WO2008101469A2 - Pharmaceutical preparation for treating metastases - Google Patents

Abstract

The invention relates to novel pharmaceutical preparations and their use for the production of drugs for treating metastases, based on liposomes. These preparations are liposomal formulations. According to the invention, these formulations comprise at least one substance having an anti-tumor effect and at least one substance having an anti-coagulation effect, in addition to potentially present further agents. Particularly they are made of base lipids and optionally further components, such as helper lipids, membrane stabilizers, and charge carriers, and may have a modified surface for improving their effectiveness.

Description

 Pharmaceutical preparation for the control of metastases

The invention relates to a pharmaceutical preparation for combating metastases based on liposomes. Areas of application of the invention are the medicine and the pharmaceutical industry.

The cause of a still high mortality rate in tumor diseases - despite improved early detection and therapy of the primary tumor - the metastasis, which is hardly influenced by local surgical or radiotherapeutic treatment, and with systemic chemotherapy only conditionally. The success of tumor therapy thus depends essentially on a comprehensive prevention of metastasis, which is triggered by circulating tumor cells. Metastasis can occur via two processes. On the one hand, a single circulating tumor cell undergoes a complex cascade of different, causally dependent adhesion processes before it can be fixed in a distal tissue ¹ . The initial step of adhesion is mediated by the endothelial receptor E-selectin, which recognizes the carbohydrate ligands sialyl Lewis "(sLe ^x ) and sialyl Lewis ^a (sLe ^a ) found on various metastatic cancer cells, through ligand-receptor interactions weak affinity binding of the tumor cell to the endothelium as a prerequisite for the subsequent steps ("rolling along" the membrane, release of further mediators, firm adhesion and invasion into the surrounding tissue). Finally, the tumor cell nests in the secondary tissue, initiates the formation of a neovascular blood system and proliferates ² .

In addition to this mechanism described for single circulating lines, on the other hand, the spontaneous formation of tumor cell microthrombi is relevant for the metastasis process ³ . Especially in narrow capillary systems (lungs, lymph nodes), these complexes can contribute to a local accumulation of the tumor cells, which in turn leads to metastasis through individual cells ⁴ . In addition, several studies have shown that activated platelets in particular attach to tumor cells and thus contribute to the shielding of immune reactions and the formation of microthrombi ⁵ . As a way to block the metastasis cascade, there are several targets that play an essential role in this process. These include the adhesion molecules on the endothelial surface and / or the ligands that are expressed by the tumor cell. These targets can be switched off ⁷ by competitive binding of suitable inhibitors (antibodies, carbohydrates) ⁶ or by inhibition of its expression with antisense or gene knockout strategies. Other approaches are aimed at inhibiting angiogenesis. This also essential process of formation of new blood vessels to supply the newly forming metastases follows the adhesion cascade.

Despite these advances in understanding metastasis, the worldwide search for new cancer drug-targeting and anti-cancer drugs has not yet led to routine use.

Compared to the prior art, therefore, has the task to develop a novel anti-tumor drug on the basis of liposomes for the prevention of metastasis of tumors.

This object is achieved according to claims 1 and 12 of the present invention, the subclaims are preferred variants.

The heart of the invention is a pharmaceutical preparation comprising liposomal formulations containing at least one substance with antitumor effect and at least one substance with anticoagulant activity and optionally other substances.

An important starting point of the invention is the elimination of the formation of aggregates of tumor cells with each other or from tumor cells and platelets, which is considered an essential prerequisite of tumor cell engraftment. This is to be achieved by surface-modified, drug-containing liposomes. In addition, under certain conditions, the adhesion of the tumor cells to the blood vessel wall can be prevented by liposomes as an alternative step of a secondary tumor cell colonization. The substances with antitumor action used in the context of the invention are in particular those substances which negatively influence or inhibit the formation, growth and / or spread of tumors. For this purpose, substances from the group of cytostatics, such as alkylating agents, mitotic inhibitors, cytostatic antimetabolites, antibiotics, hormones and hormone antagonists, as well as alkylphospholipids come into consideration. Particularly suitable are alkyl phospholipids, such as tetradecylphosphatidylcholine (TPC), hexadecylphosphatidylcholine (HPC), heptadecylphosphatidylcholine (HpPC), octadecylphosphatidylcholine (OPC), hexadecyl (1, 1-dimethyl-piperidin-4-yl) -phosphate (HPP), octadecyl (1, 1-dimethyl-piperidin-4-yl) -phosphate (OPP), hexadecylphosphatidylserine (HPS), hexadecylphosphatidylethanolamine (HPE) or edelfosine, as they can be conveniently incorporated into the membrane of the liposomal formulation due to their structure.

As substances with anticoagulant effect are used: substances from the drug group of aggregation inhibitors, such as dipyridamole, acetylsalicylic acid, ticlopidine, resveratrol, anticoagulants, such as heparin, low molecular weight heparin, heparinoids, clopidogrel, the vitamin K antagonists, such as warfarin or phenprocoumone.

Particularly suitable substances with anticoagulant activity in the context of the invention have proven to be the active substances dipyridamole and acetylsalicylic acid.

The liposomal formulation used to implement the invention is constructed from known ingredients. It consists of base lipids and additionally preferably contains helper lipids, membrane stabilizers and charge carriers, whereby a particularly favorable stability of the preparation is achieved.

As base lipids in the context of the invention are in particular soya phosphatidylcholine, egg phosphatidylcholine (EggPC), hydrogenated phosphatidylcholine (HePC), dimyristoyl-phosphatidylcholine (DMPC), (dipalmitoyl-phosphatidylcholine) (DPPC), distearyl-phosphatidylcholine (DSPC), stearyl palmitoyl-phosphatidylcholine (SPPC), palmitoyl-stearyl-phosphatidylcholine (PSPC), dioleyl-phosphatidylcholine (DOPC), palmitoyl-oleyl-phosphatidylcholine (POPC) or sphingomyelin.

Helper lipids in the context of the invention are preferably dioleyl phosphatidylethanolamine (DOPE) and dioleyl phosphatidylcholine (DOPC).

As a membrane stabilizer for the realization of the invention, cholesterol (CH) is used.

The charge carriers used are preferably negative charge carriers, such as e.g. Dicetyl phosphate (DCP), stearylamine (SA), phosphatidic acid (PA), phosphatidylglycerol (PG) or monosialoganglioside.

It has proven to be useful to also add a substance for modifying the surface. Thus, the liposomal formulation can be adapted to the requirements of a targeting.

The substance used for modifying the surface of the liposomal formulation is preferably polyethylene glycol-phosphatidylethanolamine (PEG-PE). However, PEG sterols, sialyl Lewis ^x -Peg-PE, sialyl Lewis ^A -PEG-PE, sialyl Lewis ^x -Peg sterols or sialyl Lewis ^A -PEG sterols are also effective. These compounds increase the hydrophilicity of the liposomal surface, thereby sterically shielding the vesicles from the monocyte / phagocyte system and preventing the vesicles from being recognized as foreign bodies and eliminated by the immune system. In addition, such a surface increases the affinity for platelets, whereby a nonspecific targeting effect is achieved. By additional conjugation of specific ligands such as Lewis ^{x or A} specific targeting to the site of action (platelets, endothelial membrane) is further increased.

In a particularly suitable embodiment of the invention, the pharmaceutical preparation of the constituents phosphatidylcholine (30-35 .mu.mol), octadecyl (1.1-dimethyl-piperidin-4-yl) -phosphate (10-15 .mu.mol), cholesterol (10-15 .mu.mol ), Po! yethylenglykol ₂ ooo-phosphatidylethanolamine (2-3 .mu.mol), dicetyl phosphate (5-8 .mu.mol) and dipyridamole 15-20 .mu.mol) produced (. Particularly suitable are liposomal formulations in which the liposomes have an average diameter smaller than 400 nm and the size distribution has a polydispersity index smaller than 0.4 to reduce the elimination by the monocyte / phagocytic system, thereby causing accumulation in the aggregates Platelets and tumor cells is promoted.

The liposomes are preferably prepared by classical methods, ie via lipid film hydration to produce the multilamellar vesicles, then extrusion to obtain small vesicles of defined size.

More specifically, the stock solutions of the individual substances are mixed together and a lipid film is prepared by evaporation or other suitable methods. From this one obtains by the application of known methods, e.g. Rehydration, multilamellar vesicles, which are then subsequently converted by suitable methods, preferably extrusion, into a homogeneous suspension of small vesicles having a diameter of about 100-150 nm.

The pharmaceutical preparation is suitable for the manufacture of a medicament for the treatment of diseases such as thromboembolic disorders and coagulation disorders, preferably when metastatic tumor diseases occur in this context.

The pharmaceutical preparation for the treatment of metastatic liposomal-based tumors according to the invention is completely novel.

It could be shown that the combination liposomes contained in the pharmaceutical preparation are stable over a sufficient period of time, inhibit aggregate formation, inhibit the adhesion of tumor cells to the endothelial surface and endothelial components, influence the distribution of metastases in animal experiments, and restrict metastasis and thus the metastasis number and the metastasis Reduce total tumor weight.

The liposomes used have significant advantages over other drugs. When using surface-modified liposomes, an accumulation in tumor cell platelet aggregates is achieved, whereby a targeted Release of the trapped drugs in close local proximity to the targets is achieved.

A second important advantage is the possibility of the simultaneous inclusion of several active ingredients with different active principles in the liposomes, whereby a targeted and simultaneous enrichment of all drugs (even with different pharmacokinetics of the individual components) is achieved in tumor cells and this can be attacked efficiently.

Novel and original to the combination liposomes according to the invention is that they have a modified surface and have included a cytostatic and an anticoagulant. This synergistically prevents both the binding to the endothelium (metastasis cascade) and the formation of tumor cell aggregates (embolic model). Despite the complexity of preparation of these vesicles, these liposomes can be prepared and modified in their properties (charge, stability and binding of ligands) and adapted to the requirements of a specific targeting.

By combining blockage of binding ability, cytotoxic effect and inhibition of complex formation by an anticoagulant in a vesicle, a strong, additive or even synergistic effect occurs.

The advantage of this new drug system is that it can be used on a variety of tumors, thereby reducing the high metastatic mortality of tumors by synergistically inhibiting the process of metastasis through various mechanisms:

- Reduction of aggregation capacity of platelets.

- Blocking the binding ability of circulating tumor cells

Suppression of the formation of tumor cell or tumor cell / thrombocyte aggregates,

- Killing of tumor cells by release of Kanzerostatika in the immediate vicinity of the cell.

The pharmaceutical preparation according to the invention represents a completely novel approach to the inhibition of metastasis, which promises extraordinarily high efficacy through the complex and local approach. The invention will be explained in more detail by exemplary embodiments.

Exemplary embodiments 1 - 7

It is shown in Examples 1-6 how various vesicles are prepared which have one or more active agents included in effective amount. This is followed by the characterization of physicochemical properties, storage stability and in vitro biological activity.

Example 1: Liposomes (vesicles with a diameter <200 nm and uniform size distribution) are prepared by known methods. In these vesicles, a cytostatic, z.Bsp. included the alkylphospholipid octadecyl-1, 1-dimethylpiperidin-4-yl-phosphate (OPP), doxorubicin or mitoxantrone to specifically damage tumor cells. In addition, an anticoagulant (AK) is encapsulated in these liposomes to dissolve tumor cell aggregates. Size and size distribution (quasi-elastic light scattering) and the current drug concentration (HPTLC) are determined. In addition, the stability of the liposomes at 4 ⁰ C in buffer and incubated at 37 ° C is characterized in serum over size changes and drug release.

A typical approach is explained in the following example:

The following components are pipetted as stock solutions into a 50 ml round-bottomed flask: 31.2 μmol phosphatidylcholine (PC), 12 μmol octadecyl- (1.1-dimethylpiperidin-4-yl) -phosphate (OPP), 12 μmol cholesterol (CH), 2 , 8 μmol polyethylene glycol ₂ ooo-phosphatidylethanolamine (PEG-PE), 6 μmol dicycetic phosphate (DCP) and 15.9 μmol dipyridamole (DIP). The lipid film is prepared by evaporation of the solvent at 42 ⁰ C in vacuo and then resuspended with 2 ml of PBS, pH 7.4 again. The suspension becomes yellowish turbid and consists of MLV, which are shaken overnight. The MLVs are then repeatedly forced through a mini extruder containing two pooled polycarbonate filters of 400 nm pore diameter. After 19-fold extrusion, completely large unilamellar vesicles (LUVs) with a mean diameter of 131 ± 28 nm are present. The suspension has a polydispersity index (PI) of 0.14 ± 0.07. The non-included DIP is separated by size exclusion chromatography on a Sephadex column (G50 fine). The content of PC is determined by high performance thin-layer chromatography (HPTLC) and after column separation is 16.4 ± 0.1 μmol / ml, while about 150 ± 24 μg DIP / ml liposomes are included.

The liposomes can be stored in the refrigerator for at least 8 weeks without their properties changing significantly (maximum size increase of 6% and a rise in the polydispersity index PI by a maximum of 30%).

Example 2: Liposomes with dipyridamole and mitoxantrone

In a typical approach, liposomes having a total lipid content of 42.4 μmol / ml are prepared by a known method. For 21, 6 .mu.mol PC, 12 .mu.mol CH, 6 .mu.mol DCP, 2.8 .mu.mol PEG, 8 .mu.mol dipyridamole and 4 mg mitoxantrone in the solvent mixture dichloromethane / methanol (7: 3, v / v) dissolved, the solvent removed in vacuo to obtain a thin lipid film. This is resuspended in PBS (without Ca, Mg), the suspension shaken overnight at room temperature to obtain multilamellar vesicles.

The liposomes are extruded 19 times by means of mini extruders equipped with two polycarbonate membranes with a pore diameter of 400 nm to obtain a uniform size distribution of the liposomes. Subsequently, the liposomes are passed through a Sephadex G50fine column to separate by size exclusion chromatography the unencapsulated DIP and mitoxantrone.

The size and polydispersity index (PI) of the liposomes is determined by PCS. The mean size of the liposomes is 123 nm, the PI for these liposomes being 0.134.

The trapped MTX is determined by HPTLC. It is up to 850 μg / ml liposomal suspension for these liposomes. This corresponds to an inclusion rate of 20%.

Example 3: Liposomes with dipyridamole and doxorubicin

In a typical approach, liposomes having a total lipid content of 32 μmol / ml are prepared by a known method. For this purpose, first 15.6 mM PC, 6 mM CH, 3 mM DCP, 1.4 mM PEG and 8 mM dipyridamole are dissolved in a round bottom flask in an organic solvent and then the solvent is removed in vacuo to obtain a thin lipid film. This is in citrate buffer pH 3.8 resuspended, the suspension shaken overnight at room temperature

To obtain multilamellar vesicles (MLV).

The MLV are extruded 19 times by means of mini extruders equipped with two polycarbonate membranes with a pore diameter of 400 nm, to obtain small vesicles with a uniform size distribution.

Doxorubicin is encapsulated via the well-known pH-jumping technique. For this purpose, 800 .mu.l of the liposome suspension are brought to a pH value of 7.6 by means of NaOH solution and subsequently heated in a water bath at 60 ⁰ C. Then be

Added 140 μl solution containing 3.5 mg doxorubicin in physiological

Saline, contains. Unencapsulated doxorubicin is by

Size exclusion chromatography on Sephadex G50fine separated.

The size and polydispersity index (PI) of the liposomes is determined by PCS. The mean size of the liposomes is 182 nm, with these liposomes having a value of 0.263.

The trapped doxorubicin is determined by HPTLC. It is up to 950 μg / ml liposomal suspension for these liposomes.

Example 4: Stability of the liposomes

After preparation of the liposomes they are stored at 4 ° C. Under these

Conditions did not become significant over a period of more than 100 days

Change in mean liposome size and polydispersity index.

The changes were only 7% (increase in diameter) or 11%

(Polydispersity index).

Example 5: Efficacy of liposomes is in vitro

The effectiveness of the liposomes is characterized in vitro in various test systems. The influence of liposomes on the adhesion of tumor cells and platelets is determined under static conditions in the binding assay. In addition, the influence of liposomes on the aggregation behavior of tumor cells in the absence and presence of platelets, and the complex formation between tumor cells and platelets as an essential step of metastasis is used as test systems in vitro. The following are some selected examples of test systems:

Example 5.1. Inhibition of platelet aggregation by liposomes

100 μl of platelet-rich plasma (PRP) with a platelet count of 2.5 × 10 ⁸ / ml are added per well to a 96-well plate and activated with 0.05 × thrombin / well. In some wells, the activation is omitted in order to be able to carry unactivated platelets as a control. In order to inhibit aggregation, in the next step the liposomes (100 nmol total lipid) or the free substances are added in equimolar amounts and the increase in transmission is measured photometrically at λ = 595 over a period of 120 minutes.

Activation of the platelets with thrombin leads to a decrease in extinction and an increase in transmission as a result of increasing platelet aggregation. The results are given as a percentage change in transmission with respect to the first measured value (t = 0, transmission = 0%) calculated according to formula (I).

EMW ⁼ absorbance at the measured time (t = x min) absorbance at the time of the first measurement (t = 0)

Result: Inhibition of aggregation by the combination liposomes can be achieved by 40%, while no inhibition is observed by control liposomes (see Figure 1). DlP as a free drug inhibits platelet aggregation by 30% at a concentration of 10 nmol / μl (data not shown).

Example 5.2. Inhibition of the adhesion of platelets to immobilized tumor cells

5.2.1 Labeling of platelets with calcein-AM:

2.5χ10 ⁸ platelets / ml are mixed with 50 ul Calcein-AM stock solution 0.5 mM, in DMSO) and incubated for 90 min at 37 ⁰ C. Subsequently, the platelets are washed three times with buffer solution (preferably with TH buffer, pH 7.4). washed. The fluorescence-labeled platelets are obtained by centrifuging the cell suspension at 1000 g ^-1 for 10 min each and can be quantified by measuring the fluorescence intensity in the fluostar at an excitation wavelength of λe _x = 490 nm and the emission wavelength λ = 530 nm over a standard curve ,

5.2.2 Adhesion test

In a 96-well plate, 100 .mu.l of a suspension of 1x10 ⁶ tumor cells / ml RPMI medium with 10% FCS pipetted and incubated overnight for attachment in the incubator at 37 ⁰ C and 5% CO ₂ . Thereafter, the medium is withdrawn and the attached cells are washed with PBS. To block unspecific binding sites, the cells are incubated for one hour with 100 .mu.l of a 1% BSA solution at RT. Then the supernatants are removed and the cells washed again with PBS. In the next step, 100 μl of the suspension with 2.5 × 10 ⁸ / ml platelets are then added to each well and the platelets (PRP) are activated by additional addition of 0.05 μl thrombin. In order to inhibit the adhesion, in the next step the tumor cells and platelets are incubated with liposomes (100 nmol total lipid / well) or 10 μl of a 1 M EDTA solution / well as control. In parallel, the seeding and serial dilution of fluorescently labeled platelets into a black Maxi-Sorp plate (Nunc) and their activation by 0.05 μ thrombin are performed to obtain a standard series for the quantification of bound platelets.

After 1 h of incubation at 37 ^° C., the supernatants are removed from the test plate and the cells are washed twice with PBS. Then the addition of 50 .mu.l lysis buffer / well into the control plate and of 50 .mu.l lysis buffer and 50 .mu.l PBS per well of the experimental plate. The plates are shaken for 3 minutes before the content of the test plate is transferred well to well in the measuring plate and the measurement in the fluostar at an excitation wavelength of λε _x = 490 nm and the emission wavelength λ = 530 nm is performed. The number of bound platelets is determined by the standard curve and is given in percent of added cells.

Lysis buffer: Add 2.5 g sodium dodecyl sulfate (SDS) with 1 ml 10 N NaOH and make up to 50 ml with PBS, filter. Result: The activation-related increase in the adhesion of platelets to HT29 colon carcinoma cells (Figure 2A) and to MT-3 breast cancer cells (Figure 2B) can be completely prevented by combination liposomes.

Example 5.3. Inhibition of the adhesion of tumor cells to immobilized endothelial components by liposomes

5.3.1 Fluorescent labeling of the cells:

Tumor cells (MT3 and MT1 breast cancer cells, and Lewis lung lung carcinoma cells) are fluorescently labeled as described in 2.2.1, but the marker used is 282 nmol Hoechst 33258/75 cm ² culture flask. The cells thus labeled are taken up in PBS (Ca, Mg) and adjusted to 1, 2 × 10 ⁶ cells / ml.

5.3.2 Immobilization of Adhesion Components:

100 μl of Ca / Mg-PBS containing the endothelial constituents to be investigated each are pipetted into a 96-well immunosorb plate with 200 ng of collagen IV or 1 μg of fibronectin and left to adhere overnight at 4 ° C. Thereafter, the supernatant is withdrawn and incubated at room temperature for one hour to block unspecific binding sites with 100 .mu.l of a 1% BSA solution. Finally, it is washed with PBS.

5.3.3 Determination of adhesion inhibition

The inhibition of the adhesion of the tumor cells by liposomes is then carried out as follows.

Into the wells are 50 μl of the Hoechst 33258 labeled tumor cells and 100 nmol

Given combination liposomes. It is incubated for ¹ h at 37 ^° C. before unbound tumor cells are removed by washing with buffer. Then 50 μl PBS and 50 μl lysis buffer are added to the wells with tumor cells, and it becomes 3

Shaken for a few minutes.

In parallel, a standard series of tumor cells in a black maxi-sorp

Carried plate, which are also dissolved by 50 ml lysis buffer. To this

Plate, the lysates of the incubation plate are then transferred well by well and then the fluorescence measurement in the SLT Fluostar microplate reader at% E _X = 355 nm and XEM = 460 nm. The quantification of the bound tumor cells then takes place via the mean fluorescence intensity from the standard series for each tumor cell line.

Result: Figure 3 shows the results for the inhibition of the adhesion of MT3, MT1 and Lewis lung and tumor cells. The combined liposomes inhibit the adhesion of MT3 to collagen and to fibronectin by 65 and 52%, respectively. For MT1 breast cancer cells, inhibition levels of 34% and 6% were achieved.

Example 5.4. Inhibition of complex formation from tumor cells and platelets by combination liposomes in vitro.

Tumor cells in the exponential growth phase are washed twice with Ca ²⁺ / Mg ²⁺ -PBS and incubated with 94 nmol of the Hoechst dye 33258 in 2 ml of Ca ^2-1 VMg ²⁺ - PBS for 30 min at 37 ⁰ C. After removal of the dye, the cells are washed with Ca ^2-1 VMg ²⁺ PBS and harvested by trypsinization, taken up in 2 ml of medium, centrifuged at 200 g ^'1 for 2 min and then dissolved in 2 ml Ca ² -VMg ²⁺ . PBS resuspended. The cell suspension is then adjusted to a defined cell number.

In the experiment, 1x10 ⁴ MT3 tumor cells labeled with Hoechst 33258, 2.5 × 10 ⁶ platelets and 10 nmol liposomes were taken up in 100 μl Thyrode buffer (TH buffer), activated with 0.01 μl thrombin and for 20 min under room temperature with uniform motion incubated. The mixture is then diluted to 2 ml with TH buffer and applied to slides with the cytospin centrifuge at 1500 rpm for 20 min. Thereafter, the samples are covered with a drop of mounting medium (Fluorescent Mounting Medium) and a coverslip bubble-free. The samples can then be evaluated under the fluorescence microscope (Axiophot, Zeiss). In order to visualize the tumor cells stained with Hoechst 33258, filters with an excitation wavelength of λ = 365 nm and an emission wavelength of λ = 395 nm are used.

For the results summarized in Table 1, 2 slides with 2 image sections were counted per experiment, whereby 5 experiments were carried out. Table 1: Inhibition of tumor cell complex formation in the presence of platelets by combination liposomes in vitro

MT3 cells ¹ MT3 cells ² aggregates ³ MT3 cells in aggregates ⁴ In the presence of: n ± SD n + SD [%]

+ THR 81 ± 35 2.3 ± 2.0 * 18.1 ± 13.0 *

+ aTHR 140 ± 65 5.9 ± 3.2 31.3 + 13.9

+ aTHR + Kon-L 157 ± 56 6.9 ± 2.9 35.6 ± 11.0

+ aTHR + OPP-L 140 ± 48 5.1 ± 2.1 29.1 ± 10.0

+ aTHR + DIP-L 103 ± 61 3.4 ± 3.0 * 23.7 ± 13.4

+ aTHR + DIP / OPP-L 82 ± 34 1.8 ± 1.5 * 16.9 ± 11.7 *

1 'MT3 cells are labeled with the indicated components (abbr .: THR: platelets; aPt: activated platelets; Kon-L: blank control; OPP-L; DIP-L and DIP / OPP-L: liposomes with encapsulated dipyridamole, OPP or both active ingredients).

2 Number of MT3 cells in the scanned microscopic image.

3 Mean number of aggregates with more than five MT3 tumor cells.

4 Mean number of MT3 cells [in%] with respect to total number of tumor cells.

* Significantly different from category "MT3 cells + aTHR" (Student's t-test, p <0.05).

Result: Combination liposomes completely prevent the aggregation (complex formation) of the tumor cells, whereas they increase to 172% without the addition of liposomes when the platelets are activated.

Example 6: Compatibility study for the combination liposomes

In each case 6 Nudemäuse per group were once injected intravenously combination liposome with 50, 100 or 200 nmol total lipid and recorded as a characteristic of toxic effects, the general behavior, hematological and body weight changes. The changes in the dose range studied were low. There was no change in behavior. The change in body weight was between 5 and 7% and was therefore normal and not active. Only a clear leukopenia (maximum after 2 h with reduction by 80%) and thrombopenia (maximum after 6 h with reduction by 30%) was observed, both effects were normalized again after 24 hours. In the next example, liposome formulations are tested in therapeutic studies.

Example 7: Therapeutic efficacy of the liposomal formulations in the MT3 breast cancer model

The antimetastasis effect is tested in the mouse model in different tumor models with different potential for metastasis (eg in the human HT29 colon carcinoma and MT3 and the MT1 breast cancer model).

Selected examples of therapeutic trials:

Example 7.1. Influence of the time of liposome application on the inhibition of metastasis development in MT3 mouse breast cancer

5x10 ⁶ MT3 breast cancer cells from the cell culture are injected iv at time t = 0 in 4 NMRl nu / nu mice per treatment group. In addition to the control group, which receives a single saline solution, a group at the time - 6h (based on the tumor cell injection), another group at the times -6, -4 and -2 h, and a group at the times -6, - 2, +2 and +4 h each treated with 100 nmol total lipid liposomes per 20 g mouse iv. After 29 days, the animals are killed, autopsied and the weights of the tumors and lungs determined.

Result: The results are shown in Figures 4 and 5 and show that a single treatment 6 hours before tumor cell injection showed the strongest inhibition in the number of visible metastases (Figure 4) and in the weight of the extrapulmonary metastases (Figure 5) becomes. Despite shifting the pattern of metastasis from lung to more muscle metastases, the total number of metastases is greatly reduced and no other organs are affected.

Example 7.2. Influence of the dose of liposomes on the inhibition of metastasis development in the mouse

The experiment is carried out according to 7.1 with the modification that is treated only at -6 h, but with different dosages of Liposomes. The mice receive 50, 100 or 200 nmol total lipid combination liposomes per 20 g of mouse.

As shown in Figures 6 and 7, a single treatment with 100 nmol combination liposomes / mouse can significantly reduce metastasis. In some animals, lung metastases no longer occur. While no other organs are affected, muscle metastases occur in some mice.

Example 7.3. Inhibition of metastasis development in MT3 mouse breast cancer

5x10 ⁶ MT3 breast cancer cells are injected iv at t = 0 in 6 NMRI nu / nu mice per treatment group. Six hours prior to tumor cell injection, the mice of the control group receive physiological saline once, while the mice in the other groups receive 100 nmol of total lipid of the specified liposomes or corresponding amounts of active ingredients iv injected. At the end of the experiment, the animals are killed, an autopsy performed and the weights of the tumors and the metastatic number in the lung are determined.

Result: Treatment with DIP-OPP liposomes reduces the total number of metastases by 79.1%, from an average of 9.6 per animal to 2.0 (Figure 8). The inhibitory effect is also shown in the comparison of the scores, which take into account not only the number but also the size of the metastases. While the average score in the control group is 151, only an average score of 40 is obtained after treatment with the indicated DIP-OPP liposomes. This corresponds to an inhibitory effect of 73.4%. All other liposomes or the unencapsulated active ingredients have no or a significantly lower effect (Figure 9).

Example 7.4. Inhibition of metastasis development in MT1 mouse breast cancer

5x10 ⁶ MT1 breast cancer cells are injected iv at t = 0 in 6 NMRI nu / nu mice per treatment group. Six hours before tumor cell injection, the mice of the control group receive a single saline solution while the mice in the second group receive combination liposomes injected with 100 nmol of total lipid iv. At the end of the experiment, the animals are killed, an autopsy and determined the metastatic number in the lung and outside the lungs.

Result: By treatment with DIP-OPP-liposomes, the mean

Lung metastasis reduced from 81.3 to 10.8 (equivalent to 86.7%)

(Figure 10).

Example 7.5. Inhibition of metastasis development in HT29 colon carcinoma in the mouse

2.5x10 ⁶ HT29 colon carcinoma cells are injected iv at time t = 0 in 6 NMRI nu / nu mice per treatment group. Six and two hours before tumor cell injection, the control group receives saline while the mice in the second group receive liposomes injected with 100 nmol of total lipid iv. At the end of the experiment, the animals are killed, autopsied and the weights of the tumors and the number of metastases determined.

Result: In a typical experiment, the animals in the control group had an average metastatic count of 19, which occurred in the lung (14.75), the heart (3.75) and the muscle (0.5). Treatment with DIP-OPP liposomes reduced the total number of metastases to 7.5 and thus by 40% (Figure 11).

All the features illustrated in the foregoing description, the appended claims and the drawings may be relevant to the realization of the invention in its various forms both individually and in any combination. Legend to the pictures

Figure 1: Inhibition of aggregation of platelets by combination liposomes in plasma

Shown are the mean transmission ± S.D. 80 min after activation with respect to the first measured value (transmission = 0%). 2.5x107 platelets in plasma (PRP) were activated with 0.05 units thrombin and 100 nmol liposomes added. The change in absorbance in the platelet suspension was measured at a wavelength of 595 nm. Each value represents the mean +/- S.D. for a quadruple determination of at least 10 independently performed experiments.

Platelets: activated platelets without liposomes as controls.

Kon Lip: aggregation in the presence of control liposomes.

Comb-Lip: aggregation in the presence of DIP-OPP-liposomes (combination liposomes).

(*): significantly different from control (p <0.05).

Figure 2: Inhibition of activated platelet adhesion to immobilized tumor cells by combination liposomes

1 × 10 ⁵ tumor cells / well (A: HT 29 colon carcinoma, B: MT3 breast cancer) were immobilized in a 96-well plate and after treatment with BSA with 2.5 × 10 ⁷ calcein-AM-labeled platelets (with / without thrombin activation ) and 100 nmol DIP-OPP liposomes (combination liposomes) at 37 ° C. After cell lysis, the fluorescence intensity of the bound platelets was measured at: λE _X : 490 nm and λεivi: 530 nm.

Each value represents the mean +/- S.D. for a quadruple determination of at least 8 independently performed experiments.

Platelets: Platelets without liposomes as controls Kon Lip: Adhesion in the presence of control liposomes. Comb Lip: Adhesion in the presence of DIP-OPP liposomes. (*): significantly different from control (p <0.05).

Figure 3: Inhibition of the adhesion of tumor cells to immobilized collagen and fibronectin through combination liposomes

Collagen or fibronectin were immobilized in a 96-well cell culture plate. Subsequently, ⁶ × 10 ⁴ tumor cells / well were added and incubated with 100 nmol TL liposomes for 1 hour at 37 ° C.

After cell lysis of the bound tumor cell, the proportion of bound

Tumor cells quantified by fluorescence measurement at: λ _Ex : 355 nm and XEM: 460 nm. Given the percent inhibition of adhesion in the presence of liposomes compared to the control. Each value represents the mean +/-

S.D. for a quadruple determination of at least 5 independently performed

Experiments.

MT3: Human breast cancer cells

MT1: Human breast cancer cells

LL: Murine lung carcinoma

Figures 4 and 5: Influence of the schedule and the time of treatment with liposomes on the formation of metastases in the mouse

5 ^χ 10 ⁶ MT3 breast cancer cells were injected iv at t = 0 in 4 NMRI nu / nu mice per treatment group. The control group received physiological saline once, while the mice in the other groups at the indicated times (based on the tumor cell injection) in each case 100 nmol

Total lipid to liposomes per 20 g mouse i.v. injected. After 29 d were the

Killed animals, autopsied and determined the weights of the tumors and the metastatic number.

Fig. 4: Number of visible metastases

Fig. 5: Weight of extrapulmonary metastases

Figures 6 and 7: Influence of the liposome dose on the formation of metastases in the mouse

5x10 ⁶ MT3 breast cancer cells were injected iv at time t = 0 in 4 NMRI nu / nu mice per treatment group. Six hours before tumor cell injection, the control group received a single saline solution while the mice in the other groups received the indicated amounts (total lipid) of liposomes injected iv. At the end of the experiment, the animals were killed, an autopsy made and the weights of the tumors as well as the metastasis! in the

Lungs determined.

Fig. 6: Number of visible metastases

Fig. 7: Weight of extrapulmonary metastases

Figures 8 and 9: Comparison of the efficacy of different liposomes on mouse metastasis development in MT3 breast cancer

5x10 ⁶ MT3 breast cancer cells were injected iv at t = 0 in 6 NMRI nu / nu mice per treatment group. Six hours prior to tumor cell injection, the control group received saline once, while the mice in the other group received the indicated liposomes injected with 100 nmol of total lipid or corresponding amounts of active ingredients iv. At the end of the experiment, the animals were killed, an autopsy was performed and the weights of the tumors and the number of metastases in the lung were determined. Fig. 8: Number of pulmonary and extrapulmonary metastases Fig. 9: Scores of the metastases

Liposomes: DIP / OPP-L: combination liposomes with dipyridamole and OPP

DIP-L: liposomes with dipyridamole

OPP-L: liposomes with OPP

CON-L: Empty control liposomes

OPP: free octadecyl- (1, 1-dimethyl-piperidin-4-yl) -phosphate

DIP: free dipyridamole

Scores [indicated is the diameter (in mmy.core): 1: 1; 1-2: 8; 2-3: 27; 3-4: 64; 4-5: 125; 5-8: 512.

Figure 10: Inhibition of metastasis by combination liposomes in the MT1 breast cancer model in vivo

NMRI nu / nu mice were injected iv with 5x10 ⁶ MT1 breast cancer cells at time t = 0. Six hours before tumor cell injection, the mice were treated with physiological NaCl solution (control group) or combination liposomes with 100 nmol total lipid iv. After 48 days, the animals were killed, autopsied and metastatic numbers determined in the lung and outside the lungs.

Six mice were used per group. * Significantly different from control group (P <0.05) Figure 11: Inhibition of metastasis by liposomes in the HT29 colon carcinoma model in vivo

NMRI nu / nu mice were injected with 2.5x10 ⁶ HT29 colon carcinoma cells at time t ^ O. Six and two hours before, the mice were given physiological saline (control) or liposomes with 100 nmol of total lipid iv. 8 mice were used per group. After 36 days, the animals were killed, an autopsy performed and the weights of the tumors and the metastatic count were determined.

e

List of abbreviations

CH cholesterol

DCP dicetyl phosphate

DMPC, dimyristoyl phosphatidylcholine

DOPC, dioleyl phosphatidylcholine

DOPE dioleyl-phosphatidylethanolamine

DPPC, dipalmitoyl phosphatidylcholine

DSPC, distearyl phosphatidylcholine

EggPC, egg-P-phosphatidylcholine

HePC, hydrogenated phosphatidylcholine

HPC hexadecylphosphatidylcholine

HPE hexadecylphosphatidylethanolamine

HPP hexadecyl (1, 1-dimethyl-piperidin-4-yl) phosphate

HpPC heptadecylphosphatidylcholine

HPS hexadecylphosphatidylserine

Kon-Lip control liposomes

LUV Large unilamellar vesicles (Large unilamellar vesicles)

MLV Multilamellar Vesicles

OPC octadecylphosphaüdylcholine

OPP octadecyl (1, 1-dimethyl-piperidin-4-yl) -phosphate

PA phosphatidic acid

PE phosphatidylethanolamine

PEG polyethylene glycol

PG phosphatidylglycerol

PI polydispersity index

POPC, palmitoyl-oleayl-phosphatidylcholine

PSPC ₁ palmitoyl-stearyl-phosphatidylcholine

SA stearylamine

SPPC, stearyl palmitoyl phosphatidylcholine

TPC, tetradecylphosphatidylcholine bibliography

¹ Kannagi, R. (1997) Glycoconj J 14: 577-584. Saiki, I., et al. (1996) Int J Cancer 65: 833-839.

² Engers, R. and Gabbert, HE (2000) J Cancer Res Clin Oncol 126: 682-692. Ley, K. (1996) Cardiovasc Res 32: 733-742. McEver; RP (1997) Glycoconj J 14: 585-591.

³ Obana, S. et al. (2002) Kobe J. Med. 48: 43-54. de Mascarel, I. et al. (1998) Eur. J. Cancer. 34: 58-65.

⁴ Li, CY. et al. (2000) Cancer Metastasis Rev. 19: 7-11.

⁵ Borsig, L. et al. (2001) Proc Natl Acad. USA 98: 3352-3357. Yu, Y. et al. (2002) J Cancer Res Clin Oncol 128: 283-287. Ma, YQ and Geng, JG (2002) J Immunol 168: 1690-1696.

⁶ Gege, C. et al. (2000) Chemistry 6: 111-122. Ikegami-Kuzuhara, A. et al, (2001) Br. J. Pharmacol. 134: 1498-1504. Ito, I. et al. (2001) Am. J. Kidney Dis. 38: 265-273. Stahn, R. et al. (2000) Tumor Biol 21: 176-186.

⁷ Stahn, R. et al. (2001) Ce // Mol Life 58: 141-147. Chen, HH et al. (2003) Int J Cancer 103: 169-176. Magdolen.V. (2003) Recent Results Cancer Res. 162: 43-63.

Claims

claims A pharmaceutical preparation comprising a liposomal formulation which at least one substance with antitumour activity and - At least one substance with anticoagulant effect, as well - optionally contains other substances. 2. Pharmaceutical preparation according to claim 1, characterized in that the liposomal formulation comprises at least one base lipid, preferably all of the following constituents: Based lipid, Helper lipid, Membrane stabilizers, Charge carrier and optionally a substance for modifying the surface. 3. A pharmaceutical preparation according to claim 2, wherein as base lipids soy phosphatidylcholine, EggPC (egg phosphatidylcholine), HePC (hydrogenated phosphatidylcholine), DMPC (dimyristoyl phosphatidylcholine), DPPC (dipalmitoyl phosphatidylcholine), DSPC (distearyl phosphatidylcholine), SPPC (Stearyl palmitoyl phosphatidylcholine), PSPC (palmitoyl stearyl phosphatidylcholine), DOPC (dioleyl phosphatidylcholine), POPC (palmitoyl oleyl phosphatidylcholine) or sphingomyelin. 4. Pharmaceutical preparation according to claim 2-3, wherein DOPE (dioleyl-phosphatidylethanolamine) or DOPC (dioleyl-phosphatidylcholine) are used as helper lipids. 5. Pharmaceutical preparation according to claim 2-4, wherein CH (cholesterol) is used as membrane stabilizer. 6. Pharmaceutical preparation according to claim 2-5, wherein a negative charge carrier is used as charge carrier. 7. A pharmaceutical preparation according to claim 2-6, wherein DCP (dicetyl phosphate), SA (stearylamine), PA (phosphatidic acid), PG (phosphatidylglycerol) or Monosialogangliosid be used as a negative charge carrier. 8. A pharmaceutical preparation according to claim 2-7, wherein as a substance for modifying the surface of the liposomal formulation PEG-PE (polyethylene glycol phosphatidylethanolamine), PEG sterols, sialyl Lewis ^x - Peg-PE, sialyl-Lewis ^A -PEG-PE , Sialyl Lewis ^x -peg sterols or sialyl Lewis ^A - PEG sterols. 9. Pharmaceutical preparation according to claim 1-8, wherein the diameter of the liposomes of the liposomal formulation is less than 400 nm and the size distribution has a polydispersity index of less than 0.4. 10. Pharmaceutical preparation according to claim 1-9, wherein as substance groups with antitumor effect Cytostatic agents such as alkylating agents, mitotic inhibitors, cytostatic antimetabolites and antibiotics, hormones and hormone antagonists, and alkylphospholipids used. 11. Pharmaceutical preparation according to claim 1-10, being used as substance groups with anti-tumor effect alkylphospholipids. 12. A pharmaceutical preparation according to claim 1-11, wherein as alkylphospholipids TPC (tetradecylphosphatidylcholine), HPC (hexadecylphosphatidylcholine), HpPC (heptadecylphosphatidylcholine), OPC (octadecylphosphatidylcholine), HPP (hexadecyl (1, 1-dimethyl-piperidin-4-yl) phosphate), OPP (octadecyl (1,1-dimethyl-piperidin-4-yl) -phosphate), HPS (hexadecylphosphatidylserine), HPE (hexadecylphosphatidylethanolamine) and / or edelfosine. 13. Pharmaceutical preparation according to claim 1-12, wherein as active ingredient groups with anticoagulant action aggregation inhibitors (dipyridamole, acetylsalicylic acid, ticlopidine, resveratrol); Anticoagulants (heparin, low molecular weight heparin, heparinoids, clopidogrel). Vitamin K antagonists (warfarin, phenprocoumon) are used. 14. Pharmaceutical preparation according to claim 1-13, wherein preferably dipyridamole and / or acetylsalicylic acid is used as active ingredient. 15. Pharmaceutical preparation according to claim 1-14, which preferably contains the constituents phosphatidylcholine (30-35 μmol), octadecyl- (1,1-dimethylpiperidin-4-yl) -phosphate (10-15 μmol), cholesterol (10-15 μmol) , Polyethylene glycol coboo phosphatidyl ethanolamine (2-3 μmol), dicetyl phosphate (5-8 μmol) and dipyridamole (15-20 μmol). 16. Use of a pharmaceutical preparation according to claim 1-15 for the manufacture of a medicament for the treatment of metastatic tumor diseases. 17. Use according to claim 16 for the treatment of thromboembolic disorders. 18. Use according to claim 16 for the treatment of coagulation disorders.