BR112014033118B1 - PHARMACEUTICAL COMBINATIONS OF A CDK4/6 INHIBITOR AND A BRAF INHIBITOR, AND THEIR USES - Google Patents

Abstract

PHARMACEUTICAL COMBINATIONS OF A CDK4/6 INHIBITOR AND A B-RAF INHIBITOR. The present invention relates to a pharmaceutical combination comprising: (a) a CDK4/6 inhibitor, (b) a B-Raf inhibitor and optionally (c) a MEK 1/2 inhibitor; combined preparations and pharmaceutical compositions thereof; uses of such a combination in the treatment of proliferative diseases; and methods of treating an individual suffering from a proliferative disease comprising administering a therapeutically effective amount of such a combination.

Description

FIELD OF INVENTION

[001] A combination of a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, a B-Raf kinase inhibitor and optionally a mitogen-activated protein kinase (MEK 1/2 or MEK) inhibitor which It is used to treat proliferative diseases. The present invention also relates to the uses of such a combination in the treatment of proliferative diseases; to pharmaceutical compositions of the combination of agents and methods of treating an individual suffering from a proliferative disease comprising administering a therapeutically effective amount of such combination to the individual.

BACKGROUND OF THE INVENTION

[002] Tumor development is closely associated with genetic alteration and deregulation of cyclin-dependent kinases (Cyclin-Dependent Kinase - CDKs) and their regulators, suggesting that CDK inhibitors may be useful anticancer therapeutic agents. The function of CDKs is to phosphorylate and, thus, activate or deactivate certain proteins. The catalytic step mediated by CDKs involves a phosphate transfer reaction from ATP to the macromolecular enzyme substrate. Several groups of compounds (reviewed, for example, in Fischer, P. M. Curr. Opin. Drug Discovery Dev. 2001, 4, 623-634) have been found to possess antiproliferative properties by virtue of CDK-specific ATP antagonism.

[003] Mediation at the molecular level of CDK/cyclin complex activity requires a series of stimulatory and inhibitory phosphorylation or dephosphorylation events. CDK phosphorylation is carried out by a group of CDK-activating kinases (CAKs) and/or kinases such as wee1, Myt1 and Mik1. Dephosphorylation is carried out by phosphatases such as cdc25(a & c), pp2a or KAP.

[004] Activity of the CDK/cyclin complex can also be regulated by two families of endogenous cellular proteinaceous inhibitors: the Kip/Cip family or the INK family. INK proteins specifically bind to CDK4 and CDK6. p16ink4 (also known as MTS1) is a potential tumor suppressor gene that is mutated or deleted in a large number of primary cancers. The Kip/Cip family contains proteins such as p21Cip1, Waf1, p27Kip1 and p57kip2, where p21 is induced by p53 and is capable of inactivating the CDK2/cyclin (E/A) complex. Abnormally low levels of p27 expression have been observed in breast, colon, and prostate cancers. Conversely, overexpression of cyclin E in solid tumors has been shown to correlate with poor patient prognosis. Overexpression of cyclin D1 has been associated with esophageal, breast, squamous cell, and non-small cell lung carcinomas.

[005] The fundamental roles of CDKs and their associated proteins in coordinating and driving the cell cycle in proliferating cells were described above. Some of the biochemical pathways in which CDKs play a fundamental role have also been described. The development of monotherapies for the treatment of proliferative disorders, such as cancers, that use therapeutics either generically targeting CDKs or targeting CDKs is therefore potentially highly desirable. Thus, there is a continuous need to find new therapeutic agents for the treatment of human diseases. CDK4/6 inhibitors useful in the present combinations are generally and specifically described in PCT Published Patent Application WO2010/020675, which is incorporated herein by reference.

[006] Protein kinases represent a large family of proteins, which play a central role in regulating a wide variety of cellular processes and maintaining control over cellular function. Abnormal kinase activity has been observed in many disease states, including benign and malignant proliferative disorders, as well as diseases resulting from inappropriate activation of the immune and nervous systems.

[007] The Ras-Raf-MEK-ERK signaling pathway transmits signals from receptors on the cell surface to the nucleus and is essential for cell proliferation and survival. Since 10-20% of human cancers harbor an oncogenic Ras mutation and many human cancers have activated growth factor receptors, this pathway is an ideal target for intervention.

[008] The Raf family of serine/threonine kinases includes three members: C-Raf (or Raf-1), B-Raf and A-Raf. B-Raf activating alleles have been identified in ~70% of melanomas, 40% of papillary thyroid carcinomas, 30% of low-grade ovarian carcinomas, and 10% of colorectal cancers. The majority of B-Raf mutations are found within the kinase domain, with a single substitution (V600E) accounting for 80%. Mutant B-Raf proteins activate the Raf-MEK-ERK pathway, either through high kinase activity for MEK activation or through C-Raf. The B-Raf inhibitor in the present combination therapy inhibits cellular processes involving the B-Raf kinase, blocking the signal cascade in these cancer cells and ultimately inducing cell stasis and/or death. B-Raf inhibitors useful in the present combinations are generally and specifically described in PCT Published Patent Application WO2011/025927, which is incorporated herein by reference.

[009] MEK is also one of the main proteins in the RAS/RAF/MEK/ERK pathway, which signals cell proliferation and survival and is often activated in tumors that have mutations in the RAS or RAF oncogenes or in receptor tyrosine kinases of growth. Although only rarely mutated in cancer, inhibitors of the MEK1 and MEK2 proteins have also been targeted for small molecule inhibition by virtue of their central position in the signaling cascade to the RAS/RAF/MEK signal transduction pathway.

[0010] Optional MEK 1/2 inhibitors suitable for use in the present combinations are known in the art. MEK 1/2 inhibitors useful in the present invention include PD325901, PD-181461, ARRY142886/AZD6244, ARRY-509, RO4987655 /R-7167, GSK1120212 or AS703026.

[0011] In an important embodiment, MEK 1/2 inhibitors include compounds described in document WO03/077914, which is incorporated herein by reference in full, in particular a compound of Formula (II) or (III): or pharmaceutically acceptable salts thereof (hereinafter referred to as compounds C and D, respectively) and the compounds described in WO05/051906, WO05/023251, WO03/077855, US20050049419 and US7235537, which are incorporated herein by reference in full , encompassing N3-alkylated benzimidazoles and other similar heterocyclic derivatives as MEK 1/2 inhibitors for the treatment of proliferative diseases.

SUMMARY OF THE INVENTION

[0012] The present invention relates to a therapeutic combination comprising: (a) a CDK4/6 inhibitor and a B-Raf inhibitor useful for separate, simultaneous or sequential administration to an individual in need thereof for treatment or prevention of a proliferative disease.

[0013] The present invention especially relates to a therapeutic combination comprising: (a) a CDK4/6 inhibitor of the Formula: or a pharmaceutically acceptable salt thereof (hereinafter referred to as Compound A), (b) a B-Raf inhibitor of Formula: or a pharmaceutically acceptable salt thereof (hereinafter referred to as Compound B), and (c) optionally a MEK 1/2 inhibitor.

[0014] Hereinafter, the combinations of Compound A and Compound B and the triple combination of Compound A, Compound B and a MEK 1/2 inhibitor will be said to be a COMBINATION OF THE INVENTION.

[0015] The present invention particularly relates to a COMBINATION OF THE INVENTION useful for separate, simultaneous or sequential administration to an individual who needs it for the treatment or prevention of a proliferative disease.

[0016] The present invention also relates to a COMBINATION OF THE INVENTION for use in preparing a pharmaceutical composition or medicine for the treatment or prevention of a proliferative disease in an individual in need thereof.

[0017] The present invention also relates to the use of a COMBINATION OF THE INVENTION for the preparation of a pharmaceutical composition or medicine for the treatment or prevention of a proliferative disease.

[0018] The present invention relates to a method of treating an individual who has a proliferative disease comprising administering, to said individual, a COMBINATION OF THE INVENTION in an amount which is jointly therapeutically effective against a proliferative disease.

[0019] The present invention further provides a commercial package comprising, as therapeutic agents, a COMBINATION OF THE INVENTION, together with instructions for simultaneous, separate or sequential administration for use in delaying the progression or treatment of a proliferative disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a graphical representation of the results obtained in Example 2 demonstrating greater durability of the response to the COMBINATION OF THE INVENTION.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to a therapeutic combination comprising: (a) a CDK4/6 inhibitor of the Formula: or a pharmaceutically acceptable salt thereof, and (b) a B-Raf inhibitor of Formula: or a pharmaceutically acceptable salt thereof, for simultaneous, separate or sequential administration.

[0022] The present invention also relates to a triple pharmaceutical combination that further comprises a MEK 1/2 inhibitor.

[0023] Thus, the present invention further relates to a therapeutic combination comprising (a) a CDK4/6 inhibitor of the Formula: or a pharmaceutically acceptable salt thereof (b) a B-Raf inhibitor of Formula: or a pharmaceutically acceptable salt thereof, and (c) a MEK 1/2 inhibitor for simultaneous, separate or sequential administration.

[0024] In an important embodiment of this aspect of the invention, the MEK 1/2 inhibitor is Compound C or Compound D, particularly Compound D, or pharmaceutically acceptable salts thereof.

[0025] THE COMBINATION OF THE INVENTION is, in particular, for use in the treatment or prevention of a proliferative disease.

[0026] General terms used here are defined with the following meanings, unless explicitly indicated otherwise.

[0027] The terms "comprising" and "including" are used here in their open, non-limiting sense and unless otherwise indicated.

[0028] The terms "a" and "an" and "the" and "a" and similar references in the context of the description of the invention (especially in the context of the following claims) should be understood as encompassing both the singular and the plural, unless otherwise indicated here or in clear contradiction to the context. Where the plural form is used for compounds, salts and the like, this is taken to also mean a single compound, salt or the like.

[0029] The term "combination", "therapeutic combination" or "pharmaceutical combination", as used herein, defines a fixed combination in a dosage unit form or a kit of parts for combined administration wherein Compound A and Compound B (and optionally a MEK 1/2 inhibitor) can be administered independently at the same time or separately within time intervals that allow the combination partners to show a cooperative, e.g., synergistic, effect.

[0030] The term "pharmaceutical composition" is defined herein to refer to a mixture or solution containing at least one therapeutic agent to be administered to an individual, for example, a mammal or human, in order to prevent or treat a particular disease or condition that affects the mammal.

[0031] The term "pharmaceutically acceptable" is defined herein to refer to those compounds, materials, compositions and/or dosage forms that are, within the scope of good medical judgment, suitable for contact with the tissues of an individual, e.g. , a mammal or human, without excessive toxicity, irritation, allergic response and other problematic complications commensurate with a reasonable benefit/risk ratio.

[0032] The term "a combined preparation" is defined herein to refer especially to a "kit of parts" in the sense that the combination partners (a) and (b), and optionally (c), as defined above, they can be dosed independently or through the use of different fixed combinations with different amounts of the combination partners, that is, simultaneously or at different points of time. The parts of the kit of parts can then, for example, be administered simultaneously or chronologically staggered, that is, at different time points and with equal or different time intervals for any part of the kit of parts. The proportion of the total amounts of combination partner (a) to combination partner (b) (and, if applicable, to combination partner (c)) to be administered in the combined preparation can be varied, for example, in order to address the needs of a subpopulation of patients to be treated or the needs of a single patient.

[0033] The term "coadministration" or "combined administration", as used herein, is defined to encompass the administration of the selected therapeutic agents to a single patient and is intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

[0034] The term "treat" or "treatment", as used herein, comprises a treatment that alleviates, reduces or improves at least one symptom in an individual or affects the delay of progression of a disease. For example, the treatment may be to lessen one or several symptoms of a disorder or completely eradicate a disease, such as cancer. Within the meaning of the present invention, the term "treat" also denotes interrupting, delaying the onset (i.e., the period before clinical manifestation of the disease) and/or reducing the risk of developing or worsening a disease. The term "protect" is used herein to mean preventing, delaying or treating, or all, as appropriate, the development or continuation or worsening of a disease in an individual.

[0035] The term "therapeutically active joint" or "joint therapeutic effect" means that the therapeutic agents can be administered separately (in a chronologically staggered manner, especially a sequence-specific manner) at preferred time intervals in the warm-blooded animal, especially human being, to be treated, while still showing a (preferably synergistic) interaction (joint therapeutic effect). Whether this is the case can, inter alia, be determined by monitoring blood levels which show that both compounds are present in the blood of the human being treated, at least during certain time intervals.

[0036] The term "pharmaceutically effective amount" or "clinically effective amount" or "therapeutically effective amount" of a combination of therapeutic agents is an amount sufficient to confer an observable improvement over the baseline clinically observable signs and symptoms of the disorder treated with the combination.

[0037] The term "individual" or "patient", as used herein, includes animals which are capable of suffering or being affected with cancer or any disorder that involves, directly or indirectly, cancer. Examples of individuals include mammals, for example, humans, dogs, cows, horses, pigs, sheep, goats, cats, rats, rabbits and transgenic non-human animals. In the preferred embodiment, the subject is a human being, for example, a human being who is suffering from, at risk of suffering from, or is potentially capable of suffering from cancer.

[0038] The term "about" or "approximately" will have the meaning of within 10%, more preferably within 5%, of a given value or range.

[0039] Compound A and/or Compound B and/or the optional MEK 1/2 inhibitor can be administered in free form or in pharmaceutically acceptable salt form.

[0040] A "pharmaceutically acceptable salt", as used herein, unless otherwise indicated, includes salts of acidic and basic groups that may be present in the compounds of the present invention. The compounds of the present invention which are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of the present invention are those that form non-toxic acid addition salts, that is, salts that contain pharmaceutically acceptable anions, such as acetate, benzoate, bromide, chloride, citrate, fumarate, hydrobromide, hydrochloride, iodide, lactate, maleate, mandelate, nitrate, oxalate, salicylate, succinate and tartrate.

[0041] Unless otherwise specified or clearly indicated by the text, reference to therapeutic agents useful in the COMBINATION OF THE INVENTION includes both the free base of the compounds and all pharmaceutically acceptable salts of the compounds.

[0042] The present invention also relates to a combination, such as a combined preparation or a pharmaceutical composition, which comprises (a) Compound A and (b) Compound B and, optionally, Compound D.

[0043] The present invention particularly relates to a COMBINATION OF THE INVENTION useful for the treatment or prevention of a proliferative disease in an individual in need thereof. In this embodiment of the present invention, the COMBINATION OF THE INVENTION is used for the treatment of a proliferative disease comprising administering to the subject a combination therapy comprising an effective amount of a CDK 4/6 inhibitor selected from COMPOUND A and an effective amount of a B-Raf inhibitor selected from Compound B and, optionally, an effective amount of a MEK 1/2 inhibitor, especially a MEK 1/2 inhibitor selected from Compound D. Preferably, these inhibitors are administered in therapeutically effective dosages which, when combined, confer a beneficial effect. Administration can be separate, simultaneous or sequential.

[0044] In one embodiment, the proliferative disease is cancer. The term "cancer" is used herein to mean a broad spectrum of tumors, including all solid tumors and hematologic malignancies. Examples of such tumors include, but are not limited to, benign or malignant tumors of the brain, lung (in particular small cell lung cancer and non-small cell lung cancer), squamous cell, bladder, gastric, pancreatic, breast, head and neck, renal, kidney, ureter, ovarian, prostate, colorectal, esophageal, testicular, gynecological (e.g., uterine sarcomas, carcinoma of the fallopian tubes, endometrium, cervix, vagina, or vulva), thyroid, pancreas, bone, skin , melanoma, uterine, ovarian, rectal, anal, colon, testicles, Hodgkin's disease, esophagus, small intestine, endocrine system (e.g., thyroid, parathyroid, or adrenal glands), soft tissue sarcomas, urethra, penis, leukemia, lymphomas , tumors of the central nervous system, sarcomas, myeloma, biliary, liver, neurofibromatosis, acute myeloid leukemia (Acute Myeloid Leukemia - AML), myelodysplastic syndromes (MyeloDysplastic Syndrome - MDS) and Kaposi's sarcoma.

[0045] In another embodiment of the present invention, the proliferative disease is melanoma, lung cancer (including Non-Small Cell Lung Cancer (NSCLC)), colorectal cancer (ColoRectal Cancer - CRC), breast, kidney cancer such as, for example, renal cell carcinoma (RCC), liver cancer, endometrial cancer, acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) , thyroid cancer, particularly papillary thyroid cancer, pancreatic cancer, neurofibromatosis or hepatocellular carcinoma.

[0046] In another embodiment of the present invention, the proliferative disease is a solid tumor. The term "solid tumor" means in particular melanoma, breast cancer, ovarian cancer, colorectal and generally gastrointestinal tract cancer, cervical cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer). small), head and neck cancer, bladder cancer, prostate cancer or Kaposi's sarcoma. The present combination inhibits the growth of solid tumors and liquid tumors as well. Furthermore, depending on the type of tumor and the particular combination used, a decrease in tumor volume may be achieved. THE COMBINATION OF THE INVENTION described herein is also suitable for preventing the metastatic spread of tumors and the growth or development of micrometastases. THE COMBINATION OF THE INVENTION described here is suitable for the treatment of patients, especially poor prognosis patients having metastatic melanoma, colorectal cancer or pancreatic cancer.

[0047] In another embodiment, the proliferative disease is melanoma or colorectal cancer.

[0048] THE COMBINATION OF THE INVENTION is particularly useful for the treatment of cancers that have a genetic alteration in the RAS/RAF/MEK signal transduction pathway such as, for example, a B-Raf mutation or gene amplification.

[0049] In an important embodiment, the cancer to be treated is characterized by a B-Raf mutation, for example, colorectal cancer with a B-Raf mutation and melanoma with a B-Raf mutation. In particular, the B-Raf mutation is a V600 mutation, for example, a V600E, V600K or V600G mutation.

[0050] The nature of proliferative diseases is multifactorial. Under certain circumstances, drugs with different mechanisms of action can be combined. However, considering just any combination of therapeutic agents that have different mode of action will not necessarily lead to combinations with advantageous effects.

[0051] Administration of a pharmaceutical combination of the invention may result not only in a beneficial effect, for example, a synergistic therapeutic effect, for example, in relation to alleviating or delaying the progression of or inhibiting symptoms, but also in other beneficial effects. surprising, for example, fewer side effects, more durable response, an improved quality of life or a decrease in morbidity, compared to a monotherapy applying only one of the pharmaceutical therapeutic agents used in the combination of the invention.

[0052] An additional benefit is that lower doses of therapeutic agents of the COMBINATION OF THE INVENTION can be used, for example, so that dosages can not only often be lower, but can also be applied less frequently or can be used more frequently. in order to decrease the incidence of side effects seen with either combination partner individually. This is in accordance with the wishes and requirements of the patients to be treated.

[0053] It can be shown, through established test models, that a COMBINATION OF THE INVENTION results in the beneficial effects described hereinbefore. Those skilled in the art are fully capable of selecting a relevant test model to prove such beneficial effects. The pharmacological activity of a COMBINATION OF THE INVENTION can, for example, be demonstrated in a clinical study or in an animal model essentially as described below.

[0054] By determining a synergistic interaction between one or more components, the ideal range for effect and absolute dose ranges of each component for effect can be definitively measured by administering the components over different ranges of weight/weight ratios and doses for patients in need of treatment. For humans, the complexity and cost of conducting clinical studies in patients may make the use of this form of testing as a primary synergy model impractical. However, the observation of synergy in one species can be predictive of the effect in other species and there are animal models, as described here, to measure a synergistic effect and the results of such studies can also be used to predict ranges of effective dose ratios. and the absolute doses and plasma concentrations required in other species by applying pharmacokinetic/pharmacodynamic methods. Correlations established between tumor models and the effects observed in humans suggest that synergy in animals can be demonstrated by xenotransplantation models.

[0055] In one aspect, the present invention provides a synergistic combination for administration to humans comprising (a) Compound A and (b) Compound B, in a combination range (weight/weight) that corresponds to the ranges observed in a model of tumor. Suitably, the ratio range in humans corresponds to a selected non-human range of between 50:1 to 1:50 parts by weight.

[0056] According to another aspect, the present invention provides a synergistic combination for administration to humans comprising Compound A and Compound B, where the dose range of each component corresponds to the synergistic ranges suggested in a tumor model or clinical study adequate. In general, compound A is administered at a dose in the range of 10 mg to 2000 mg per day and compound B is administered at a dose in the range of 10 mg to 1000 mg per day, for example, 50 mg to 600 mg per day. day.

[0057] In the COMBINATION OF THE INVENTION, Compound A is preferably administered in a dose of 100 to 900 mg/day, preferably between 200 and 900 mg/day, for example, 200, 400, 700 or 900 mg/day day; compound B is preferably administered in a dose of 150 to 600 per day, preferably 400 to 600 per day, in particular 450 or 600 mg/day; and, as the optional MEK 1/2 inhibitor, compound D is administered at a dose of 15 to 150 mg/day, preferably administered on a BID schedule, e.g., 15 to 60 mg BID, e.g., 45 mg BID.

[0058] It is an object of the present invention to provide a pharmaceutical composition comprising the COMBINATION OF THE INVENTION which is jointly therapeutically effective against a proliferative disease. In this composition, the combination partners can be administered in a single formulation or unit dosage form, administered concurrently but separately, or sequentially administered via any suitable route. The unit dosage form may also be a fixed combination.

[0059] Pharmaceutical compositions for administration separately from the combination partners or for administration in a fixed combination, that is, a single galenic composition comprising the COMBINATION OF THE INVENTION, can be prepared in a manner known per se and are those suitable for enteral administration , such as administration orally or rectally, and parenterally to mammals (warm-blooded animals), including humans, comprising a therapeutically effective amount of at least one pharmacologically active combination partner alone, for example, as indicated above, or in combination with one or more pharmaceutically acceptable carriers, especially suitable for enteral or parenteral application.

[0060] The new pharmaceutical composition may contain from about 0.1% to about 99.9%, preferably from about 1% to about 60% of the therapeutic agent(s).

[0061] Pharmaceutical compositions suitable for combined therapy for enteral or parenteral administration are, for example, those in unit dosage forms, such as sugar-coated tablets, tablets, capsules or suppositories or ampoules. If not otherwise indicated, these are made in a manner known per se, for example by means of various conventional processes of mixing, grinding, direct compression, granulation, sugar coating, dissolving, freeze-drying processes, melt granulation or manufacturing techniques readily apparent to those skilled in the art. It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not, in itself, constitute an effective amount, since the required effective amount can be achieved by administering a plurality of units. dosage.

[0062] In one embodiment, the present invention also relates to a COMBINATION OF THE INVENTION for use in preparing a pharmaceutical composition or medicine for the treatment or prevention of a proliferative disease in an individual in need thereof.

[0063] According to the present invention, a therapeutically effective amount of each of the combination partners of the COMBINATION OF THE INVENTION can be administered simultaneously or sequentially and in any order and the components can be administered separately or as a fixed combination. For example, the method of treating a proliferative disease according to the invention may comprise (i) administering agent (a) in free form or pharmaceutically acceptable salt and (ii) administering agent (b) in free form or pharmaceutically acceptable salt. acceptable (and optionally agent (c) in free form or pharmaceutically acceptable salt, simultaneously or sequentially in any order, in jointly therapeutically effective amounts, preferably in synergistically effective amounts, e.g., daily or intermittent dosages corresponding to the amounts described herein The combination partners of the COMBINATION OF THE INVENTION may be administered separately at different times during the course of therapy or concurrently in divided or individual combination forms. The invention, therefore, is to be understood as encompassing all such simultaneous or alternating treatment regimens and the term "administration" should be interpreted accordingly.

[0064] The effective dosage of each of the combination partners employed in the COMBINATION OF THE INVENTION may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the condition being treated and the severity of the condition being treated. Thus, the dosage regimen of the COMBINATION OF THE INVENTION is selected according to a variety of factors, including the route of administration and the patient's renal and hepatic function.

[0065] The optimal proportions, individual and combined dosages and concentrations of the combination partners (a) and (b) of the COMBINATION OF THE INVENTION that confer efficacy without toxicity are based on the kinetics of availability of the therapeutic agents to target sites and are determined using processes known to those skilled in the art.

[0066] Effective dosing of each of the combination partners may require more frequent administration of one of the compounds compared to the other compound(s) in the combination. Therefore, to allow for appropriate dosage, packaged pharmaceutical products may contain one or more dosage forms that contain the combination of compounds and one or more dosage forms that contain the combination of one of the compounds but not the other(s). compound(s) of the combination.

[0067] When the combination partners, which are used in the COMBINATION OF THE INVENTION, are applied as marketed as individual drugs, their dosage and mode of administration may be in accordance with the information provided in the package leaflet of the respective marketed drug. , if not otherwise mentioned here.

[0068] The optimal dosage of each of the combination partners for the treatment of a proliferative disease can be determined empirically for each individual using known methods and will depend on a variety of factors including, but not limited to, the degree of advancement of the disease; the individual's age, body weight, general health, sex and diet; the time and route of administration; and other medications the individual is taking. Optimal dosages can be established using routine testing and procedures that are well known in the art.

[0069] The amount of each combination partner that can be combined with the carrier materials to produce a single dosage form will vary depending on the individual treated and the particular mode of administration. In some embodiments, unit dosage forms containing the combination of agents as described herein will contain amounts of each agent in the combination that are typically administered when the agents are administered individually.

[0070] The dosage frequency may vary depending on the compound used and the particular condition to be treated or prevented. Patients can generally be monitored for therapeutic efficacy using assays appropriate to the condition being treated or prevented, which will be familiar to those skilled in the art.

[0071] The present invention relates to a method of treating an individual having a proliferative disease comprising administering, to said individual, a COMBINATION OF THE INVENTION in an amount which is jointly therapeutically effective against a proliferative disease. In particular, the proliferative disease to be treated with a COMBINATION OF THE INVENTION is a melanoma or colorectal cancer, particularly a melanoma with a B-Raf mutation or colorectal cancer, for example, a melanoma or colorectal cancer with a B-Raf V600 mutation. In addition, treatment may include surgery or radiotherapy.

[0072] The present invention also relates to the COMBINATION OF THE INVENTION for use in the treatment of a proliferative disease, in particular cancer.

[0073] The present invention further provides a commercial packaging comprising, as therapeutic agents, the COMBINATION OF THE INVENTION, together with instructions for simultaneous, separate or sequential administration for use in delaying the progression or treatment of a proliferative disease in an individual in need of the same.

[0074] The following examples illustrate the invention described above; however, they are not intended to limit the scope of the invention in any way. The beneficial effects of the pharmaceutical combination of the present invention can also be determined by other test models known to those skilled in the relevant art.

Example 1

[0075] To evaluate the sensitivity of Compound A and efficacy of the combination of compounds A and B in HMEX1906 primary human melanoma xenografts sensitive to Compound B. Since each agent induced tumor stasis or regression in previous studies, the objective of the present study is to investigate the time-to-regrowth after treatment with the COMBINATION OF THE INVENTION.

[0076] The tumors are minced/crushed into a cell line type suspension (homogenized tumors). Add 7 mL of matrigel and 1.5 mL of HBSS. Warm the suspension in the palm until the Matrigel is thick and implant into the right flank with an 18G sc needle of hairless female mice. Treatment starts 16 days after implantation.

[0077] Compound A shows significant antitumor activity in the B-rad-mutated HMEX1906 xenotransplant model with T/C stasis = 7% at 150 mg/kg, qd and 76% regression at a dose of 250 mg/kg, qd. Compound B shows 57% regression at a dose of 3 mg/kg, bid. THE COMBINATION OF THE INVENTION shows 91% regression on day 46 of study [31 days of dosing].

[0078] The vehicle group and single agent Compound A [150 mg/kg] were dosed for 31 days and ended after the last dose. Compound A [250 mg/kg], single agent Compound B and the COMBINATION OF THE INVENTION were dosed for 45 days and monitored for delay in tumor growth after the last dose [59 study days].

[0079] No significant body weight loss was observed in the treated animals.

Example 2

[0080] Objective: To evaluate the durability of the response to Compound A, Compound B and the combination of Compounds A and B on HMEX2613 primary human melanoma xenotransplants (with BRAF mutation). Objective of the study is to investigate the time to regrowth to 500 mm3 after treatment with individual agents and the COMBINATION OF THE INVENTION.

[0081] Tumor xenografts are implanted into hairless female mice in a manner similar to that described in Example 1. Treatment is initiated 19 days post-implantation and continued for 11 days in the groups dosed with vehicle and single agent Compound A or 18 days in the groups dosed with the single agent Compound B and the COMBINATION OF THE INVENTION. Formulation No.1: 0.5% methyl cellulose Formulation No.2: Compound A in 0.5% methyl cellulose Formulation No.3: Compound B in 0.5% carboxy methyl cellulose/0.5 Tween 80 %

[0082] Tumor xenografts are implanted into hairless female mice in a manner similar to that described in Example 1. Treatment is initiated 19 days post-implantation and continued for 11 days in the groups dosed with vehicle and single agent Compound A or 18 days in the groups dosed with the single agent Compound B and the COMBINATION OF THE INVENTION.

[0083] The results are graphically represented in Figure 1.

[0084] Compound A shows moderate antitumor activity in the Braf-mutated HMEX2613 xenotransplantation model with a T/C of 45%.

[0085] Compound B and the COMBINATION OF THE INVENTION show complete regression of the tumor.

[0086] The group with vehicle and single agent Compound A was dosed for 11 days and then humanely euthanized. Single agent Compound B and the Compound B/Compound A combination were dosed for 18 days and monitored for tumor regrowth after the last dose.

[0087] Single agent Compound B shows tumor regrowth 5 days after the last dose and 37% of animals without tumor at the end of the study [5 of 8 tumors grew again].

[0088] THE COMBINATION OF THE INVENTION shows a growth delay of 26 days after the last dose and 62% of animals without tumor at the end of the study [3 of 8 tumors grew again].

[0089] No significant loss of body weight was observed in the treated animals.

Example 3

[0090] Objective: To evaluate the efficacy of the COMBINATION OF THE INVENTION in the HMEX1906 primary melanoma model that is cultured in the presence of and is resistant to 5 mg/kg of Compound B (HMEX1906-R5).

[0091] Drug formulation: Compound A is formulated in 0.5% MC/0.5% Tween 80 and Compound B was formulated in 20% PEG300/3% ETPGS.

[0092] Tumor xenografts are implanted into hairless female mice in a manner similar to that described in Example 1. The mice were not dosed with Compound B after tumor implantation.

[0093] Mice were assigned to the following groups 18 days post-implantation of the tumor with an average volume of 266 mm3 and average body weight of 25 grams.

[0094] Groups: 10 mice/group, via PO, dose volume of 0.2 mL

[0095] Group 1: Vehicle, 0 mg/kg bidx14

[0096] Group 2: Compound A, 250 mg/kg qdx21

[0097] Group 3: Compound B, 5 mg/kg bidx21

[0098] Group 4: Compound A at 250 mg/kg qd x21 + Compound B at 5 mg/kg bid x21 Results: * 3 mice were sacrificed due to the large tumor volume

Example 4

[0099] Materials and Methods - Compound stocks are prepared in DMSO at a final concentration of 10 mM. Working stocks are serially diluted in appropriate cell culture medium in 3-fold increments to achieve final assay concentrations ranging from 2.7 μM to 1.2 nM for Compounds B and D and 10 μM to 4.6 nM for Compound A.

[00100] Cell lines, cell culture, cell viability measurements - A-375 and WM-266-4 cells were purchased from the American Type Culture Collection (ATCC). A-375 cells were grown in DMEM medium (ATCC) and WM-266-4 cells were grown in EMEM medium (ATCC), both supplemented with 10% fetal bovine serum (Gibco) and incubated at 37°C/5 % CO2. Cell lines engineered to express commonly occurring alleles indicative of resistance were purchased from Novartis-Emeryville. These resistant models include A-375 cells that express mutant MEK1P124L, truncated p61-BRAFV600E, or mutant NRASQ61K and WM-266-4 cells that express mutant MEK1C121S, truncated p61-BRAFV600E, or mutant NRASQ61K. These cells were cultured in the appropriate parental medium with selection marker G418 and in the presence of 5 μM LFE158 (MEK mutants) or LIH720 (truncated p61-BRAFV600E).

[00101] Plate Layout, Cell Distribution, and Compound Addition - For screening, cells were seeded in 80 μl of medium in 384-well plates (Thermo Scientific, cat No. 4332) at cell densities of 500 (375-A ) or 750 (WM-266-4) per cavity using a MultiDrop Combi (Thermo-Fisher) with an 8-channel pattern cassette. To promote an even distribution of cells throughout the cavity, cells were quickly centrifuged at 1000 rpm and incubated at room temperature for 30 minutes. All plates were incubated at 37 °C, 5% CO2 for 24 h before compound addition. Compound stock was prepared fresh in appropriate culture medium and added using a PAA robot equipped with a Pin 200 nl tool. In a minimum of three replicate wells, effects of a single agent and the combination after 72 hours were assessed by quantifying cellular ATP levels via Cell Titer Glo (Promega), according to the manufacturer's protocol and through microscopy imaging. For imaging, cells were fixed to plates and permeabilized with a solution of 10% PFA, 0.3% TX-100 in PBS via a WellMate dispenser with controlled delivery rates. Cell nuclei were stained with Hoechst 33342 (H3570, Invitrogen) and all necessary washing steps were performed by a BioTek washer.

[00102] Automated Image Analysis - Images from the InCell Analyzer 2000 (GE Healthcare, 28-9534-63) were in TIFF format and had a size of 2048 x 2048 pixels, capturing the entire well of a 384-well plate. An automated image analysis pipeline was established using custom-made scripts in the open source statistical programming language R and functions from the BioConductor package EBImage. The objective was to quantify the number of viable nuclei (cells) per well as an approximation for cell viability. The pipeline was comprised of seven steps: (I.) leveling the image to reduce the number of intensity peaks, (II.) applying a threshold function to separate the foreground (signal) from the background (noise), (III .) identification of local maxima in the foreground that serve as seeds for the kernels, (IV.) filtering of nearby local maxima, (V.) propagation of kernels of remaining local maxima, and (VI.) extraction of object features from the propagated nuclei (numbers of nuclei, size characteristics and intensity characteristics). As a last step (VII.), to exclude debris (e.g., fragmented nuclei) from the count, objects identified in DMSO- and staurosporine-treated cavities were used to obtain characteristic distributions for viable and fragmented nuclei, respectively. These were used to define breakpoints of differentiation between viable and fragmented nuclei. The number of fragmented cores was subtracted from the total number of objects identified and the result was reported as the final count for that cavity.

[00103] Data normalization - Data comprised triplicate measurements for each treatment condition (compound), 42 replicates of DMSO-treated wells and duplicates of staurosporine-treated wells. Data were normalized to the median of DMSO measurements and summarized by calculating the median of the triplicates. The data was imported into Chalice to calculate compound synergies. Results:

Claims (10)

1. Pharmaceutical combination, characterized by the fact that it comprises: (a) a CDK4/6 inhibitor of Formula: or a pharmaceutically acceptable salt thereof, and (b) a B-Raf inhibitor of Formula: or a pharmaceutically acceptable salt thereof, for simultaneous administration. 2. Pharmaceutical combination, according to claim 1, characterized by the fact that it further comprises a MEK 1/2 inhibitor. 3. Pharmaceutical combination, according to claim 1, characterized by the fact that the MEK 1/2 inhibitor is a compound of Formula: or a pharmaceutically acceptable salt thereof. 4. Pharmaceutical combination, according to any one of claims 1 to 3, characterized by the fact that it is for use in the treatment of a proliferative disease in an individual in need thereof. 5. Pharmaceutical combination, according to any one of claims 1 to 3, characterized by the fact that it is for use in the preparation of a medicine for treating a proliferative disease. 6. Pharmaceutical combination according to claim 4, characterized by the fact that the proliferative disease is melanoma, lung cancer (including non-small cell lung cancer (NSCLC)), colorectal cancer (CRC), breast cancer, kidney cancer, renal cell carcinoma (RCC), liver cancer, acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), thyroid cancer, pancreatic cancer, neurofibromatosis or hepatocellular carcinoma. 7. Pharmaceutical combination according to claim 6, characterized by the fact that the proliferative disease is defined by a B-Raf mutation. 8. Pharmaceutical combination according to claim 6, characterized by the fact that the proliferative disease is defined by a B-Raf V600 mutation. 9. Pharmaceutical combination according to claim 6, characterized by the fact that the proliferative disease is melanoma or colorectal cancer. 10. Use of the combination, as defined in any one of claims 1 to 3, characterized by the fact that it is for manufacturing a medicine for treating a proliferative disease.