WO2004037286A2

WO2004037286A2 - Combination of a trioxopyrimidine compound inhibitor and an anti-tumor agent, and uses thereof

Info

Publication number: WO2004037286A2
Application number: PCT/EP2003/011682
Authority: WO
Inventors: Matthias Arlt; Sabine Bauer; Hans-Willi Krell; Achim Krueger
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2002-10-23
Filing date: 2003-10-22
Publication date: 2004-05-06
Also published as: WO2004037286A3; AU2003274059A1; AU2003274059A8

Abstract

A pharmaceutical combination consisting of a trioxopyrimidine with inhibitory activity against matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) and an antibody against urokinase receptor (anti-uPAR antibody) is effective in the treatment and prevention of metastasis.

Description

Combination of a trioxopyrimidine compound inhibitor and an anti-tumor agent, and uses thereof

The present invention relates to combinations and compositions consisting of a trioxopyrimidine compound in combination with an antibody against urokinase receptor and methods for the treatment of patients with solid, metastasized or non- metastasized tumors using said combinations and compositions.

Introduction

In modern clinical oncology, the biggest challenge for the successful treatment of patients is the problem of metastasis rather than the primary tumor itself. For tumor cells to be able to spread and form distant metastases several prerequisites have to be fulfilled. Among these one of the most important ones is the ability to grow invasively into the surrounding tissue, intravasate into the blood- or lymphatic vessel system and finally to extravasate and seed in the target tissue.

Recently a new class of molecules, namely the proteases, was identified to play a major role in this process. With the help of these enzymes tumor cells break down extra-cellular matrix proteins which are major constituents of connective tissue and basal membranes. Among these proteases the matrix metalloproteases (MMPs), and, more specifically, MMP-2 and MMP-9 (= gelatinases A and B) were identified as major contributors in this process of matrix degradation (Johansson, N., et al, Cell. Mol. Life Sci. 57 (2000) 5-15). In addition, especially MMP-9 was found to play an important role in the formation of new blood vessels, a process called angiogenesis, which is essential for a tumor to establish and uphold a sufficient supply with nutrients and oxygen (Vu, T.H., et al., Cell 93 (1998) 411-422). Not surprisingly, indeed MMP-2 and/or MMP-9 were found to be over-expressed by a large proportion of individual tumors irrespective of histological origin.

Inhibition of MMPs, either with the naturally occurring Tissue Inhibitors of Metalloproteases (TIMPs), or with low molecular weight inhibitors, resulted in impressive anti-tumor and anti-metastatic effects in animal models (Brown, P.D., Medical Oncology 14 (1997) 1-10). Most of the low-molecular weight inhibitors of MMPs are derived from the hydroxamic acid compound class and inhibit MMPs in a broad manner, being not selective for MMP-2 and MMP-9, the key MMPs in tumor invasion, metastatic spread, and angiogenesis. However, MMP inhibiting molecules from another structural class, the trioxopyrimidines, have been described, e.g. in WO 97/23465 and WO 01/25217, which are incorporated by reference. This class of compounds is extremely potent, and highly selective, with an almost exclusive specificity for MMP-2, MMP-9, while sparing most other members of the MMP family of proteases.

Several MMP inhibitors, predominantly of the hydroxamic acid substance class with broad substrate specificity were, and in part still are, in clinical testing. All of the published clinical results with these inhibitors were disappointing, showing little or no clinical efficacy (Fletcher, L., Nature Biotechnology 18 (2000) 1138-1139). The reason for this lack of efficacy in the clinic most likely is the fact that patients could not be given high enough doses for anti-tumor or anti-metastatic activity because of the side effects associated with these broadly acting inhibitors. These dose-limiting side effects were predominantly arthralgias and myalgias (Drummond, A.H., et al., Ann. N.Y. Acad. Sci. 878 (1999) 228-235). As a possible way to circumvent this problem, the combination of MMP inhibitors with classical cytostatic/cytotoxic compounds was evaluated in animal studies. Indeed, in these experiments, MMP inhibitors, in combination with cytostatic/cytotoxic drugs, showed enhanced tumor inhibiting efficacy (Giavazzi, R., et al., Clin. Cancer Res. 4 (1998) 985-992). In addition, International Patent Application WO 02/089824 shows the combination of trioxopyrimidine based gelatinase inhibitors and cytotoxic/cytostatic compounds such as cisplatin, Paclitaxel, Gemcitabine or Etoposide.

However, for an efficient treatment of metastasis, there is still a great demand for potent therapeutic agents.

The invention provides the combination of trioxopyrimidine-based gelatinase inhibitors with an antibody against urokinase receptor for prevention and treating metastases.

Surprisingly, it was found that a combination of trioxopyrimidine-based inhibitors which are highly selective for MMP-2 and MMP-9 in combination with antibodies binding to the urokinase receptor (uPAR) synergistically inhibits the forming of metastases.

Trioxopyrimidines according to the invention are compounds from a well-known structural class and are known to have an inhibitory activity against MMP-2 and MMP-9 (also known as gelatinase inhibitors). Such compounds are described in, for example, US Patent Nos. 6,242,455 and 6,110,924; WO 97/23465 and WO 01/25217 and are effective and highly selective. According to the invention, the trioxopyrimidine compounds inhibit the proteolytic activity of fibrosarcoma cells like HT 1080 (CCL 121) for at least about 20% in an amount of 20 μg/ml.

According to the invention, the following compounds are particularly preferred:

5-Biphenyl-4-yl-5-[4-(4-nitro-phenyl)-piperazin-l-yl]pyrimidine-2,4,6-trione (Compound I)

5-(4-Phenoxy-phenyl)-5-(4-pyrimidin-2-yl-piperazin-l-yl)-pyrimidine-2,4,6- trione (Compound II)

Antibodies against the urokinase receptor, preferably monoclonal antibodies, are also described in the state of the art (e.g., US Patent No. 5,519,120). Particularly preferred are antibodies, which bind uPAR at an epitope located outside residues 1- 87 of uPAR, and which inhibit the binding between uPA and uPAR. Such antibodies inhibit the proteolytic activity of fibrosarcoma cells like HT 1080 (CCL 121) for at least about 30% in an amount of 80 μg/ml. Such preferred antibodies are described in, for example, US Patent No. 6,025,142. Particularly preferred are the monoclonal antibodies described in this US patent and produced by hybridoma cell lines 1.H2.10A3 and 1.C8.26A3, which were deposited at the DSM in July 27, 1994 under the terms and conditions of the Budapest Treaty and were assigned the numbers DSM ACC 2178 and DSM ACC 2179.

The combination according to the invention has to be administered over several weeks, or months, to the patient in need of such a therapy. It is preferred to begin the therapy according to the invention before or immediately during, the beginning of therapy of the primary tumor. The trioxopyrimidine compound is administered enterally or parenterally, preferably orally, with doses ranging between 0.5 mg/kg and 100 mg/kg. Administration of the anti-uPAR-antibody is preferably performed by enterally or parenterally in a liquid or solid form. Both partners of the combination therapy can be contained in separate package format or together in a kit. Such a kit contains the i.v. preparations of the antibody, e.g. ampoules and a suitable formulation of the trioxopyrimidine compound (preferable tablets). The antibody is administered at the very beginning in a higher dosage, followed by lower dosages. The initial dose is preferably between 50 and 100 mg/kg, followed by two doses per week of 10 to 50 mg/kg.

Preferred application schemes are:

Trioxopyrimidine:

50 mg/kg or 100 mg/kg - 2x per day

Anti-uPAR-antibody:

Loading dose 12 mg/kg once, followed by a maintenance dose of 6 mg/kg every other week or

Initial dose: 50 mg/kg, 80 mg/kg, or 100 mg/kg

Subsequent doses: 20 mg/kg, 30 mg/kg, or 40 mg/kg - 2x per week.

The antibody is preferably administered to the mammal with a carrier. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of a pharmaceutically acceptable carrier include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered. The antibody can be administered to the mammal by injection (e.g., intravenous, intraperitoneal, subcutaneous, intra-muscular), or by other methods such as infusion that ensure its delivery to the bloodstream in an effective form. For the trioxopyrimidines all forms of administration come into consideration such as, for example, tablets, capsules, coated tablets, syrups, solution, suspensions etc. Water, which contains additives such as stabilizers, solubilizers and buffers that are usual in injection solutions, is preferably used as the injection medium.

The following examples, references, and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Description of the Figures

Figure 1 Inhibition of the proteolytic activity of HT 1080 fibrosarcoma cells by treatment with Compound I, Compound II, Compound III (comparison) and H2-(anti-uPAR)-antibody as single agents and in combination.

Figure 2 Reduction in number of lung metastases in the different treatment groups in a setting of experimental metastasis.

Figure 3 Proportion of mice with metastasis-free lungs in the different treatment groups in a setting of experimental metastasis.

Figure 4 Tumor weights of primary tumor in the different treatment groups after 28 days. Treatment started at day 10 (staging day).

Figure 5 Reduction in spontaneous lung metastasis in the different treatment groups after 28 days. Treatment started at day 10 (staging day).

Figure 6 Proportion of mice with metastasis-free lungs in the different treatment groups after 28 days. Treatment started at day 10 (staging day).

Figure 7 Reduction in spontaneous liver metastasis after 28 days.

Treatment started at day 10 (staging day). Figure 8 Comparison of survival in the H2 antibody group, Compound II group and the combination group compared to the control. (Further explanation: see Example 4.)

Example 1

Influence of trioxopyrimidine and H2-(anti-uPAR)-antibody treatment on

HT1080 fibrosarcoma cells

The cell line HT1080 (ATCC CCL 121) displays high expression levels of uPA/uPAR and the gelatinases that are mainly involved in the metastatic process. Therefore this cell line has been chosen for the in vitro and in vivo studies.

In the in vitro studies 25,000 HT1080 cells were seeded in the wells of a 96-well microtiter plate in 100 μl DMEM with 10 % FCS. After 24 hours incubation under cell culture conditions the medium was removed and the adherent cells were washed with PBS (including Ca2+ and Mg2+). Then the different trioxopyrimidine-type inhibitors (Compound II, Compound I), Compound III and the anti-uPAR-antibody (H2 antibody, 1H2.10A3) were added in DMEM (200 μl/well) without phenolred and without FCS. The experimental setting and the concentrations were as follow:

Compound II alone 20 μg/ml

Compound I alone 20 μg/ml

Compound III alone 20 μg/ml

H2-(anti-uPAR)-antibody alone 80 μg/ml

Compound II + H2-antibody 20 μg/ml + 80 μg/ml

Compound I + H2-antibody 20 μg/ml + 80 μg/ml

Compound III + H2-antibody 20 μg/ml + 80 μg/ml

10 μl plasminogen (0.5 units/ml) and 10 μl of the fluorescein conjugated DQ- Gelatin substrate (1 mg/ml) were added to each well. After 24 hours incubation under cell culture conditions the assay was measured at 485/535 nm (Ex/Em). Results:

Clear inhibition of the proteolytic activity of the HT1080 fibrosarcoma cells occurred when cells were treated with the single agents Compound I, Compound II, Compound III or H2-(anti-uPAR)-antibody (see Fig. 1). Compared to the untreated control Compound III reduced the proteolytic activity of the fibrosarcoma cells about approximately 5 %, Compound II about approximately 20 %, Compound I about approximately 35 % and the H2-(anti-uPAR) -antibody about approximately 30 %.

The combination of the trioxopyrimidine-type gelatinase inhibitors with the anti- uPAR-antibody led to a stronger inhibition of the proteolytic activity than treatment with the single agents (see Fig.l). By combining the H2-(anti-uPAR)- antibody with Compound II the inhibitory effect was increased to 37 % (H2-Ab alone: 30 % ; Compound II alone: 20 %) and the combination of H2~(anti-uPAR)- antibody with Compound I caused a cumulative inhibition of 48 % (H2-Ab alone: 30 % ; Compound I alone: 35 %).

Compound III ((E)-2(R)-[l(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'- isobutyl-2'-(methanesulphonyl)-4-methylvalerohydrazide) is a substance which has a selectivity for MMP-8 and TACE (TNF-α convertase) but not for MMP-2 and MMP-9.

Example 2

Influence of trioxopyrimidine and H2-(anti-uPAR)-antibody treatment on lung and liver metastasis (experimental metastasis assay)

The cell line HT1080 displays high expression levels of uPA/uPAR and gelatinases. These components are mainly involved in the metastatic process. The treatment group has been chosen as follows: Table 1:

Overview of experiment:

Table 2:

HT1080 AN K15-1, a lacZ-tagged human fibrosarcoma cell line was injected at day 0 in CD1 nu/nu mice (females, 9 week old, average 25 g) after a period of one week. Tumor cell injection was performed with lxlO⁶ cells in 200μl PBS i.v. into the tail vein of each mouse. Fitness and mortality was observed each day.

Treatment started at day minus 1 (one day before tumor cell inoculation). Mice were randomized into treatment groups and received the first treatment epretreatment') with the respective agents (agent combination). The compounds and the antibody were formulated for application in vivo using the compound vehicle or the H2 vehicle (antibody) as solvent.

Compound vehicle:

Sodium carboxymethyl cellulose (high viscosity grade, 2 mg/ml), propylparaben (0.1 mg/ml), methylparaben (0.9 mg/ml) dissolved in distilled water.

H2 vehicle:

0.1 mol/1 phosphate buffered saline (PBS), pH 7.5.

Compound II and the compound vehicle were given orally twice a day for 28 days starting from day -1 (one day before tumor cell inoculation). Application volume was 200μl per mouse (8 ml/kg). MAK 1.H2.10A3 and the H2-vehicle were given i.p. twice a week for 28 days starting from day -1. Application volume of the loading dose was 1 ml per mouse (40 ml/kg). Application volume of the treatment dose was 300μl per mouse (12 ml/kg).

At day 28 all mice were euthanized with CO₂ and analyzed for body weight. Blood samples, tissue samples of lung and liver were collected and snap-frozen in liquid nitrogen. Lungs and livers were fixed in 2% formaldehyde 0.2% glutaraldehyde lx PBS.

Evaluation of lung and liver metastasis:

Lung metastases were counted on the organ surface, by identification of the blue color of the colonies formed by the lacZ- tagged injected cell line after X-gal staining of the whole organs. Metastasis load in the liver was investigated by quantitative PCR of Alu-sequences. For statistics, Mann-Whitney Rank Sum Test (SPSS Statistical Algorithm, 2^nd ed. , 1991, ISBN 0-918469-89-9) was used for comparison of the number of metastases, Fisher-Exact-Test (two-sided) (Sachs, L., Angewandte Statistik, 6 ed., Springer, 1984) for comparison of the number of metastases free lungs.

Metastases were counted on the surface of the lungs after X-gal staining. The data are plotted as relative values (%) in comparison to the control. Results:

Liver metastasis:

In all treatment groups as compared to the control, there was a clear trend towards reduction of tumor formation regarding the metastasis load in the livers, determined optically after X-Gal staining and by quantitative PCR for Alu- sequences. The effect of combination treatment more pronounced compared to the single treatments.

Lung metastasis:

In all treatment groups, the number of lung metastases was significantly reduced as compared to the vehicle control (Compound II, p = 0.0370 ; H2 antibody, p = 0.0121, Compound II + H2, p = 0.0012). The combined treatment with the H2 antibody and Compound II led to a stronger reduction of the number of lung metastases as the single agents alone (see Fig.2).

In addition, the percentages of mice with metastasis-free lungs are plotted in the treatment groups and in the control group (see Fig. 3). All treatment groups exhibited a certain percentage of mice without any lung metastases. Compared to the control, treatment with H2 antibody alone protected 10% of the mice (p = 0.57) and treatment with Compound II alone protected 23% of the mice (p = 0.11) against lung metastasis. A statistically significant protection against metastasis of lungs was achieved only by combined treatment with H2 antibody and Compound II (43%, p = 0.0037).

In summary, both Compound II and H2 (uPAR-antibody) alone significantly decrease the number of metastases. However, the combination of Compound II and H2 antibody surprisingly decreases the number of metastases to a greater extent compared to the single agent treatments alone. Example 3

Influence of trioxopyr-rnidine and H2-(anti-uPAR)-antibody treatment on primary tumor growth and spontaneous lung and liver metastasis

The aim of this study was to evaluate the inhibitory effect on primary tumor growth as well as the spontaneous metastasis in a staged tumor setting. The treatment groups were chosen as follows:

Table 3:

Overview of experiment:

Table 4:

HT1080 AN K15-1, a lacZ-tagged human fibrosarcoma cell line were injected at day 0 in CD1 nu/nu mice (females, 9 week old, average 25 g) after an adaptation period of 1 week. Tumor cell injection was performed with lxlO⁶ cells in 200μl PBS s.c. into the right flank of each mouse. Fitness and mortality was observed each day. Tumor size was measured two times per week.

Gelatinase inhibitor Compound II suspension was prepared freshly every day before substance application, lyophilized H2 antibody was dissolved in 14.3 ml PBS and stored at 4°C as aliquots with an concentration of 7 mg/ml .

Treatment started at day 10 (staging day), mice were randomized into treatment groups and received the first treatment with the respective agents (agent combination). Compound II and the vehicle of the compound were given orally twice a day for 18 days starting from day 10. Application volume was 200μl per mouse (8 ml/kg). H2 antibody and the H2 -vehicle were given i.p. twice a week for 18 days starting from day 10. Application volume of the loading dose was 300μl per mouse (12 ml/kg). Application volume of the treatment dose was 200μl per mouse (8 ml/kg).

At day 28 all mice were euthanized with CO₂ and analyzed for body and tumor weight. Blood samples, tissue samples of the primary tumor, lung and liver were collected and snap-frozen in liquid nitrogen. Lungs and livers were fixed in 2% formaldehyde 0.2% glutaraldehyde lxPBS.

Evaluation of tumor weight inhibition:

Tumor weight inhibition was calculated as follow:

Mp treated TWI% = 100 - X 100

MP untreated

Evaluation of spontaneous metastasis to lung and liver:

Lung metastases were counted on the organ surface, by identification of the blue color of the colonies formed by the lacZ- tagged injected cell line after X-gal staining of the whole organs. Metastasis-load in the liver was investigated by quantitative PCR of Alu-sequences. The data are plotted as relative values (%) in comparison to the control.

For statistics, Mann- Whitney Rank Sum Test was used for comparison of the number of metastases, Fisher-Exact-Test (two-sided) for comparison of the number of metastases free lungs.

Results:

Primary tumor:

Treatment with Compound II alone and in combination with H2 antibody led to a tumor weight inhibition of 43.0% and 48.5%, in contrast to uPAR antibody treatment (see Fig. 4).

Lung metastasis:

In contrast, lung metastasis determined optically after X-Gal staining was reduced in all treatment groups as compared to the control (see Fig. 5).

In addition, the percentages of mice with metastasis-free lungs are plotted in the treatment groups and in the control group (see Fig. 6). All treatment groups exhibited a certain percentage of mice without any lung metastases. Compared to the control, treatment with H2 antibody alone protected 16% of the mice and treatment with Compound II alone protected 11% of the mice against lung metastasis. Again treatment with both agents in combination led to the strongest protection (24%).

Liver metastasis:

In all treatment groups as compared to the control, there was a clear trend towards reduction of tumor formation regarding the metastasis load in the livers, determined by quantitative PCR for Alu-sequences (see Fig. 7). The effect of combination treatment more pronounced compared to the single treatments.

Example 4

Influence of trioxopyrimidine and H2-(anti-uPAR)-antibody on survival

The aim of the experiment was to determine if a treatment with H2 antibody or with the gelatinase inhibitor Compound II or both compounds in combination prolongs the survival time of mice in the fibrosarcoma xenotransplant model HT1080-lacZ. The experimental metastasis setting was used for the animal study design to examine if the combined treatment is more efficacious compared to single agents alone.

Rationale:

The combination of both compounds was more efficacious than the single agents alone in reduction metastasis to lung and liver. In addition only the combined treatment significantly protected mice against lung metastasis (43%, p = 0.0037).

Treatment groups:

Table 5:

Overview of experiment:

HT1080 AN K15-1 was injected at day 0 in CD1 nu/nu mice (females, 9 week old, average 25 g) after a period of 1 week. Tumor cell injection was performed with lxlO⁶ cells in 200μl PBS i.v. into the tail vein of each mouse. Fitness and mortality was observed each day.

Treatment started at day minus 1 (one day before tumor cell inoculation), mice were randomized into treatment groups and received the first treatment ('pretreatment') with the respective agents (agent combination). Compound II MBP and the Ro-vehicle were given orally twice a day for 52 days starting from day -1 (one day before tumor cell inoculation). Application volume was 200μl per mouse (8 ml/kg). H2 antibody and the H2-vehicle were given i.p. twice a week for 52 days starting from day -1. Application volume of the loading dose was 300μl per mouse (12 ml/kg). Application volume of the treatment dose was 200μl per mouse (8 ml/kg).

All moribund mice were euthanized with CO2. At day 57 all surviving mice were euthanized with CO2 and analyzed for body weight. Blood samples, tissue samples of lung and liver were collected and snap-frozen in liquid nitrogen. Lungs and livers were fixed in 2% formaldehyde 0.2% glutaraldehyde.

A comparison of survival in the H2 antibody group, Compound II group and the combination group compared to the control is shown in Fig. 8.

In the vehicle-treated control group (solid line) the first mouse was moribund at day 22. Until day 30, 50% of the mice in the control group were dead. To this time point in the treatment groups all mice were still alive. Over the whole time course of the study, all treatment groups revealed survival of more than 50%. The first mouse in the H2 antibody treatment group (dashed line) became moribund at day 34, and the first mice of the Compound II treatment group (dotted line) only at day 39. At this time point, 75% or 90% of mice, respectively, had already died in the control group. At day 43, the last moribund mice in the control group (solid line) were euthanized, while 78% of mice treated with the H2 antibody, 80% of mice treated with Compound II and 100% of mice treated with both agents (dash-dotted line) were still alive. Treatment of the remaining animals continued for one more week, followed by another week of close health monitoring without any further treatment. During this period no more mice died.

List of References

Brown, P.D., Medical Oncology 14 (1997) 1-10

Drummond, A.H., et al., Ann. N Acad. Sci. 878 (1999) 228-235

Fletcher, L, Nature Biotechnology 18 (2000) 1138-1139

Giavazzi, R., et al., Clin. Cancer Res. 4 (1998) 985-992

Johansson, N., et al., Cell. Mol. Life Sci. 57 (2000) 5-15

Rasheed, S., et al., Cancer 33 (1974) 1027-1033

Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Sachs, L., Angewandte Statistik, 6 ed., Springer, 1984 SPSS Statistical Algorithm, 2^nd ed. , 1991, ISBN 0-918469-89-9 US 5,519,120 US 6,025,142 US 6,110,924 US 6,242,455

Vu, T.H., et al., Cell 93 (1998) 411-422 WO 01/25217 WO 02/089824 WO 97/23465

Claims

Patent Claims

1. A pharmaceutical combination consisting of a trioxopyrimidine with inhibitory activity against matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) and an antibody against urokinase receptor (anti-uPAR antibody).

2. Pharmaceutical combination according to claim 1, characterized in that the anti-uPAR antibody binds to an epitope of uPAR located outside amino acids 1-87 of uPAR and inhibits the binding between uPA and uPAR.

3. Method for the preparation of a pharmaceutical combination for use in treating a patient in need of an anti-metastasis therapy by administration of an effective amount of a trioxopyrimidine compound with inhibitory activity against MMP-2 and MMP-9, characterized in that the pharmaceutical combination is formulated to provide an anti-uPAR antibody for the use in combination with said trioxopyrimidine compound and said anti-uPAR antibody is in a form to enable administration.

4. A pharmaceutical combination in the form of a pre-packed unit for use in treating a patient in need of an anti-metastasis therapy, characterized by comprising separate portions of each of a trioxopyrimidine compound with inhibiting activity against MMP-2 and MMP-9 in a first container and an anti-uPAR antibody in a second container, whereby the amount of compounds in each container is effective for treatment and prevention of metastasis.

5. Use of a trioxopyrimidine having an inhibitory activity against matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) and an antibody against urokinase receptor (anti-uPAR antibody) for the preparation of a pharmaceutical combination.

6. Method for treating a subject (mammal, wherein the preferred mammal is a human) in need of an anti-metastasis therapy, characterized by administering to said subject an effective amount of (i) a trioxopyrimidine compound having inhibiting activity against MMP-

2 and MMP-9 and (ii) an anti-uPAR antibody.