaneous reduction of systemically related side effects to achieve. These are special applications of the magnetic fluids developed for broad technical areas, the so-called ferrofluids.

Attempts to carry out targeted chemotherapy with magnetic, drug-laden albumin microparticles with the aid of magnetic fields are described by KJ Widder et al. in Eur. J. Cancer Clin. Onkol., Vol. 19, pp. 135-139 (1983) and PK Gupta and CT. Hung in N. Willmot and J. Daly (eds.), "Microspheres and Regional Cancer Therapy", pp. 71-116, CRC Press, Boca Rayton (FL) (1991). The active ingredient lies in the core coated with albumin before, but what makes phagocytosis necessary to release the drug.

DE-A-19 624 426 describes magnetic particles which comprise a core with nanocrystalline magnetic particles and a shell made of polymers with such reactive groups which are capable of covalent bonding or of ion exchange. It is proposed to use therapeutic agents such as e.g. to bind the cytostatics doxorubicin or mitoxantrone, for example via ion exchange reactions, to the magnetic particles and then to apply them intravenously in the form of a dispersion. With reference to other prior art documents, DE-A-19 624 426 initially mentions that - due to the structure and structure of those magnetic particles - e.g. is to be injected intra-arterially, but this should lead to considerable problems in clinical practice. The actual teaching of DE-A-19 624 426 therefore distances itself from such an application route and, according to the task, only considers intravenous application.

A.S. Luebbe et al. in Cancer Res., Vol. 56, pp. 4694-4701 (1996) preclinical experiments on animals using magnetic field-assisted drug targeting when administered intravenously. Furthermore, A.S. Luebbe et al. in Cancer Res., Vol. 56, pp. 4686-4693 (1996) describes studies on the biocompatibility of magnetic particles loaded with 4'-epidoxorubicin.

However, the actual therapeutic effectiveness of magnetic particles loaded with therapeutic substances has not yet been described. In particular, the abovementioned prior art does not reflect the conditions which are required for an efficient and effective regional or local therapy of diseases while at the same time minimizing systemically caused side effects. It was therefore an object of the present invention to provide technical means and ways to improve the regional or local therapy of diseases.

According to the invention, the object is achieved in that magnetic particles, which comprise a magnetic substance in combination with a therapeutic substance, are used for regional therapy of illnesses supported by magnetic fields by means of intra-arterial application.

The present invention and the preferred embodiments are explained in more detail below with reference to the accompanying figures.

1 shows the magnetic resonance image representation of tumors (VX-2 carcinoma) of rabbits after intraarterial (FIG. 1A) or intravenous (FIG. IB) application of magnetic particles and after 60 minutes of application of an external magnetic field (magnetic resonance 6 hours afterwards).

2 shows a histological section through the VX-2 door immediately after the magnetic field-assisted regional therapy according to the present invention.

FIGS. 3A, 3B and 3C show enlarged areas of the sections shown as fields A, B and C in FIG. 2.

4 shows the reversible binding of a therapeutic substance via ionic interaction to ionically charged groups of the polymer shell of the magnetic particles according to an embodiment of the present invention.

FIGS. 5A to 5G illustrate the effect of the regional therapy according to the invention, which is supported by a magnetic field using magnetic particles compared to various control groups and comparative samples.

6 shows an example of a side effect (leukocyte values) in connection with the inventive, field-assisted, regional therapy using magnetic particles in comparison with control groups and comparative samples.

FIG. 7A shows the absence of further side effects in the system according to the invention, in contrast to the occurrence of side effects in a control group that can be seen in FIG. 7B.

Fig. 8 shows schematically the quantitative enrichment of ⁵⁹ Fe with and without magnetic support. (Magnetic Targeting) in different tissues and organs.

9A shows the quantitative determination of the distribution of the active ingredient mitoxantrone by means of HPLC analysis with regard to the released active ingredient after intraarterial application of the active ingredient without exposure to magnetic fields in different tissues and organs.

9B shows the quantitative determination of the distribution of the active ingredient mitoxantrone by means of HPLC analysis with regard to the released active ingredient after intraarterial application according to the invention. of the active ingredient with magnetic field effects in different tissues and organs.

10A shows the distribution of the active ingredient mitoxantrone in percentages after intra-arterial application of the active ingredient without exposure to a magnetic field, based on the amount of mitoxantrone applied in different tissues and organs.

FIG. 10B shows the distribution of the drug mitoxantrone in percentages by fiction, _j jemäßer intraarterial Application of the active ingredient with magnetic field action based on the amount of mitoxantrone applied in different tissues and organs.

11 shows the quantitative determination of the distribution of the active ingredient mitoxantrone by means of HPLC analysis with regard to the released active ingredient after intravenous administration of the active ingredient in different tissues and organs.

12 shows the distribution of the active ingredient mitoxantrone in percentages after intravenous administration of the active ingredient based on the applied amount of mitoxantrone in different tissues and organs.

An essential criterion of the concept according to the invention is that the magnetic particles, which comprise the magnetic substance and the therapeutic substance combined, are present in an embodiment for intra-arterial application. It has surprisingly been found that the local enrichment at the desired destination and the regional effectiveness of the therapeutic substance are significantly improved and, at the same time, harmful side effects are minimized if intra-arterial administration is provided and not intravenously as in the prior art described at the beginning ,

The significantly better regional enrichment of magnetic particles loaded with a therapeutic substance for the targeted, regional therapy in the case of an intra-arterial application is compared to that in FIG. 1A

Result of an intravenous application (FIG. IB) in the form of magnetic resonance image representations using the example of a tumor in the rear limbs (VX-2 carcinoma) of rabbits shown. The magnetic resonance image was recorded 6 hours after magnetic particles, which were loaded with a cytostatic, were applied either intra-arterially or intravenously in suitable liquids and then an inhomogeneous magnetic field, the pole of which was aimed at the tumor site, was applied for 60 minutes. The tumor in the medial thigh area of the rear limbs is indicated by a dotted, oval line, the area marked with an "f" indicating the head of the thigh bone (femur). The defined extinction signals of the magnetic resonance imaging, which are visible in FIG. 1A and are restricted to the tumor area, confirms the high concentration of the magnetic particles loaded with the therapeutic substance in a stable state even 6 hours after application and targeted enrichment by means of magnetic force, during the comparison with the intravenous application shows almost no signal extinction in FIG.

As part of experimental investigations with magnetic

Particles that were radioactively labeled for quantitative detection were demonstrated that even 80% to 90% of the magnetic particles used according to the invention could be concentrated in the tumor or in the peritumor area when administered intra-arterially. This demonstrates the significant advantage of the present invention, according to which a reduced dose, preferably a substantially reduced dose of the corresponding therapeutic substance, is required for the same effectiveness of the conventionally applied therapeutic substance, which leads to considerably fewer side effects. Because of this excellent accumulation in the target area, a complete remission of the tumor could be achieved according to the invention using the example of a tumor even with a single application. No embolization due to the magnetic particles used was observed.

Furthermore, it was found that with the configuration according to the invention for intraarterial application of the magnetic particles loaded with the therapeutic substance, an excellent distribution of the magnetic particles through the entire tumor is reached. This is shown on the basis of the histological findings shown in FIGS. 2 and the associated FIGS. 3A, 3B and 3C. In the procedure according to the invention, the high amounts of magnetic particles are achieved over the entire tumor area, as can be seen from the overall sections of the tumor in FIG. 2 by means of the accumulations of the brown-black visible magnetic particles. There are closely localized areas with accumulations of magnetic particles of particularly high density, but also magnetic particles scattered and distributed over the entire tumor in lower density. A more detailed examination of the area marked as field A in FIG. 2 further shows (see FIG. 3A) that the blood vessel that supplies the tumor has a high intramural concentration of the magnetic that is oriented towards the magnetic field

Has particles. In the interstitial area of the tumor tissue (see field B, which is shown enlarged in FIG. 3B) there are also numerous, highly concentrated accumulations of magnetic particles (represented by arrows in FIG. 3B). Finally, the transition area between muscles and tumor tissue shown in FIG. 3C also shows a large number of highly concentrated collections of magnetic particles, which can be seen as black condensation inside and outside the tumor.

So that the magnetic particles for regional therapy of diseases can be administered via intra-arterial application, the magnetic particles are usually in a form suitable for intra-arterial application, suitably in a liquid known to the person skilled in the art and designed for infusion or perfusion. The liquid should be biocompatible and not immunogenic, especially sterile and pyrogen-free.

The magnetic particles can be used in view of the combination between the magnetic substance and the therapeutic see substance according to the invention basically in the form of two alternative configurations. In the first case the therapeutic substance is reversibly bound to the surface of the magnetic particles with a magnetic core substance, whereas in the second alternative the therapeutic substance is firmly connected to the magnetic substance. While the first-mentioned embodiment is intended for molecular substances that develop their therapeutic effect after the reversible binding has been separated, the second, alternative embodiment was primarily designed for radiation therapy, in which it is not the direct action of a dissociated molecular substance, but rather the emission a biologically effective radiation arrives.

In both configurations of the magnetic particles, it is advantageous to coat a core in which the magnetic substance is present with a polymer. Such a basic structure and modification options for magnetic particles are known per se, and so far reference can be made to known literature and sources of disclosure relating to the structure and production of corresponding magnetic particles or magnetic liquids (ferrofluids), in particular those in the present Disclosure with publications to be included in "Encyclopedia of Physical Science and Technology", Vol. 9, p. 321 ff., Academic Press, New York (1992) and DE-A-19 624 426 mentioned at the outset. to use a polymer as a covering material which is compatible in aqueous media, in particular in a physiological environment, and is used for stabilization in the aqueous medium and during use.

Examples of suitable polymers include: starch, starch esters or ether derivatives, their degradation products such as dextrins, furthermore dextrans, other polysaccharides, pectins, proteins such as albumin, casein, collagen, gelatin and their derivatives and Degradation products, as well as synthetic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acids or the like. In particular, starch, starch degradation products and derivatives, dextrans, dextrins, serum albumin and polyvinyl alcohol can be mentioned as particularly suitable polymers for the construction of the casing for the above-mentioned purpose.

The particle size of the magnetic particles, which can be determined by means of dynamic light scattering, is set within the scope of the invention with regard to the desired target location. It has been found that an excellent compromise can be achieved between the avoidance of a tendency to embolism, good mobility through the vascular system in connection with excellent enrichment and distribution at the desired target location with sufficient orientation and maneuverability by the magnetic field to be applied after intra-arterial application if the particle size is in the range from 20 nm to 500 nm and in particular in the range from 50 nm to 200 nm. The particle size is set in a manner known per se (see above sources), primarily by setting suitable manufacturing conditions for the magnetic particles (nanoparticles).

It was demonstrated that the procedure according to the invention made it possible to achieve complete remission of the tumor after the short duration of a single treatment. It turned out to be particularly advantageous that only a minor part, usually already at 10-50% of the conventionally systemically applied dose of the therapeutic substance used in each case, was able to achieve complete remission of the tumor without the occurrence of side effects. Depending on the dose, a 50% reduction in tumor volume resulted after 4 to 6 days (at a dose that corresponds to 20% of the conventionally systemically applied dose, there was a 50% reduction after 3 - 12 days (on average 6 days); in the case of a dose of 50% of the conventional systemically applied dose, there was a 50% reduction after 3 - 6 days (mean: 4.2 days). In particular due to the possibility of using the concept according to the invention to significantly reduce the dose of the therapeutic substance and, if necessary, to use only a single application, the side effects associated with systemic therapy are minimized or completely suppressed. None of the treated individuals developed side effects such as hair loss (alopecia), ulcers (ulcers) or muscle atrophy, and the general condition (weight, food intake, feces and urine output, activity) remained throughout the 3-month observation period compared to the physiological conditions of healthy individuals - viduen normal. No significant changes in serum iron levels or leukocyte levels were found. In contrast, the values and results in comparison and control groups were significantly worse. In the case of application of the therapeutic substance without magnetic substance, remission could be achieved at high doses (at 100% of the conventionally systemically used dose after 33 days and at 75% of the conventionally systemically used dose after 36 days), but hair loss developed ( Alopecia) after 33 days. There was also a blue-green urine stain, the muscles became atrophic, and the hairless area developed skin inflammation and ulceration. A reduction in the therapeutic substance used alone to 20 or 50% of the conventional systemically applied dose could not bring about remission of the tumor, and metastases developed on average after 48

Days. The side effects were less in this case, but the treatment was associated with weight loss and leukocytopenia. The other comparison group with the magnetic substance alone (ferrofluids) showed an extremely bad course of the disease, in which the tumor volume increased progressively, with palpable, increased Lymph nodes (metastases) after 45 days. The comparison with an intravenous application (20 or 50% dose of the therapeutic substance) showed only a very low tumor remission, whereby the volume reduction of the tumor was not statistically significant in comparison to the control group. In these investigations, the comparison conditions were identical in each case (ie identical magnetic substance (starch-coated iron oxide particles; particle diameter 100 nm); application of an inhomogeneous magnetic field with a maximum magnetic flux density of, for example, 1.7 Tesla and a corresponding magnetic field gradient).

The above-mentioned first embodiment of the magnetic particles used according to the invention is described in more detail below.

With this configuration, there are clear advantages if the therapeutic substance is reversibly bound to the polymer shell described above via ionic interaction, in which case the polymer applied to the magnetic core particles bears ionically charged groups. Because the therapeutic effects can be influenced particularly favorably, it is preferred according to the invention that the degree of association in the reversible bond between the therapeutic substance and the ionically charged groups of the polymer shell is set by suitable ambient conditions. These suitable conditions include, in each case individually or in combination, the temperature, the pH and in particular the osmolality. With these factors, a very rapid dissociation of the therapeutic substance from the magnetic carrier particles can be achieved in a favorable manner, with the result that the therapeutic substance after localization in the target location, e.g. a tumor that can act freely very quickly and to a therapeutically very efficient, abrupt increase in active

Active ingredient concentration leads. For example, after the intra-arterial application and the subsequent localization and enrichment using the magnetic field concentrated at the target site at a point in time at which the magnetic particles at the target site are sufficiently accumulated (approximately after 30 minutes to a few hours) the physiological ambient conditions are changed, that a dissociation of the ionic bond of the therapeutic substance is favored by the magnetic carrier. For example, depending on the type of ionic interaction, the desired dissociation can be promoted by a local temperature device or by the supply of physiologically tolerable infusion solutions, which are adjusted according to the respective factors such as pH and / or osmolality, preferably on the same intra-arterial solution Application point are fed.

Depending on the type of therapeutic substance to be used, the polymer of the shell can be designed or modified with ionically charged groups or ionizable groups in such a way that the ionic interaction can take place in a reversible manner. If the therapeutic substance has, for example, anionic groups, such as, for example, carboxylate, sulfate, sulfonate, phosphate or similar groups, the shell polymer should carry correspondingly positively charged or ionizable groups, for example due to the presence or incorporation of primary, secondary or tertiary groups Amino groups or quarterly ammonium groups or by imino groups can be realized. On the other hand, there is the possibility, in the case of therapeutic substances which have positively charged or ionizable groups such as primary, secondary or tertiary amino groups or quaternary ammonium groups, the correspondingly reversely charged or ionizable groups such as carboxylate, sulfate, sulfonate, phosphate or similar groups in the To provide or install envelope polymer. A particularly favorable combination is achieved when the polymer shell carries negatively charged phosphate groups and the therapeutic substance is reversibly bound to the phosphate groups via one or more amino or ammonium groups, because on the one hand, a stable association between the substance combination initially exists when the loaded magnetic particles are provided, but on the other hand enables rapid dissociation under physiological environmental conditions and at the same time very good tolerability and compliance with natural systems is realized. Starch polymers esterified with phosphate groups have proven to be very suitable polymer substances for coating magnetic cores.

Although the choice of therapeutic substance is fundamentally not subject to any restrictions, the concept according to the invention is best used if the therapeutic substance is to have a regional or local effect. Particularly suitable therapeutic substances include: low molecular weight, synthetic or natural or semi-synthetic drugs, antibodies, in particular monoclonal antibodies and genetically engineered, preferably human or partly human antibodies, furthermore peptides, angiogenesis factors, hormones, lymphokines and cytokines, lectins and oligonucleotides and DNAs such as Antisense oligonucleotides or DNA therapeutically used DNA substances, each as isolated DNA, in the form of

Plasmids or vectors or forms enclosed in suitable carrier systems can be present. A particularly useful area of application of the present invention is regional therapy against tumors, but also the treatment of local inflammation, local arteriosclerosis, local

Infections, especially fungal infections, and the like. In addition to a gene therapy approach, therapeutic substances, in particular antitumor agents, in particular cytostatics, and substances which can emit high-energy radiation or can emit by activation can be considered here. It is a very suitable cytostatic For example, to call mitoxantrone, which can be bonded to magnetic particles in a stable but inexpensively reversibly dissociable manner. The structure of such a magnetic particle is shown in FIG. 4 by way of example in the case of magnetic nanoparticles modified with phosphate groups.

The therapeutic substance used as an emitter of high-energy radiation can generally be a biologically active radiator and is preferably an α and / or a γ radiator or such a substance which, by activation, for example by a neutron source, is converted into an α and / or γ-emitter can be converted or transferred. In this context, boron or boron compounds can be mentioned as very promising therapeutic substances, which can be converted into an α-emitter by means of neutron activation. The use of boron or boron compounds can be an excellent way to realize with the inventive magnetic ^"carrier system, is described in detail below, and which thereby enabled Strahlenthera- pie is both very targeted as a local therapy feasible, and therefore offers itself as side effects of therapy.

In this embodiment, the therapeutic substance need not be reversibly associated on the surface of the magnetic particles, as was described above in the context of the first embodiment of the magnetic particles for the invention. When using a high-energy radiation or a therapeutic substance that can be activated for this purpose, it is preferred that the substance is integrated directly into the magnetic core of the particles or that the magnetic core is coated with the therapeutic substance. This can easily be achieved, for example, by forming suitable magnetic iron-substance alloys or compounds in the first case a solid chemical connection between the magnetic substance and the therapeutic substance are provided, the term "substance" including the element capable or activatable for emission of high-energy radiation, for example magnetic iron-boron alloys, iron borides, iron borates or magnetic, mixed oxides or mixed oxides such as boron ferrites, boron magnetites, or by coating magnetic particles of, for example, ferrites, magnetites or similar double oxides with the metal, the metal oxide or another compound of the

Element that is capable of or can be activated to emit intense radiation, e.g. with boron oxide, boric acid, borate ions or boric acid ester. In the alloy and connection examples mentioned above, alternatively or further elements, which may have a favorable influence on the magnetization, the material properties or the production, can be present in chemical compound, e.g. Nickel, cobalt, rare earth elements, silicon and / or carbon.

Otherwise, the above general descriptions for the first embodiment of the magnetic particles apply accordingly, in particular with regard to the optionally provided coating with suitable polymers, the preferably set particle size of the magnetic particles and the use of a preferred dose, which is reduced compared to the conventionally used radiation dose, the over the targeted regional therapy according to the invention can be implemented.

The magnetic field device that can be used for therapy support should generate an inhomogeneous magnetic field. For favorable target orientation of the magnetic particles, particularly inhomogeneous magnetic fields generating electromagnets with a pole maximum of up to 2.5 Tesla, preferably up to 2.0 Tesla, in particular in the range from over 1.0 Tesla to 1.8 Tesla, are suitable, starting from from the specified pole maximum sets a correspondingly strong magnetic flux gradient. Such strong magnetic fields are very efficient in combination with the magnetic particles described above. The pole maximum mentioned above should preferably occur at a distance of up to 20 mm, more preferably up to 15 mm, from the pole piece of the electromagnet. With such a configuration, there are particularly favorable magnetic flux and orientation conditions and in particular correspondingly favorable gradients that support the accumulation of the magnetic particles in the desired target organ or tissue. In order to focus the magnetic particles more efficiently in deeper areas of the body, for example in the case of pancreatic carcinoma, rotating magnetic fields can also be used advantageously.

The present invention is explained in more detail below with the aid of examples, which, however, are not to be interpreted as restrictive.

Examples and comparative examples

The magnetic particles used in the examples and comparative examples (magnetic nanoparticles for liquid compositions / ferrofluids [FF]) were produced in accordance with DE-A-19 624 426 (available from chemicell, Berlin, Germany). They consist of a colloidal dispersion of magnetic particles, which can be obtained by wet chemical processes from iron oxides and hydroxides to produce special multi-domain particles. The particles were encased in starch polymers modified with phosphate groups in order to stabilize them under various physiological conditions and to enable ionic binding to the therapeutic substance. The cytostatic agent mitoxantrone, which is cationic or positively ionizable, was used as a therapeutic substance Carries groups and is capable of reversibly binding to the starch polymers of the coating of the magnetite particles modified with esterified phosphate groups, with the formation of reversible ionic interactions.

Table 1 below shows the characteristics of the exemplary ferrofluids used.

Table 1

The combination used as a representative example between mitoxantrone and magnetic particles carrying phosphate groups is shown schematically in FIG. 1, the relevant chemical structures of the therapeutic substance and of the phosphate groups being shown in the figure broken down and enlarged. The positively charged NH ⁺ groups of the hydrochloride forms of mitoxantrone (MTX-HCl) are associated with the phosphate groups of the starch derivative at a pH of 7.4. The loaded ferrofluids contained 6.5 mg mitoxantrone per 10 ml of the liquid Composition. Since the binding of the therapeutic substance was reversible via the ionic interaction, the dissociation could be adjusted by means of suitable factors of the physiological environmental conditions such as pH, osmolality and temperature by changing the blood electrolyte concentration according to the specific requirements and the desired dissociation. In the experiments described below, the therapeutic substance almost completely dissociated after 60 minutes.

The efficacy tests were carried out on a meaningful animal model, namely in VX-2 squamous cell carcinoma established in rabbits (A. Hough et al., Am. J. Pathol., 87, p. 537 (1977); CS Galasko and DS Muckle, Br. J. Cancer, 29, p. 59 (1974)). After implantation in soft tissue, the tumor grows rapidly with increasing vascularity in the area. The animals soon develop (within 2 - 3 weeks) central tumor necrosis, locoregional lymph node metastases and hematogenous metastases in the lungs. Fragments of vital VX-2 were used to transmit the tumor.

Tissue with a size of 1 mm was taken from the tumor periphery of donor animals, transferred to a special medium (RPMI 1640, 2.0 g / 1 NaHC0 ₃ , L-glutamine) and immediately under sterile conditions in the rear limbs of anesthetized recipient rabbits (total number n = 26) implanted in the supply area of the femoral artery. Chemotherapy was started when the tumors reached a volume of approximately 3500 mm ³ . For chemotherapy, the animals were anesthetized with an intramuscular injection of 35 mg / kg body weight ketamine and 5 mg / kg body weight xylazine, then the thigh-related artery was cannulated and an indwelling catheter (0.8 mm diameter) was removed after the thighs had been removed. related vein from the saphenic nerve about 2 cm distal to the groin-related groove. Mitoxantrone-bound ferrofluids (FF-MTX) in different doses, mitoxantrone alone (MTX) or ferrofluids alone (FF) were administered by perfusion over a period of 10 min. For comparison, mitoxantrone-bound ferrofluids were also administered intravenously in various doses. No therapeutic substance was used in a control group.

Table 2 below shows an overview of the experimental protocol, "n" denotes the number of tumor-bearing animals examined in each group. The "Dose" column indicates the proportion of therapeutic substance compared to the normal systemic mitoxantrone dose (10 mg / m ² ) that was administered by a single application. The intra-arterial application was via the femoral artery, the intravenous application via the ear vein. The external magnetic field was concentrated over the tumor.

Table 2

To generate an inhomogeneous, external magnetic field, an electromagnet with a magnetic flux density in the

Maximum of 1.7 Tesla used. The magnetic flux density was focused on the tumor region with a specially adapted pole shoe, which was brought into contact with the surface of the tumor. The magnetic flux density in the area of the tumor surface was about 1.7 Tesla and about 10 mm below the tip of the pole piece about 1.0 Tesla. The magnetic field was concentrated over the tumor during the application of the infusion solution described above and applied for a total of 60 min.

For blood testing, blood samples were taken once a week by venipuncture and centrifuged at 2000 x g for two hours. Immediately after taking the blood, measurements of various parameters in clinical chemistry (iron, alanine aminotransferase, aspartate ainotransferase, γ-glutamyl transferase, alkaline phosphatase and lactate dehydrogenase) and blood count parameters (cell values of whole blood and differential blood) were carried out.

The statistical evaluation was as follows:

The tumor volume was calculated using the formula for elliptical masses (1/6 πa ² b, a = width of the horizontal axis, b = length of the vertical axis). Volume changes were expressed as percentages related to 100% tumor volume at baseline. Statistical analyzes of the relative tumor volumes were performed using the one-time sample t-test (with a conservative, fixed value of 100% for the control group) and a t-test for two independent samples (Welch test). For blood parameters (absolute values), the t-test was used for two independent samples. The resulting bilateral p-values were considered significant at 0.05 or less. The result was significant at values from 0.01 to 0.05 and highly significant at values below 0.01. The p-values were calculated on a Microsoft Excel version 97 using the "Statistical Package for Social Sciences" (SPSS), version 9.0.

The results for the groups shown in Table 2 above are shown graphically in the respective FIGS. 5A to 5G, the development of the mean tumor volume (with the maximum and minimum values shown as bars) is plotted against the time curve. The term "metastases" means the beginning of metastasis formation. Accordingly, "alopecia" means the beginning of the hair loss found. "Treatment" indicates the initial, one-time treatment.

The results of groups la and 1b according to the invention with the intra-arterial application of a dose of only 20% or 50% of cytostatic agent (mitoxantrone), compared to the systemically used dose, which were reversibly bound to magnetic particle carriers by means of ionic interaction , show a highly significant reduction in tumor volume after only a few days (see FIGS. 5A and 5B). Neither metastasis nor alopecia were observed. In contrast, intraarterial administration of mitoxantrone alone was associated with poor results, since a reduced dose compared to systemic doses (20 or 50%) did not lead to tumor remission but led to metastasis, whereas higher doses (75 or 100%) did Reduced tumor volume, but started much later over time and led to alopecia. (see FIG. 5C) Ferrofluids alone showed practically no therapeutic effects. The corresponding FIG. 5D shows a progressive increase in the tumor volume with palpable, enlarged lymph nodes in the groin area (metastases). Furthermore, the comparison with the intravenous application (groups 4a and 4b, FIGS. 5E and 5F) showed no statistically significant tumor remissions in comparison to the intraarterial application according to the invention. 5G shows in relation to comparison group 5 the importance of the support by a magnetic field, in which the procedure was as in groups la and lb according to the invention, but without applying the external magnetic field. Further tumor growth and metastasis formation could be prevented, however no remission of the tumor was observed. At the time of treatment, less than 5% of the animals showed a low necrotic fraction in the area of the tumor.

The results on the local and systemic side effects are summarized in FIGS. 6A and 6B. In control group 6, the general condition deteriorated during the observation period. The tumor-infected animals developed pneumonia, which explains the increase in the number of leukocytes observed in FIGS. 6A and 6B with respect to the control group. In contrast, there were no significant changes in the leukocyte values in groups la and lb according to the invention (see FIGS. 6A and 6B). The other parameters examined also remained normal in the observation period of three months. 7A clearly shows the normal appearance of the hind legs, which is also visible from the outside, after the tumor treatment of the group according to the invention. In the comparison groups, some strong side effects were observed, such as the alopecia symptoms mentioned above, urine discoloration, inflammation symptoms, ulceration and weight loss. In contrast to the externally normal appearance in the group according to the invention (cf. FIG. 7A), the strong side effects of an intra-arterial application with the cytostatic alone (comparison group 2) can clearly be seen in FIG. 7B.

For histological assessment and for magnetic resonance imaging (see FIGS. 3 and 1), the tumor was removed for 60 min immediately after the single application of 50% FF-MTX in the femoral artery and the application of the magnetic field, and in 3, 7% formalin fixed. 5 μm thick paraffin sections of the tumor were cut and stained with hematoxylin and eosin. The histological staining led to the histological findings shown in FIGS. 2 and 3A to 3C. In addition, magnetic resonance image analysis was carried out on four tumor-bearing animals after a corresponding application and 6 hours after application of the external magnetic field. The images were carried out at 1.5 Tesla using a clinical magnetic resonance scanner (ACS-NT; Philips). A fat suppressing Tl-weighted turbospin echo sequence was used for the image display (TR 535, TE 20, echo train sounds 5). This led to the magnetic resonance image depictions shown in FIGS. 1A and IB.

Attempts to quantify the magnetic particles or released active substances in various tissues and organs were also carried out. The results will be explained below with reference to FIGS. 8 to 12.

FIG. 8 shows the quantitative enrichment of ⁵⁹ Fe with and without magnetic field support (magnetic drug targeting). With the influence of a magnetic field, ⁵ times (equivalent to magnetic particles) could be accumulated in the tumor 34 times more, and even more than 236 times more peritumally. This is very important especially for the sano situation (ie the tumor has been completely recorded, including the tumor extensions). There is also a much lower accumulation in the liver, spleen and bone marrow. The data in FIG. 8 always relate to activity (Bq) / gram of tissue.

FIGS. 9A and 9B show the HPLC analyzes with regard to the released active substance mitoxantrone after intraarterial application without exposure to a magnetic field (FIG. 9A) and with

Magnetic field exposure (Fig. 9B) specified. The relative indication of mitoxantrone (μg) / gram of tissue results in a 6-fold greater accumulation in the tumor, a 9-fold greater peritumoral and a 5-fold greater accumulation in the lymph nodes affecting the same side in the case of the invention Application (Fig. 9B) compared to the comparison values without magnetic field (Fig. 9A).

The comparison shown in FIGS. 10A and 10B results in relation to the amount of mitoxantrone applied

(5 mg / m ² body surface) in the tumor a 120-fold stronger enrichment with intra-arterial application with magnetic field action compared to intra-arterial application without magnetic field action. Peritumoral results in a 30-fold greater concentration of active ingredient.

The comparative experiments shown in FIGS. 11 and 12 show that the intravenous administration of the active ingredient mitoxantrone results in almost no accumulation in the corresponding muscle area. On the other hand, there is a strong concentration in the liver, kidney and spleen.

Claims

claims

1. Magnetic particles, which comprise a magnetic substance in combination with a therapeutic substance, for the magnetic field-assisted, regional therapy of diseases by means of intra-arterial application.

2. Magnetic particles according to claim 1, characterized in that it comprises a core in which the magnetic substance is present and a shell made of a polymer.

3. Magnetic particles according to claim 1 or 2, characterized in that the particle size of the magnetic particles is greater than 50 nm and less than 200 nm.

4. Magnetic particles according to one of the preceding claims, characterized in that the therapeutic substance is present in one dose, calculated on the basis of the particle-bound amount, which corresponds to 10 to 50% of the conventionally systemically applied dose.

5. Magnetic particles according to claim 2, characterized in that the therapeutic substance is reversibly bound via ionic interaction to the polymer shell which carries ionically charged groups.

6. Magnetic particles according to claim 5, characterized in that the polymer shell carries negatively charged phosphate groups and that the therapeutic substance is reversibly bound to the phosphate groups via one or more amino or ammonium groups.

7. Magnetic particles according to claim 5 or 6, characterized in that the polymer shell is formed from starch polymers esterified with phosphate groups.

8. Magnetic particles according to one of claims 5 to 7, characterized in that the degree of association of the reversible bond between the therapeutic substance and the ionically charged groups of the polymer shell is set by conditions of temperature, pH and / or osmolality.

9. Magnetic particles according to one of the preceding claims with a cytostatic agent as a therapeutic substance.

10. Magnetic particles according to claim 9, characterized in that the cytostatic is mitoxantrone.

11. Magnetic particles according to one of claims 1 to 8 with a DNA substance as a therapeutic substance.

12. Magnetic particles according to one of claims 1 to 8, characterized in that the therapeutic substance is a biologically active radiator or can be converted into a biologically active radiator by activation.

13. Magnetic particles according to claim 12, characterized in that the therapeutic substance is an α and / or a γ emitter or can be converted into an α and / or a γ emitter by activation.

14. Magnetic particles according to claim 13, characterized in that the therapeutic substance is boron or a boron compound which can be converted into an α-radiator by means of neutron activation.

15. Magnetic particles according to one of claims 12 to 14, characterized in that the therapeutic substance is integrated in the magnetic core of the particles, or that ^" the magnetic core is coated with the therapeutic substance.

16. Magnetic particles according to one of the preceding claims, characterized in that the therapy is supported by an inhomogeneous magnetic field with a pole maximum of over 1.0 Tesla to 2.0 Tesla.

17. Magnetic particles according to claim 1, characterized in that the magnetic particles are present in a liquid designed for infusion or perfusion.

18. Use of magnetic particles according to one of claims 1 to 17 for regional therapy against tumors.

19. Use of magnetic particles according to one of claims 1 to 17 for regional therapy against local infections.

20. Use of magnetic particles according to one of claims 1 to 17 for regional therapy against local inflammation.