HK1166004A - Therapeutic combination comprising a plk 1 inhibitor and an antineoplastic agent - Google Patents

Description

Therapeutic combination comprising a PLK1 inhibitor and an antineoplastic agent

The present invention relates generally to the field of cancer therapy and more specifically provides an anti-tumor combination comprising a PLK1 inhibitor and one or more anti-tumor agents, which combination has a good anti-tumor effect.

Cancer is a major cause of death in humans; surgery, radiation therapy and chemotherapy are useful approaches to combat cancer. In particular, combination chemotherapy, which is intended to treat cancer by the combined or combined use of more than one drug, is a widely accepted treatment for neoplastic diseases, such as cancer. There have been several and still ongoing efforts to select increasingly effective and safe antitumor combinations for administration to patients suffering from cancer. In order to reduce the toxic effects of the individual drugs when used alone, and because in some cases the combination has greater efficacy than either agent alone, enhancing the antitumor efficacy of known antitumor compounds by administering them in combination with one or more different antitumor agents is a strongly felt need in the field of anticancer therapy.

Expression of PLK1 was seen in all proliferating normal tissues, whereas overexpression was observed in a range of tumors including breast, prostate, ovarian, lung, gastric and colon cancers. Once PLK1 was depleted by RNAi in cancer cells, inhibition of proliferation and reduced viability (which resulted in cell cycle arrest at the 4N DNA content stage followed by apoptosis) was observed. Although 4 different PLKs family members are described in humans, this inhibition of enzyme activity or depletion of PLK1 is sufficient to induce G2/M cell cycle arrest and apoptosis in tumor cell lines and tumor regression in xenograft models. Furthermore, for other PLKs, the function of tumor suppressor genes has been described, and it has been reported that PLK2 and PLK3 (but not PLK1) are expressed in non-proliferating, differentiated post-mitotic cells (e.g., neurons), suggesting that PLK 1-specific compounds may have better safety (see, e.g., Strebhardt K, et al, Nat Rev Cancer 2006; 6 (4): 321-30).

The use of mitotic inhibitors in cancer therapy is a widely accepted clinical strategy for the treatment of a wide range of human cancers (see, e.g., Jackson JR, et al, Nature reviews cancer 2007; 7, 107-117). The taxanes (paclitaxel and docetaxel) and vinca alkaloids (vincristine and vinblastine) act by stabilizing or destabilizing microtubules (with catastrophic consequences in cell progression through mitosis). They are first-line therapeutics (vinca alkaloids) in several tumor types, including hodgkin's disease, non-hodgkin's lymphoma, testicular cancer, neuronal cell tumors, and Wilms tumor, and second-line therapeutics (taxanes) in cisplatin-refractory ovarian cancer, breast cancer, lung cancer, and esophageal cancer. However, certain toxicities of these agents (e.g., peripheral neuropathy) are often observed due to the role of microtubules in processes such as cell migration, phagocytosis, and axonal transport.

Pyrazoloquinazolines described and claimed in patent application WO2008074788 (nervaano Medical Sciences Srl.) are potent inhibitors of PLK1 and are therefore suitable for the treatment of proliferative disorders, in particular cancer.

The compound of formula (I) is one of the compounds described and claimed in the patent applications indicated above. Its preparation, pharmaceutical compositions containing it and its use in medicine are also described and claimed in this application.

It has now been surprisingly found that the antitumor effect of the compounds of formula (I) is greatly enhanced when administered in combination with known antitumor agents.

The present invention provides novel combinations comprising (a) a compound of formula (I) and (b) one or more known antineoplastic agents. These combinations are particularly useful in the treatment of proliferative disorders, particularly cancer.

More specifically, the combinations according to the invention are very suitable as antitumor agents in therapy and do not have the drawbacks associated with the currently available antitumor agents in terms of toxicity and side effects.

The object of the present invention is therefore a combination which comprises (a) a compound of the formula (I)

And (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, alkylating or alkylating-like agents, intercalating agents, topoisomerase I or II inhibitors, antimitotic agents, kinase inhibitors, proteasome inhibitors, and antibodies inhibiting growth factors or their receptors, wherein the active ingredients of the combination are present in each case in free form or in the form of a pharmaceutically acceptable salt or any hydrate or solvate thereof.

Another aspect relates to a combination according to the invention for simultaneous, separate or sequential use.

Another aspect relates to a combination according to the invention for use in the treatment or delay of progression of a proliferative disorder.

Another aspect of the present invention relates to the use of a combination according to the present invention in the manufacture of a medicament for the treatment or delay of progression of a proliferative disorder, wherein said use comprises the simultaneous, sequential or separate administration of said therapeutic combination to a subject in need thereof.

Furthermore, the present invention relates to a pharmaceutical composition comprising a combination according to the invention in admixture with a pharmaceutically acceptable carrier, diluent or excipient.

Another aspect relates to a pharmaceutical composition according to the invention for use in the treatment or delay of progression of a proliferative disorder.

Another aspect of the invention relates to the use of a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of a proliferative disorder or for delaying the progression of a proliferative disorder, wherein said use comprises the simultaneous, sequential or separate administration of said pharmaceutical composition to a subject in need thereof.

Furthermore, the present invention relates to a method of treating a proliferative disorder, said method comprising administering to a subject in need thereof simultaneously, sequentially or separately a combination of the invention.

Another aspect relates to a method of treating a proliferative disorder, said method comprising administering to a subject in need thereof simultaneously, sequentially or separately a pharmaceutical composition of the invention.

Another aspect relates to a method of reducing side effects caused by antineoplastic therapy with an antineoplastic agent in a mammal, including a human, in need thereof, said method comprising administering to said mammal a combined preparation comprising (a) a compound of formula (I) as defined in claim 1 and (b) one or more antineoplastic agents selected from the group consisting of an antimetabolite agent, an alkylating or alkylating-like agent, an intercalating agent, a topoisomerase I or II inhibitor, an antimitotic agent, a kinase inhibitor, a proteasome inhibitor and an antibody that inhibits a growth factor or its receptor, in amounts effective to produce a synergistic antineoplastic effect.

The present invention also provides a commercial kit comprising: in a suitable container means (a) a compound of formula (I) as hereinbefore defined, and (b) one or more antineoplastic agents selected from the group consisting of an antimetabolite agent, an alkylating or alkylating-like agent, an intercalating agent, a topoisomerase I or II inhibitor, an antimitotic agent, a kinase inhibitor, a proteasome inhibitor and an antibody which inhibits a growth factor or a receptor thereof.

In the kit according to the invention, (a) a compound of formula (I) as defined above, and (b) one or more antineoplastic agents selected from the group consisting of an antimetabolite agent, an alkylating or alkylating-like agent, an intercalating agent, a topoisomerase I or II inhibitor, an antimitotic agent, a kinase inhibitor, a proteasome inhibitor and an antibody inhibiting a growth factor or its receptor, are present in a single container means or in different container means.

Another embodiment of the present invention is a commercial kit comprising a pharmaceutical composition as described above.

The kit according to the invention is intended for simultaneous, separate or sequential use in antitumor therapy.

The kit according to the invention is intended for use in anticancer therapy.

According to a preferred embodiment of the invention, the combination of the invention comprises a compound of formula (I) and an antimetabolite selected from the group consisting of 5-fluorouracil, azacytidine, capecitabine, cytarabine, gemcitabine, pemetrexed, methotrexate, edatrexate, hydroxyurea, fludarabine and mercaptopurine.

Preferably, the antimetabolite for use in the present invention is gemcitabine or cytarabine.

According to another preferred embodiment of the invention, the combination according to the invention comprises a compound of formula (I) and an alkylating or alkylating-like agent selected from the group consisting of nitrogen mustards (nitrogen mustards, cyclophosphamide, ifosfamide, melphalan and chlorambucil), aziridines (thiotepa), nitrosoureas (carmustine, lomustine, semustine), triazenes (dacarbazine and temozolomide) and platinum derivatives (cisplatin, oxaliplatin, carboplatin and satraplatin).

More preferably, the alkylating or alkylating-like agent for use in the present invention is cisplatin.

According to another preferred embodiment of the invention, the combination of the invention comprises a compound of formula (I) and an intercalator; preferably, the intercalating agent is bleomycin.

According to another preferred embodiment of the invention, the combination of the invention comprises a compound of formula (I) and a topoisomerase I inhibitor selected from topotecan, SN-38, CPT11 and 9-nitrocamptothecin.

More preferably, the topoisomerase I inhibitor is SN-38 or CPT 11.

According to another preferred embodiment of the invention, the combination of the invention comprises a compound of formula (I) and a topoisomerase II inhibitor selected from doxorubicin, epirubicin, idarubicin, nemorubicin, mitoxantrone, etoposide and teniposide.

Preferably, the topoisomerase II inhibitor is doxorubicin.

According to another preferred embodiment of the invention, the combination of the invention comprises a compound of formula (I) and an antimitotic agent selected from paclitaxel, docetaxel, ixabepilone, vinblastine, vincristine, vindesine and vinorelbine.

More preferably, the antimitotic agent is paclitaxel.

According to another preferred embodiment of the invention, the combination of the invention comprises a compound of formula (I) and a kinase inhibitor selected from sorafenib, dasatinib, gefitinib, erlotinib, sunitinib, imatinib, nilotinib and lapatinib.

According to another preferred embodiment, the kinase inhibitor is sorafenib or dasatinib

According to another preferred embodiment of the invention, the combination of the invention comprises a compound of formula (I) and a proteasome inhibitor; preferably, the proteasome inhibitor is bortezomib.

According to another preferred embodiment of the invention, the combination of the invention comprises a compound of formula (I) and an antibody inhibiting a growth factor or its receptor selected from bevacizumab (antibody to vascular endothelial growth factor), cetuximab, panitumumab, matuzumab, nimotuzumab (antibody to epidermal growth factor receptor), trastuzumab and pertuzumab (antibody to ErbB 2).

According to another preferred embodiment, the antibody is bevacizumab.

The term "synergistic antineoplastic effect" as used herein means inhibiting tumor growth, preferably complete tumor regression, by administering to a mammal, including a human, an effective amount of a combination of a compound of formula (I) as defined above and one or more antineoplastic agents selected from the group consisting of antimetabolite agents, alkylating or alkylating-like agents, intercalating agents, topoisomerase I or II inhibitors, antimitotic agents, kinase inhibitors, proteasome inhibitors and antibodies that inhibit growth factors or their receptors.

The term "combined preparation" as used herein is especially defined as a "kit of parts" in the sense that the combination partners (a) and (b) (as defined above) can be administered (i.e. at the same time or at different time points) independently or by using different fixed combinations with differing amounts of the combination partners (a) and (b). The components of the kit of parts may then be administered, for example, simultaneously or staggered in time (i.e., at different points in time and with equal or different time intervals for any component of the kit of parts). Very preferably, the time intervals are chosen such that the effect on the treated disease in the combined use of the parts is greater than the effect which would be obtained by use of only any one of the combination partners (a) and (b). The ratio of the total amounts of the combination partner (a) to be administered to the combination partner (b) in the combined preparation may be varied, e.g. in order to cope with the needs of a patient sub-population to be treated or the needs of the individual patient (different needs may be due to the particular disease, age, sex, weight, etc. of the patient). Preferably, there is at least one beneficial effect (e.g. a mutual potentiating effect of the combination partners (a) and (b)), in particular a synergistic effect (e.g. a stronger than additive effect, an additional beneficial effect, less side effects, a combined therapeutic effect at ineffective doses of one or both of the combination partners (a) and (b)), and very preferably a strong synergistic effect of the combination partners (a) and (b).

The term "administered" or "administering" as used herein means parenteral and/or oral administration. By "parenteral" is meant intravenous, subcutaneous, and intramuscular administration.

For the administration of the compounds of formula (I), a course of treatment of from about 5 to about 500mg per dose, 1 to 5 times per day, is generally employed.

The compounds of formula (I) may be administered in a variety of dosage forms, for example, oral tablets, capsules, sugar-or film-coated tablets, liquid solutions or suspensions; suppositories for rectal administration; injections or infusions administered parenterally (e.g., intramuscularly) or by intravenous and/or intrathecal and/or intraspinal administration.

In the methods of the invention, for the administration of an antimetabolite, preferably cytarabine or gemcitabine, a course of treatment of 200mg/m of body surface area is typically employed²To 5000mg/m²As weekly administration. More preferably, the course of therapy generally employed is administration on days 1 and 8 of a 21-day cycle or on days 1, 8 and 15 of a 28-day cycle or on day 1 of a 21-day cycleUsing about 500mg/m²To 1250mg/m²。

For the administration of an alkylating agent, preferably temozolomide, a course of treatment of 15mg/m of body surface area per day is generally employed²To 300mg/m². More preferably, a course of treatment of up to about 50mg/m per day for up to 42 consecutive days is generally employed²To 150mg/m²。

For the administration of platinum derivatives, preferably cisplatin, a course of 10mg/m of body surface area per day every 2-3 weeks is generally employed²To 100mg/m². More preferably, the course of treatment generally employed is 1 time every 2 weeks with about 30mg/m administered on day 1²To 85mg/m²。

For the administration of an intercalating agent, preferably bleomycin, a course of 1U/m2 to 100U/m2 of body surface area administered 1 or 2 times per week is typically employed.

For administration of topoisomerase I inhibitors, preferably CPT-11, a course of 1mg/m of body surface area per day for 2-10 consecutive days is generally employed²To 500mg/m². More preferably, the course of treatment generally employed is administration of about 50mg/m weekly or every 2 weeks or every 3 weeks²To 350mg/m²。

For the administration of a topoisomerase II inhibitor, preferably doxorubicin, a course of treatment generally of 0.1mg/m of body surface area per day for 2-10 consecutive days²To 500mg/m². More preferably, a course of treatment generally employed is administration of about 0.5mg/m per day for 3-5 consecutive days in a 21-day cycle²To 100mg/m²。

For administration of an antimitotic agent, preferably paclitaxel, a course of treatment of about 50mg/m of body surface area per 3 weeks of administration is typically employed²To 100mg/m²Or 30mg/m per week²And (3) starting.

For administration of a kinase inhibitor, preferably sorafenib, a course of treatment generally employed may be from 1mg to 5000 mg. More preferably, a course of treatment of from about 10mg to about 10mg is employed2000 mg. For the administration of proteasome inhibitors, preferably bortezomib, a course of treatment of 0.1mg/m of body surface area per 3 weeks of administration is generally employed²To 30mg/m²。

Finally, for the administration of an antibody inhibiting a growth factor or its receptor, preferably bevacizumab, a course of treatment generally taken may be from 0.1mg/kg to 100 mg/kg. More preferably, a course of treatment is employed wherein 1mg/kg to 20mg/kg is administered on day 1 of a 3 week cycle.

When the active components of the combined preparation according to the invention are provided together with a pharmaceutically acceptable carrier or excipient, a pharmaceutical composition is formed. Such pharmaceutical compositions constitute another embodiment of the invention.

The pharmaceutically acceptable carriers and excipients are selected to minimize the side effects of the pharmaceutical compound but not to negate or inhibit the efficacy of the compound to the point where the treatment is ineffective.

The pharmaceutically acceptable carriers or excipients used in the preparation of the pharmaceutical compositions according to the invention are well known to those skilled in the art of formulating compounds in the form of pharmaceutical compositions. For example, "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to mammals, including humans. For example, "pharmaceutically acceptable excipient" means any inert substance that is intentionally added to the formulation of a dosage form to serve as a diluent or medium for the active substance. The term includes binders, fillers, disintegrants and lubricants.

However, the combination of the invention may be used without any sustained-release adjuvant.

Pharmaceutical formulations and administration techniques may be found in "Remington's pharmacological sciences"; mack Publishing co., Easton, PA, latest edition.

Pharmaceutical compositions suitable for parenteral administration are prepared in sterile form. The sterile composition may thus be a sterile solution or suspension in a non-toxic parenterally acceptable diluent or solvent.

The amount of active ingredient contained in the pharmaceutical composition according to the invention may vary and will depend to a large extent on various factors (such as the route of administration and the vehicle).

The combination according to the invention and the pharmaceutical composition are suitable for use in anticancer therapy.

The anti-tumor treatments of the present invention are particularly useful for treating all forms of cancer, including but not limited to: cancers such as bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer including small cell lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer including squamous cell carcinoma; hematopoietic cancers of the lymphoid lineage (lymphoblastic carcinomas) including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt's lymphoma; hematopoietic cancers of the myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndromes, and promyelocytic leukemia; carcinomas of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; mesothelioma; central and peripheral nervous system cancers including astrocytomas, neuroblastoma, glioma, and schwannoma; other carcinomas including mesothelioma, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, thyroid follicular carcinoma and kaposi's sarcoma.

Due to the key role of PLK1 in the regulation of cell proliferation, the combinations and pharmaceutical compositions of the present invention are also useful in the treatment of various cell proliferation disorders, such as, for example, benign prostate hyperplasia, familial adenomatosis, polyposis, neurofibromatosis, psoriasis, (associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis and post-operative stenosis and restenosis) vascular smooth muscle cell proliferation.

The combinations and pharmaceutical compositions of the invention, as modulators of apoptosis, may also be useful in the treatment of cancer, viral infections, prevention of the development of AIDS in HIV-infected individuals, autoimmune diseases and neurodegenerative disorders.

The antitumor effect of the combined preparation of the invention is shown, for example, by the following in vitro tests which are intended to illustrate the invention without limiting it in any way.

Examples

Materials and methods:exponentially growing human pancreatic cancer (Mia-PaCa, Capan-1), human colon cancer (HCT-116), leukemia (HL60), and multiple myeloma (KMS11) cell lines were seeded with 5% CO humidified at 37 deg.C₂Incubation in the environment. Drugs were added to the experimental cultures and incubated for 72 hours at 37 ℃ in the dark. MCF7 Breast cancer cell line inoculated and humidified 5% CO at 37 ℃₂Incubation in the environment. Drugs were added to the experimental cultures and incubated at 37 ℃ for 120 hours in the dark. Scalar doses of the compound of formula (I) and the antineoplastic agent were added to the medium 24 hours after inoculation. The test was performed on two treatment schedules: simultaneous administration (both drugs were administered to cells 72/120 hours) and continuous administration (the compound of formula (I) was administered 24 hours after the other agents). The drug solution is prepared immediately after use. At the end of the treatment, cell proliferation was determined by using the intracellular adenosine triphosphate monitoring system (CellTiterGlo-Promega) of the envision (perkinelmer) reader. Inhibition activity was assessed by comparing the treatment data to control data using the test probe (AssayExplorer) (MDL) program. The dose that inhibited 50% cell growth was calculated using a sigmoidal interpolation curve. The combination index (C.I.) was calculated using a proprietary computer program for the analysis of the effects of multiple drugs based on the Chou-Talalay equation for mutually non-exclusive drugs (Adv Enzyme Regul 1984; 22: 27-55), where C.I. < 1 indicates a stronger than additive effect; C.I.: strong antagonism is more than 3; 1.3-3 antagonism; 1.2-0.8 additive effect; 0.8-0.3 synergistic effect; less than 0.3 strong synergistic effect.

Example 1: in vitro cytotoxic activity of the compound of formula (I) in combination with gemcitabine.

The results of the synergistic effect of the compound of formula (I) and gemcitabine (i.e., 0.3 < C.I. < 0.8) are reported in Table 1 for the Capan-1 human pancreatic carcinoma cell line.

TABLE 1

Example 2: the in vitro cytotoxic activity of the compound of formula (I) in combination with cisplatin.

The results of the synergistic effect of the compound of formula (I) and cisplatin (i.e., 0.3 < C.I. < 0.8) are reported in Table 2 for HCT-116 human colon carcinoma cell line.

TABLE 2

Example 3: in vitro cytotoxic activity of the compound of formula (I) in combination with SN-38.

The results of the synergistic effect of the compound of formula (I) and SN-38 (i.e., 0.3 < C.I. < 0.8) are reported in Table 3 for the HCT-116 human colon carcinoma cell line.

TABLE 3

Example 4: in vitro cytotoxic activity of the compound of formula (I) in combination with doxorubicin.

The results of the synergistic effect of the compound of formula (I) and doxorubicin (i.e., 0.3 < C.I. < 0.8) are reported in Table 4 for the HL-60 human leukemia cell line.

TABLE 4

Example 5: in vitro cytotoxic activity of the compound of formula (I) in combination with paclitaxel.

The results of the synergistic effect of the compound of formula (I) and paclitaxel (i.e., 0.3 < C.I. < 0.8) are reported in Table 5 for HCT-116 human colon carcinoma, HL60 human leukemia and KMS11 human multiple myeloma cell lines.

TABLE 5

Example 6: in vitro cytotoxic activity of the compound of formula (I) in combination with sorafenib.

The results of the synergistic effect of the compound of formula (I) and sorafenib (i.e., 0.3 < C.I. < 0.8) are shown in Table 6 to be reported for HCT-116 human colon cancer and Mia-PaCa human pancreatic cancer cell lines.

TABLE 6

Example 7: in vitro cytotoxic activity of the compound of formula (I) in combination with dasatinib.

The results of the synergistic effect of the compound of formula (I) and dasatinib (i.e. 0.3 < c.i. < 0.8) are shown in table 7 to be reported for the MCF7 breast cancer cell line after 120 hours of treatment.

TABLE 7

Example 8: in vitro cytotoxic activity of the compound of formula (I) in combination with bortezomib.

The results of the synergistic effect of the compound of formula (I) and bortezomib (i.e., 0.3 < c.i. < 0.8) are reported in table 8 for KMS11 human multiple myeloma and Mia-PaCa human pancreatic carcinoma cell line.

TABLE 8

Example 9: the in vitro cytotoxic activity of the compound of formula (I) in combination with cytarabine.

The results of the synergistic effect of the compound of formula (I) and cytarabine (i.e., 0.3 < C.I. < 0.8) are reported in Table 9 for the HL-60 human promyelocytic leukemia cell line.

TABLE 9

Example 10: the in vivo cytotoxic activity of the compound of formula (I) in combination with CPT 11.

Balb male nude mice, purchased from Harlan (Italy), were placed in cages with sterilized paper filter covers (paper filter cover), food and bedding and acidified water according to the European Community council directive No. 86/609/EEC.

HT29 human colon cancer tumor fragment was implanted subcutaneously. Treatment was initiated when tumors were palpable. The compound of formula (I) was administered by the oral route at doses of 45 and 60mg/kg daily on days 2, 3, 4, 6, 7 and 8. CPT11 was administered by intravenous route at a dose of 45mg/kg on days 1, 5, 9. In combination, the compound of formula (I) is administered on days 2, 3, 4, 6, 7 and 8, and CPT11 is administered on days 1, 5, 9. Tumor growth and body weight were measured every 3 days. Tumor growth was assessed by caliper. Two diameters were recorded and tumor weight was calculated according to the following formula: length (mm) x width²/2. The effect of anti-tumor therapy is evaluated as delaying the onset of exponential growth of tumors (see literature Anticancer drugs 7: 437-60, 1996). This delay (T-C value) is defined as the difference in the time (unit: day) required for the tumors of the treated group (T) and the control group (C) to reach a predetermined size (1 g). Synergistic effects are identified when the T-C value of the combination is > the T-C value of the individual drugs.

Toxicity was assessed on the basis of weight loss. The results are reported in table 10.

Watch 10

^*Oral treatment is carried out for 2, 3, 4, 6, 7 and 8 days

^**Treatment by intravenous route on days 1, 5 and 9

^***Treatment with a compound of formula (I) on days 2, 3, 4, 6, 7, 8; treatment with CPT11 on days 1, 5, and 9

The compound of formula (I) in combination with CPT11 produced a clear synergistic effect. The observed T-C for the compound of formula (I) in combination with CPT11 is greater than the expected simple addition of T-C from monotherapy. No toxicity was observed in any of the treatment groups.

Example 11: in vivo cytotoxic activity of the compound of formula (I) in combination with 5 FU.

Balb male nude mice, purchased from Harlan (Italy), were placed in cages with sterilized paper filter covers, food and bedding, and acidified water according to European Community guidelines No. 86/609/EEC. HT29 human colon cancer tumor fragment was implanted subcutaneously. Treatment was initiated when tumors were palpable. The compound of formula (I) was administered by the oral route at a dose of 45mg/kg daily on days 2, 3, 4, 6, 7 and 8. 5FU was administered by intravenous route at a dose of 50mg/kg on days 1, 5 and 9. In combination, the compound of formula (I) is administered on days 2, 3, 4, 6, 7 and 8, and 5FU is administered on days 1, 5 and 9. Tumor growth and body weight were measured every 3 days. Tumor growth was assessed by caliper. Two diameters were recorded and tumor weight was calculated according to the following formula: length (mm) x width²/2. The effect of anti-tumor therapy is evaluated as delaying the onset of exponential growth of tumors (see literature Anticancer drugs 7: 437-60, 1996). This delay (T-C value) is defined as the difference in the time (unit: day) required for the tumors of the treated group (T) and the control group (C) to reach a predetermined size (1 g). Synergistic effect is identified when the T-C value of the combination is > the T-C values of the individual drugs and additive effect is identified when the T-C value of the combination is equal to the T-C values of the individual drugs.

Toxicity was assessed on the basis of weight loss. The results are reported in table 11.

TABLE 11

^*Oral treatment is carried out for 2, 3, 4, 6, 7 and 8 days

^**Treatment by intravenous route on days 1, 5 and 9

^***Using the compound of formula (I) for 2, 3, 4, 6, 7, 8 daysTreatment; treatment with 5FU on days 1, 5, and 9

The T-C observed when the compound of formula (I) is combined with 5FU is similar to the expected simple addition of T-C from monotherapy, suggesting an additive effect.

Example 12: the in vivo cytotoxic activity of the compound of formula (I) in combination with bevacizumab.

Balb male nude mice, purchased from Harlan (Italy), were placed in cages with sterilized paper filter covers, food and bedding, and acidified water according to European Community guidelines No. 86/609/EEC.

HT29 human colon cancer tumor fragment was implanted subcutaneously. Treatment was initiated when tumors were palpable. The compound of formula (I) is administered by the oral route in a volume of 10ml/kg at doses of 45 and 60mg/kg daily on days 2, 3, 4, 6, 7, 8, 10, 11 and 12. Bevacizumab was administered at a dose of 20mg/kg by intraperitoneal route on days 1, 5, 9 and 13. In combination, the compound of formula (I) is administered on days 2, 3, 4, 6, 7, 8, 10, 11 and 12, and bevacizumab is administered on days 1, 5, 9 and 13. Tumor growth and body weight were measured every 3 days. Tumor growth was assessed by caliper. Two diameters were recorded and tumor weight was calculated according to the following formula: length (mm) x width²/2. The effect of anti-tumor therapy is evaluated as delaying the onset of exponential growth of tumors (see literature Anticancer drugs 7: 437-60, 1996). This delay (T-C value) is defined as the difference in the time (unit: day) required for the tumors of the treated group (T) and the control group (C) to reach a predetermined size (1 g). Synergistic effect is identified when the T-C value of the combination is > the T-C values of the individual drugs and additive effect is identified when the T-C value of the combination is equal to the T-C values of the individual drugs. Toxicity was assessed on the basis of weight loss. The results are reported in table 12.

TABLE 12

^*Oral treatment was performed on days 2, 3, 4, 6, 7, 8, 10, 11, 12.

^**Treatment was given by intravenous route on days 1, 5, 9, 13.

^***Treatment with a compound of formula (I) on days 2, 3, 4, 6, 7, 8, 10, 11, 12 and bevacizumab on days 1, 5, 9, 13.

The observed T-C when the compound of formula (I) is combined with bevacizumab is similar to the expected simple addition of T-C resulting from monotherapy. No toxicity was observed in any of the treatment groups.

Example 13: the in vivo cytotoxic activity of the compound of formula (I) in combination with cytarabine.

SCID female mice from Harlan (Italy) were placed in cages with sterilized paper filter covers, food and bedding, and acidified water according to European Community guidelines No. 86/609/EEC. Mice were injected intravenously with 0.2ml of 5X10⁶AML-PS acute myeloid leukemia cells.

The compound of formula (I) is administered by the oral route at a dose of 120mg/kg on days 2, 3, 14, 15, 26, 27, 38, 39. Cytarabine was administered by intraperitoneal route at a dose of 75mg/kg on days 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 26, 27, 28, 29, 30, 38, 39, 40, 41, 42. In combination, the compound of formula (I) is administered on days 2, 3, 14, 15, 26, 27, 38, 39 and cytarabine is administered on days 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 26, 27, 28, 29, 30, 38, 39, 40, 41, 42. Body weight was measured every 3 days. The mean time to live of the treated animals relative to the control group is measured to evaluate the antitumor therapeutic effect. The results are reported in table 13.

The antitumor efficacy of the combination group was statistically significant relative to that of the monotherapy group, suggesting that the combination therapy of the compound of formula (I) and cytarabine has potential synergy. No toxicity was observed.

Watch 13

^*Oral treatment is carried out for 2, 3, 14, 15, 26, 27, 38 and 39 days,

^**treatment was given by intraperitoneal route on days 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 26, 27, 28, 29, 30, 38, 39, 40, 41, 42.

^***Oral treatment with a compound of formula (I) is performed on days 2, 3, 14, 15, 26, 27, 38, 39 and intraperitoneal treatment with cytarabine is performed on days 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 26, 27, 28, 29, 30, 38, 39, 40, 41, 42.

Claims (25)

1. A combination comprising (a) a compound of formula (I)

And (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, alkylating or alkylating-like agents, intercalating agents, topoisomerase I or II inhibitors, antimitotic agents, kinase inhibitors, proteasome inhibitors, and antibodies that inhibit growth factors or their receptors, wherein the active ingredients of the combination are present in each case in free form or in the form of a pharmaceutically acceptable salt or any hydrate or solvate thereof.

2. The combination according to claim 1 wherein the antimetabolite agent is selected from the group consisting of 5-fluorouracil, azacytidine, capecitabine, cytarabine, gemcitabine, pemetrexed, methotrexate, edatrexate, hydroxyurea, fludarabine, and mercaptopurine.

3. The combination according to claim 2 wherein the antimetabolite agent is gemcitabine or cytarabine.

4. The combination according to claim 1, wherein the alkylating or alkylating-like agent is selected from the group consisting of nitrogen mustards (nitrogen mustards, cyclophosphamide, ifosfamide, melphalan and chlorambucil), aziridines (thiotepa), nitrosoureas (carmustine, lomustine, semustine), triazenes (dacarbazine and temozolomide) and platinum derivatives (cisplatin, oxaliplatin, carboplatin and satraplatin).

5. A combination according to claim 4 wherein the alkylating or alkylating-like agent is cisplatin.

6. The combination according to claim 1, wherein the intercalating agent is bleomycin.

7. The combination according to claim 1, wherein said topoisomerase I inhibitor is selected from topotecan, SN-38, CPT11, and 9-nitrocamptothecin.

8. The combination according to claim 7, wherein said topoisomerase I inhibitor is SN-38 or CPT 11.

9. The combination according to claim 1, wherein the topoisomerase II inhibitor is selected from doxorubicin, epirubicin, idarubicin, nemorubicin, mitoxantrone, etoposide and teniposide.

10. The combination according to claim 9 wherein said topoisomerase II inhibitor is doxorubicin.

11. The combination according to claim 1 wherein the antimitotic agent is selected from the group consisting of paclitaxel, docetaxel, ixabepilone, vinblastine, vincristine, vindesine and vinorelbine.

12. The combination according to claim 11 wherein the antimitotic agent is paclitaxel.

13. The combination according to claim 1, wherein said kinase inhibitor is selected from the group consisting of sorafenib, dasatinib, gefitinib, erlotinib, sunitinib, imatinib, nilotinib, and lapatinib.

14. The combination according to claim 13, wherein the kinase inhibitor is sorafenib or dasatinib.

15. The combination according to claim 1, wherein the proteasome inhibitor is bortezomib.

16. The combination according to claim 1, wherein the antibody inhibiting a growth factor or its receptor is selected from bevacizumab (antibody to vascular endothelial growth factor), cetuximab, panitumumab, matuzumab, nimotuzumab (antibody to epidermal growth factor receptor), trastuzumab and pertuzumab (antibody to ErbB 2).

17. The combination according to claim 16, wherein said antibody inhibiting a growth factor or its receptor is bevacizumab.

18. A pharmaceutical composition comprising a combination according to any one of claims 1 to 17 in admixture with a pharmaceutically acceptable carrier, diluent or excipient.

19. A combination according to any one of claims 1 to 17 or a pharmaceutical composition according to claim 18 for simultaneous, separate or sequential use.

20. A method of treating or delaying the progression of a proliferative disorder comprising administering to a patient in need thereof simultaneously, sequentially or separately a therapeutically effective amount of a combination according to any one of claims 1 to 17.

21. A method of treating a proliferative disorder comprising simultaneously, sequentially or separately administering to a patient in need thereof (a) a compound of formula (I) as defined in claim 1 and (b) one or more antineoplastic agents selected from the group consisting of an antimetabolite agent, an alkylating or alkylating-like agent, an intercalating agent, a topoisomerase I or II inhibitor, an antimitotic agent, a kinase inhibitor, a proteasome inhibitor and an antibody that inhibits a growth factor or a receptor thereof.

22. A method of reducing side effects caused by antineoplastic therapy with an antineoplastic agent in a mammal, including man, in need thereof, said method comprising administering to said mammal a combined preparation comprising a compound of formula (I) as defined in claim 1 and one or more antineoplastic agents selected from the group consisting of an antimetabolite agent, an alkylating or alkylating-like agent, an intercalating agent, a topoisomerase I or II inhibitor, an antimitotic agent, a kinase inhibitor, a proteasome inhibitor and an antibody that inhibits a growth factor or its receptor, in amounts effective to produce a synergistic antineoplastic effect.

23. A commercial kit comprising a combination as defined in claim 1 or a pharmaceutical composition as defined in claim 18 in a suitable container means for simultaneous, separate or sequential use thereof.

24. A combination according to claim 1 or a pharmaceutical composition according to claim 18 for use in the treatment or delay of progression of a proliferative disorder.

25. Use of a combination according to claim 1 or a pharmaceutical composition according to claim 18 in the manufacture of a medicament for the treatment or delay of proliferation disorders.