WO2020229685A1

WO2020229685A1 - Combination therapy for proliferative conditions

Info

Publication number: WO2020229685A1
Application number: PCT/EP2020/063708
Authority: WO
Inventors: Berit Johansen; Astrid Jullumstrø FEUERHERM
Original assignee: Avexxin As
Priority date: 2019-05-15
Filing date: 2020-05-15
Publication date: 2020-11-19
Also published as: GB201906867D0

Abstract

A pharmaceutical composition or combination product comprising certain polyunsaturated long-chain ketones in combination with certain protein kinase inhibitors, in particular phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitors (PI3K), such as buparlisib. The invention also relates to the use of said pharmaceutical composition or combination product for the treatment or prevention of proliferative conditions such as cancer, e.g. breast cancer or lymphoid cancer.

Description

COMBINATION THERAPY FOR PROLIFERATIVE CONDITIONS

This invention relates to a pharmaceutical composition or combination product comprising certain polyunsaturated long-chain ketones in combination with certain protein kinase inhibitors, in particular phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitors (PI3K). The invention also relates to the use of said

pharmaceutical composition or combination product for the treatment or prevention of proliferative conditions such as cancer, e.g. breast cancer or lymphoid cancer. The invention also relates to methods of treating or preventing proliferative conditions in patients comprising administration of the pharmaceutical composition or combination product of the invention to the patient.

Background

Basal-like breast cancer (BLBC), which represents ~15 % of all breast cancers, is an aggressive molecular subtype of the disease associated with poor prognosis. Most BLBCs are triple-negative (lacking expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) and thus unresponsive to currently available targeted therapies.

Leukaemias account for approximately 8% of all human malignancies. Human leukaemia can be divided into four subgroups based on the cell lineage and clinical manifestation. Acute leukaemias, acute myeloid leukaemia (AML) and acute lymphoid leukaemia (ALL), are associated with aggressive and usually fatal diseases, unless treated immediately. Both are most prevalent amongst young children. Due to their slow onset, chronic leukaemias, chronic myeloid leukaemia (CML) and chronic lymphoid leukaemia (CLL), are most commonly found in the older population.

Acute lymphoid leukaemia (ALL) represent 12% of all leukaemia cases, and approximately 80% of all leukaemia cases amongst children. The majority of ALL patients do not display an identifiable genetic or environmental cause. Genetic mutations, hereditary links and exposure to carcinogenic factors are however believed to be the most prominent risk factors. In ALL, early haemopoietic cells, called blast cells, accumulate in the bone marrow. This is also reflected in the clinical manifestation of ALL, characterised by anaemia and leucopenia as the bone marrow fails to exert its normal function.

Hence, new molecular targets for treatment of these cancers are called for. The present inventors have devised a new combination therapy that targets proliferative conditions in general and breast cancer and lymphoid cancer in particular.

The invention relies on the combination of a long chain polyunsaturated ketone compound and certain inhibitors of PI3K. The present inventors have surprisingly found that the combination of these two compounds leads to a combination therapy that works synergistically. In particular, the combination has been shown to synergistically reduce breast cancer and lymphoid cancer cell viability.

Summary of Invention

Thus, viewed from one aspect the invention provides a pharmaceutical composition comprising:

(A) a compound of formula (I):

R-L-CO-X (I) wherein R is a C 10-24 unsaturated hydrocarbon group optionally interrupted by one or more heteroatoms or groups of heteroatoms selected from S, O, N, SO, or SO2, said hydrocarbon group comprising at least 4 non-conjugated double bonds;

L is a linking group forming a bridge of 1 to 5 atoms between the R group and the carbonyl CO wherein L comprises at least one heteroatom in the backbone of the linking group; and

X is an electron withdrawing group;

or a pharmaceutically acceptable salt, or a hydrate or solvate thereof; and

(B) a compound of formula (X) a compound of formula (XI)

pilaralisib); or a compound of general formula (XII) wherein Ri is C1 -4 alkyl, such as Me or Et.

R₂ is H or Hal;

R3 is N or CH; and

R4 is H or Hal;

or a pharmaceutically acceptable salt, or a hydrate or solvate thereof of any of formulae (X) to (XII).

Preferred compounds of formula (XII) are those of formula:

duvelisib); lisib). or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

Viewed from another aspect the invention provides a combination product for simultaneous, sequential or separate use comprising:

(A) a compound of formula (I):

R-L-CO-X (I) wherein R is a C 10-24 unsaturated hydrocarbon group optionally interrupted by one or more heteroatoms or groups of heteroatoms selected from S, O, N, SO, SO2, said hydrocarbon group comprising at least 4 non-conjugated double bonds; L is a linking group forming a bridge of 1 to 5 atoms between the R group and the carbonyl CO wherein L comprises at least one heteroatom in the backbone of the linking group; and

X is an electron withdrawing group;

or a pharmaceutically acceptable salt, or a hydrate or solvate thereof; and

(B) a compound of formula (X)

a compound of formula (XI)

pilaralisib); or a compound of general formula (XII)

wherein Ri is C1 -4 alkyl, such as Me or Et.

R₂ is H or Hal;

R3 is N or CH; and

R₄ is H or Hal;

Viewed from another aspect the invention provides a pharmaceutical kit composition for simultaneous, sequential or separate use comprising a first composition comprising a compound (I) as herein defined and a pharmaceutically- acceptable diluent or carrier, and a second composition comprising a compound (X) to (XII) as herein defined and a pharmaceutically-acceptable diluent or carrier.

In particular, the invention relates to a pharmaceutical composition, combination product or kit as herein before defined in which the compound of formula (I) is:

X= CF₃ = Compound A1 or

X= CF3 = Compound A2 or a salt thereof; and

a compound of formula (X) to (XII) as hereinbefore defined or a

pharmaceutically acceptable salt, or a hydrate or solvate thereof.

Viewed from another aspect the invention provides a pharmaceutical composition or combination product as hereinbefore defined for use in the treatment or prevention of a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer.

Viewed from another aspect the invention provides a method of treating or preventing a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer in a patient in need thereof comprising administering to said patient, preferably a human, an effective amount of a pharmaceutical composition or combination product as herein before defined.

Viewed from another aspect the invention provides a method of treating, such as reducing symptoms of, or preventing a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer in a patient in need thereof comprising administering to said patient, preferably a human, an effective amount of a compound of formula (I) and simultaneously, separately or sequentially administering to said patient a compound of formula (X) to (XII) as herein before defined. In sequential administration either compound can be administered first.

Viewed from another aspect the invention provides a method of treating, such as reducing symptoms of, or preventing a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer, in a patient in need thereof comprising:

(i) identifying a patient who has received either a compound of formula

(I) or a compound of formula (X) to (XII) as herein before defined respectively;

(ii) administering to said patient an effective amount of either a compound of formula (X) to (XII) or a compound of formula (I) as herein before defined so that said patient is administered with both a compound of formula (I) and a compound of formula (X) to (XII). Viewed from another aspect the invention provides use of a composition or combination product as hereinbefore defined in the manufacture of a medicament for treating or preventing a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer.

Viewed from another aspect the invention provides a process for the preparation of a composition as hereinbefore defined comprising blending a compound of formula (I) and a compound of formula (X) to (XII) in the presence of at least one pharmaceutical excipient.

Definitions

The term Hal represents halide, e.g. Cl or F.

Where possible, the compounds of the invention, i.e. those of formula (I) to (V) or formula (X) to (XV) can be administered in salt, hydrate or solvate form, especially salt form or can be administered in non-salt form.

The invention relates both to a pharmaceutical composition in which compounds (I) and (X) to (XII) are blended together in a single composition and to a combination product such as a kit in which the active compounds are provided in separate compositions but are designed for administration simultaneously, separately or sequentially. Any method for treating or preventing a proliferative disorder as defined herein encompasses simultaneous, separate or sequential administration of the active components or administration of the composition of the invention.

A“combination” according to the invention refers to either a fixed

combination in one dosage unit form, or a kit of parts for the combined

administration where a compound of the formula (I) and its combination partner formula (X) to (XII) (also referred to as "combination partner" or "therapeutic agent") may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative and preferably a synergistic effect.

A“combination product” as used herein means a product suitable for pharmaceutical use that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" or "fixed dose" means that the active ingredients, e.g. a compound of formula (I) and its combination partner, a compound of formula (X) to (XII), are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g. a compound of formula (I) and the combination partner, a compound of formula (X) to (XII), are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the warm-blooded animal in need thereof.

All discussion below relating to preferred compounds of the invention is equally applicable to both these aspects of the invention.

Detailed Description of the Invention

This invention concerns a combination therapy of a compound of formula (I) and a compound of formula (X) to (XII). We have surprisingly found that this combination therapy results in synergy. Our results demonstrate a reduction in the viability of breast cancer cells or lymphoid cancer cells, the pharmaceutical composition or combination product offering a larger reduction than could have been expected from the use of individual compounds individually, i.e. the combination of the compounds produces an overall effect that is greater than the sum of the individual elements.

Proliferative Disorder

This invention relates to a new combination therapy for proliferative disorders. Preferably, the composition of the invention is used for the treatment of a proliferative disease selected from a benign or malignant tumor, carcinoma of the brain, kidney, liver, adrenal gland, bladder, breast, lymphatic system, stomach, gastric tumors, ovaries, colon, rectum, prostate, pancreas, lung, vagina or thyroid, sarcoma, glioblastomas, multiple myeloma, leukemia or gastrointestinal cancer.

It is especially preferred if the proliferative disorder is a mammary carcinoma or lymphoid cancer, such as chronic lymphoid leukaemia or acute lymphoid leukaemia. The composition or combination product of the invention can target specifically metatstaic breast adenocarcinoma or acute lymphoid leukaemia.

Compound A The invention relies on the therapeutic combination of a compound of formula (I) and a compound of formula (X) to (XII). The compound of formula (I) is R-L-CO-X (I) wherein R is a C 10-24 unsaturated hydrocarbon group optionally interrupted by one or more heteroatoms or groups of heteroatoms selected from S, O, N, SO, SO2, said hydrocarbon group comprising at least 4 non-conjugated double bonds;

X is an electron withdrawing group; or a salt thereof.

The group R preferably comprises 5 to 9 double bonds, preferably 5 or 8 double bonds, e.g. 5 to 7 double bonds such as 5 or 6 double bonds. These bonds should be non-conjugated. It is also preferred if the double bonds do not conjugate with the carbonyl functionality.

The double bonds present in the group R may be in the cis or trans configuration however, it is preferred if the majority of the double bonds present (i.e. at least 50%) are in the cis configuration. In further advantageous embodiments all the double bonds in the group R are in the cis configuration or all double bonds are in the cis configuration except the double bond nearest the carbonyl group which may be in the trans configuration.

The group R may have between 10 and 24 carbon atoms, preferably 12 to 20 carbon atoms, especially 17 to 19 carbon atoms.

Whilst the R group can be interrupted by at least one heteroatom or group of heteroatoms, this is not preferred and the R group backbone preferably contains only carbon atoms.

The R group may carry up to three substituents, e.g. selected from halo, Cl- 6 alkyl e.g. methyl, or C1 -6 alkoxy. If present, the substituents are preferably nonpolar, and small, e.g. a methyl group. It is preferred however, if the R group remains unsubstituted.

The R group is preferably an alkylene group. The R group is preferably linear. It preferably derives from a natural source such as a long chain fatty acid or ester. In particular, the R group may derive from arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid.

Thus, viewed from another aspect the invention employs a compound of formula (G)

R-L-CO-X (G) wherein R is a C _10-24 unsubstituted unsaturated alkylene group said group comprising at least 4 non-conjugated double bonds;

X is an electron withdrawing group or a salt thereof.

Ideally R is linear. R is therefore preferably an unsaturated C10-24 polyalkylene chain.

The linking group L provides a bridging group of 1 to 5 backbone atoms, preferably 2 to 4 backbone atoms between the R group and the carbonyl, such as 2 atoms. The atoms in the backbone of the linker may be carbon and/or be heteroatoms such as N, O, S, SO, or SO₂. The atoms should not form part of a ring and the backbone atoms of the linking group can be substituted with side chains, e.g. with groups such as C1 -6 alkyl, oxo, alkoxy, or halo.

Preferred components of the linking group are -CH₂-, -CH(Ci-6alkyl)-, -N(Ci- ₆alkyl)-, -NH-, -S-, -0-, -CH=CH-, -CO- , -SO-, -SO2- which can be combined with each other in any (chemically meaningful) order to form the linking group. Thus, by using two methylene groups and an -S- group the linker -SCH2CH2- is formed. It will be appreciated that at least one component of the linker provides a heteroatom in the backbone.

The linking group L contains at least one heteroatom in the backbone. It is also preferred if the first backbone atom of the linking group attached to the R group is a heteroatom or group of heteroatoms.

It is highly preferred if the linking group L contains at least one -CH2- link in the backbone. Ideally the atoms of the linking group adjacent the carbonyl are

-CH₂-. It is preferred that the group R or the group L (depending on the size of the L group) provides a heteroatom or group of heteroatoms positioned a, b, g, or d to the carbonyl, preferably b or g to the carbonyl. Preferably the heteroatom is O, N or S or a sulphur derivative such as SO.

Highly preferred linking groups L therefore are -NH2CH2, -NH(Me)CH2-, - SCH2-, or -SOCH2-.

The linking group should not comprise a ring.

Highly preferred linking groups L are SCH2, NHCH2, and N(Me)CH2.

Viewed from another aspect the invention employs a compound of formula

(II)

R-L-CO-X (II) wherein R is a linear C 10-24 unsubstituted unsaturated alkylene group said group comprising at least 4 non-conjugated double bonds;

L is -SCH2-, -OCH2-, -SOCH2, or -SO2CH2-; and

X is an electron withdrawing group or a salt thereof.

The group X is an electron withdrawing group. Suitable groups in this regard include O-C1-6 alkyl, CN, OCO2-C1-6 alkyl, phenyl, CHah, CHahH, CHalH2 wherein Hal represents a halogen, e. g. fluorine, chlorine, bromine or iodine, preferably fluorine.

In a preferred embodiment the electron withdrawing group is CHah, especially CF3.

Thus, preferred compounds of formula (I) are those of formula (III)

R-Y1-Y2-CO-X (III) wherein R and X are as hereinbefore defined;

Y1 is selected from O, S, NH, N(Ci-₆-alkyl), SO or SO2 and

Y2 is (CH₂)n or CH(C_{I -6} alkyl); or

where n is 1 to 3, preferably 1.

More, preferred compounds of formula (I) are those of formula (IV)

R-Y1-CH₂-CO-X (IV) wherein R is a linear C 10-24 unsubstituted unsaturated alkylene group said group comprising at least 4 non-conjugated double bonds;

X is as hereinbefore defined (e.g. CF₃); and

Y1 is selected from O, S, SO or SO2.

More, preferred compounds of formula (I) are those of formula (V)

R-S-CH2-CO-CF3 (V) wherein R is a linear C 1₀-24 unsubstituted unsaturated alkylene group said group comprising at least 4 non-conjugated double bonds.

Highly preferred compounds for use in the invention are depicted below:

where X is as hereinbefore defined such as CF₃.

The following compounds are highly preferred for use in the invention:

A2 Compound B

The second component of the composition or combination product of the invention is a compound of formula (X) to (XII) as hereinbefore defined.

In compounds of general formula (XII):

it is preferred if one of R₂ or R4 is H and the other is Hal, e.g. F or Cl. It is preferred if F is H and R₂ is Hal.

It is preferred if Ri is methyl or ethyl.

In a preferred embodiment therefore, the invention provides a structure of formula (XII) in which:

Ri is Me or Et;

R₂ is Hal;

R3 is N or CH; and

R₄ is H.

Preferred options are: or acalisib (of formula XV).

In a further embodiment, pilaralisib may be used. In a most preferred embodiment, the compound is: (X - buparlisib) or a salt thereof.

In a most preferred embodiment therefore the invention relates to a pharmaceutical composition or combination product comprising Compound A1 or Compound A2 and buparlisib.

In another embodiment, the invention relates to a pharmaceutical composition or combination product comprising Compound A1 or Compound A2 and idelalisib, duvelisib, acalisib or pilaralisib.

Alternatively, another combination product of the invention is buparlisib, Compound A1 and Compound A2.

Where possible, the compounds of the invention, i.e. those of formula (I) to (V) or formula (X) to (XII) can be administered in salt, hydrate or solvate form, especially salt form.

Typically, a pharmaceutical acceptable salt may be readily prepared by using a desired acid. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. For example, an aqueous solution of an acid such as hydrochloric acid may be added to an aqueous suspension of a compound of formula (X) to (XII) and the resulting mixture evaporated to dryness (lyophilised) to obtain the acid addition salt as a solid.

Alternatively, a compound of formula (X) to (XII) may be dissolved in a suitable solvent, for example an alcohol such as isopropanol, and the acid may be added in the same solvent or another suitable solvent. The resulting acid addition salt may then be precipitated directly, or by addition of a less polar solvent such as diisopropyl ether or hexane, and isolated by filtration. Suitable addition salts are formed from inorganic or organic acids which form non-toxic salts and examples are hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate,

trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, pyruvate, oxalate, oxaloacetate, trifluoroacetate, saccharate, benzoate, alkyl or aryl sulphonates (e.g. methanesulphonate, ethanesulphonate, benzenesulphonate or p-toluenesulphonate) and isethionate. Representative examples include trifluoroacetate and formate salts, for example the bis or tris trifluoroacetate salts and the mono or diformate salts, in particular the tris or bis trifluoroacetate salt and the monoformate salt.

Compounds of formula (I) may be manufactured using known chemical synthetic routes. It is convenient to begin synthesis from the commercially available compounds arachidonic acid (AA), EPA (all-Z-eicosa-5,8, 11 ,14,17-pentaenoic acid) or DHA (all-Z-docosa-4,7, 10, 13, 16, 19-hexaenoic acid). Conversion of the acid functionality of these compounds into, for example a -COCF₃ group can be achieved readily, e.g. by converting the carboxylic acid into its corresponding acid chloride and reacting the same with trifluoroacetic anhydride in the presence of pyridine.

Introduction of a heteroatom into the carbon chain is also achieved readily. Conveniently, for example, the starting acid is reduced to an alcohol and, if required, converted to the corresponding thiol. The nucleophilic thiol may then be reacted with a group such as BrCFkCOCFs thereby introducing the carbonyl and electron withdrawing species. Complete synthetic protocols may be found in J. Chem. Soc., Perkin Trans 1 , 2000, 2271-2276 or J. Immunol., 1998, 161 , 3421.

Synthesis methods for the preparation of compounds of formula (X) to (XII) are described in the literature. These compounds are commercially available.

The weight ratio of the compounds of formula (I) to compounds of formula (X) to (XII) in composition or combination product of the invention will be guided by intended use, the age and general health of the subject, and other parameters known to those of skill. For example, a particular weight ratio suitable for certain applications may be 10 to 90 wt% to 90 to 10 wt%, such as 30 to 70 wt% to 70 to 30 wt%.

More preferably, the amounts of each compound are determined in molar terms, and the ratio of each is 5:1 to 1 :5 moles, such as 2:1 to 1 :2 moles. Often, the compounds are used in an equimolar amount for certain applications. The amount of the compounds of the invention in the composition will often be determined by the physican depending on the dosage required.

The composition or combination product of the invention is proposed primarily for use in the treatment or prevention of proliferative disorders such as cancer.

By treating or treatment is meant at least one of:

(i). inhibiting the disease i.e. arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or subclinical symptom thereof, or

(ii). relieving or attenuating one or more of the clinical or subclinical symptoms of the disease.

By prevention is meant (i) preventing or delaying the appearance of clinical symptoms of the disease developing in a mammal.

The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician. In general a skilled man can appreciate when "treatment" occurs. It is particularly preferred if the composition or combination product of the invention are used therapeutically, i.e. to treat a condition which has manifested rather than prophylactically. It may be that the composition or combination product of the invention is more effective when used therapeutically than prophylactically.

The composition or combination product of the invention can be used on any animal subject, in particular a mammal and more particularly to a human or an animal serving as a model for a disease (e.g., mouse, monkey, etc.).

In order to treat a disease an effective amount of the active composition or combination product needs to be administered to a patient. A "therapeutically effective amount" means the amount of a composition or combination product that, when administered to an animal for treating a state, disorder or condition, is sufficient to effect such treatment. The "therapeutically effective amount" will vary depending on the composition or combination product, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated and will be ultimately at the discretion of the attendant doctor.

It may be that to treat cancer according to the invention that the composition or combination product of the invention has to be readministered at certain intervals. Suitable dosage regimes can be prescribed by a physician. The composition or combination product of the invention typically comprises the active components in admixture with at least one pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

The term "carrier" refers to a diluent, excipient, and/or vehicle with which an active compound is administered. The pharmaceutical compositions of the invention may contain combinations of more than one carrier. Such pharmaceutical carriers are well known in the art. The pharmaceutical compositions may also comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s) and so on. The compositions can also contain other active components, e.g. other drugs for the treatment of cancer.

It will be appreciated that pharmaceutical composition or combination products for use in accordance with the present invention may be in the form of oral, parenteral, transdermal, sublingual, topical, implant, nasal, or enterally administered (or other mucosally administered) suspensions, capsules or tablets, which may be formulated in conventional manner using one or more

pharmaceutically acceptable carriers or excipients. The compositions of the invention could also be formulated as nanoparticle formulations.

However, for the treatment of cancer, the composition or combination product of the invention will preferably be administered orally or by parenteral or intravenous administration, such as injection. The composition or combination product may therefore be provided in the form of an tablet or solution for injection.

The pharmaceutical composition or combination product of the invention may contain from 0.01 to 99% weight - per volume of the active material. The therapeutic doses will generally be between about 10 and 2000 mg/d ay and preferably between about 30 and 1500 mg/d ay. Other ranges may be used, including, for example, 50-500 mg/day, 50-300 mg/day, 100-200 mg/day.

Administration may be once a day, twice a day, or more often, and may be decreased during a maintenance phase of the disease or disorder, e.g. once every second or third day instead of every day or twice a day. The dose and the administration frequency will depend on the clinical signs, which confirm

maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skilled in the art. It is within the scope of the present invention to administer the combination products described herein to a subject that has been exposed to, is being exposed to, or will be exposed to one or more anti-proliferative compounds and particularly those known to be used in many anti-cancer therapies. Non-limiting examples include aromatase inhibitors, anti-estrogens, topoisomerase I or II inhibitors microtubule active compounds, alkylating compounds, histone deacetylase inhibitors, and cyclooxygenase inhibitors such as those disclosed in

W02006/122806 and references cited therein Choice of whether to combine a combination product of the invention with one or more of the aforementioned anti- cancer therapies will be guided by recognized parameters known to those of skill in the field, including the particular type of cancer being treated, the age and health of the subject, etc.

The invention is described further below with reference to the following non limiting examples and figure.

Description of Figures:

Figure 1 shows the results of combination treatment of BKM120 (mM) and Compound A2 (mM). The bars represent the mean relative viability ± SD of three biological replicates. In figure 1 the low dose BKM120 is expected to give a 15% reduction in viability. Ctrl = control treatment, set to 100% viability. * = p <0.05, ** = P <0.01 , *** = p <0.001 ,

¨ = p <0.0001.

The following compounds were used in the examples:

X= CF3 = Compound A2

Compounds B:

47;

Methods

Cell Culture. The MDA-MB-468 cell line was from ATCC. The MDA-MB-468 cell line were established from a pleural effusion of a patient with metastatic breast adenocarcinoma. The cell line is of triple negative breast cancer, and is classified as basal-like with high enrichment of cell cycle, and cell division component and pathways such as (the DNA replication reactome, G2 cell-cycle pathway, RNA polymerase, and G1 to S cell cycle components).

MDA-MB-468 cells were maintained in RPMI-1640 culturing media supplemented with 10 % FBS, 0.3 mg/mL L-glutamine and 0.1 mg/ml_ gentamicin and incubated in 37°C humidified atmosphere. The cells were sub-cultured using trypsin-EDTA 0.5 % every 3-4 days with cell density of 2-3 million cells in a T75 cm² culturing flask to ensure actively proliferating cells.

The CCRF-CEM (ATCC CCL-119) cell line is derived from T lymphoblasts from peripheral blood of a child with acute leukaemia. The cells were grown according to the recommendations of the supplier. The culture medium, RPMI- 1640, was supplemented with 10 pL/mL glutamine, 2 pL/mL gentamicin and 10 % foetal bovine serum (FBS) to make complete growth medium (CGM). The cells were incubated at 37oC in a humidified atmosphere with 5% C02. Passaging was conducted every 2 - 4 days to maintain a cell density below 2.5 ^c 1 0⁶ cells/m L, and thus ensure a continuous proliferation. Cells were allowed to acclimatise for two weeks after establishment before they were utilised for experimental purposes and discarded after three months.

Resazurin Viability Assay.

The MDA-MB-468 cells were seeded in fully supplemented medium at a density of 3 x 10³ cells/well in 96 well plates. After 24 h of cultivation, when the cells were -30-50 % confluent, the medium was replaced with serum free growth medium without gentamycin to ensure synchronization of the cells and to increase cell sensitivity to treatment. The medium was replaced with fresh serum free medium with or without compound A2 (Coegin Pharma, Norway), NVP-BKM120 (Cayman Chemicals, US), CAL-101 (Selleck Chemicals, US), XL- 147 (Cayman Chemicals, US), and I PI-145 (Cayman Chemicals, US). The cells were observed under the microscope to evaluate possible morphology changes and signs of stress before the addition of resazurin according to the manufacturer’s instructions (RnD Systems, UK). MDA-MB-468 cells were incubated for 24 h, 48 h and 72 h post treatment. Resazurin was metabolized for 1-2 h (37°C, 5% CO2) before

fluorescence was read at 544 nm excitation and 590 nm emission wavelength (BioTek Synergy HT). The experiments were performed in series of eight wells per treatment and repeated 2-3 times.

The CCRF-CEM cells were seeded at 4.0 ^c 10⁵ cells/mL 72 hours prior to the experiments to ensure that the cells were actively proliferating. Cells were seeded in 96 well plates at 5 ^c 10⁴ cells/well in 80 pL in either 10% FBS or in 0.5% FBS. The outermost wells were filled with medium instead of cells to avoid possible edge effects. The cells were incubated for 1 hour to allow them to settle prior to treatment with inhibitors. Working stocks of the inhibitors were made by diluting the inhibitors in dimethyl sulfoxide (DMSO). The final concentration of inhibitors was made by addition of medium. The concentration of DMSO in the treatments and control was standardised to 0.2%. The cells were treated with inhibitors for 24, 28, or 72 hours before adding resazurin.

Combination assay

To determine synergy, we assessed if the effect of the combination therapy was significantly greater than the effect by each monotherapy. Statistical significance between treatments were determined using Welch’s ANOVA with Tamhane’s T2 correction for multiple comparisons, p <0.05 was considered statistically significant.

For the CCRF-CEM experiments, Bliss Independence was also included to calculate a combination index (Cl) for the investigated combinations. The combination index was calculated using:

CI= EA + En— (EA X E )

EAB where Cl is the combination index, E_A represent the effect of drug A given as a monotherapy, EB represent the effect of drug B given as a monotherapy and EAB represent drugs A and B given in combination at the same concentrations as the monotherapies. A Cl of less than 1 indicates synergy and a Cl of more than 1 indicate antagonism, whereas a Cl of 1 is indicative of additivism. Example 1

Dose-dependent inhibition is obtained for several PI3K inhibitors and cPLA2a inhibitor compound A2

It has been demonstrated that PI3K and cPLA2a inhibitors both effectively reduce cell proliferation in BLBC/TNBC. Dose-responses of isoform specific PI3K inhibitors and selective cPI_A2a inhibitors in the MDA-MB-468 cells were performed prior to co-treatment experiments with PI3K inhibitors and compound A2.

In the initial experiments, compound A2 and a panel of PI3K inhibitors were included. Compound A2 inhibited cell viability of MDA-MB-468 cells by 50 % at 9 mM after 24 h, 7.5 mM after 48 h and 5 pM after 72 h (Table 1). Of the PI3K inhibitors, the pan-PI3K inhibitors BKM120 (r110a/b/d/g inhibitor) and XL- 147 (PI3K a/d/g inhibitor) were most potent (Table 1). BKM120 was found to reduce cell viability by 50 % at 8 mM after 24 h, 6 mM at 48 h and 5 pM after 72 h, whereas XL- 147 was found to reduce cell viability by 50 % in doses around 24 pM after 24 h, 13 pM at 48 h and 14 pM at 72 h. The effects of XL- 147 was most evident after 48 h treatment, no additional effects were seen after 72 h. The results of these dose- response experiments were used to determine the sub-optimal doses of each single inhibitor to be included in the subsequent co- treatment experiments. Table 1 : Calculated IC50- values of isoform specific PI3K inhibitors and the cPLA2a inhibitor compound A2 on BLBC/TNBC cell line MDA-MB-468. The values calculated after 24 h, 48 h and 72 h of treatment (n=3)

Example 2

Sub-optimal doses of cPLA2a inhibitors and each PI3K inhibitor in the co treatment experiments were identified for both cell lines. Sub-optimal doses of the cPLA2a inhibitor compound A2 was determined to be 2.5 pM, 3.0 pM and 3.5 pM in the MDA-MB-468 cell line. Sub-optimal doses of BKM120, IPI-145, CAL-101 & XL- 147 were 1 mM, 2.5 mM, 50 mM and 0.5 mM for the MDA-MB-468 cell line, respectively.

In order to find the most effective dose in the combination treatment, three different doses of compound A2 were chosen to be combined with each selective PI3K inhibitor for the MDA-MB-468 cells.

Combination treatment of compound A2 in the MDA-MB-468 cells with CAL- 101 showed a significant synergistic effect with 3.5 mM compound A2 with 50 mM CAL-101 after 24 h and 48 h of treatment. Following 24 h, 48 h and 72 h of treatment of sub-optimal doses of compound A2, showed 92 % ± 6 %, 93 % ± 6 % and 80 % ± 9 % cell viability, respectively. CAL-101 had 82 % ± 5 %, 75 % ± 3 %, 60 % ±4 %, after 24 h, 48 h and 72 h, respectively. In combination, an additional reduction of 31 % ±3 % was observed after 24 h and 30 % ± 2 reduction after 48 h of treatment (Table 2).

For the pan-PI3K inhibitor BKM120, a 40 % ± 10 % additional inhibition was observed when 3.5 mM of compound A2 were combined with 1 mM BKM120.

For the pan PI3K XL-147 a/g/d inhibitor, synergistic effects upon

combination with compound A2 were found after 24 and 48 h. After 72 h of treatment, an additional 31 % ± 4 % reduction in cell viability was observed in the MDA-MB-468 cells (Table 2).

Table 2: Viability of MDA-MB-468 cells after treatment with sub-optimal doses isoform specific PI3K inhibitors and the cPLA2a inhibitor compound A2 alone and in combination after 24 h, 48 h and 72 h. The viability was measured in percentage relative to the untreated control set to 100 %.( p-value * <0.05, P-value ** <0.01 , ordinary one-way ANOVA)

Discussion

Co-treatment of PI3K inhibitors such as BKM120 (pan-PI3K inhibitor), CAL- 101 (P1106 inhibitor) or XL- 147 (RI3Ka/g/d inhibitor) with the cPLA2a inhibitors compound A2 acts synergistically on reducing cell viability in the MDA-MB-468 cells. The observations that synergistic effects on cancer cell viability are obtained when compound A2 is combined with either of the PI3K inhibitors CAL- 100, BKM120 and XL- 147 indicate that the co-treatment strategy may be an effective treatment strategy in reducing cell viability and reducing resistance in highly aggressive breast cancer such as BLBC/TNBC. Example 3

Compound A2 and BKM120 were used in a CCRF- CEM cell line. Based on the established dose and time dependency for Compound A2 and BKM120, a number of combination experiments were performed in medium with the ideal serum concentration. Suboptimal doses of each compound, typically corresponding to 10 - 20% inhibition of viability were used to investigate possible synergy.

In Table 3, inhibition is presented as reduction in viability determined by the resazurin assay, with corresponding combination index (Cl) for selected

combinations of low dose BKM120 with Compound A2.

When assessing the Cls in Table 3, all the tested combinations of low dose

BKM120 and Compound A2 indicate synergy as the Cl values are all less than 1.

As seen in Figure 1 , the reduction in viability for this combination is statistically significant from 0.6 mM BKM120 alone. Furthermore, all combination treatments are both statistically significant from the control treatment (p <0.001), as well as from the low dose of BKM120 given as a monotherapy.

Table 3: The total inhibition for the combinations between BKM120 and Compound 1 presented as the mean ± SD with corresponding combination index (Cl)

Claims

1. A combination product comprising:

(A) a compound of formula (I):

R-L-CO-X (I) wherein R is a C _10-24 unsaturated hydrocarbon group optionally interrupted by one or more heteroatoms or groups of heteroatoms selected from S, O, N, SO, or SO₂, said hydrocarbon group comprising at least 4 non-conjugated double bonds;

X is an electron withdrawing group;

or a pharmaceutically acceptable salt, or a hydrate or solvate thereof; and (B) a compound of formula (X)

a compound of formula (XI) pilaralisib); or a compound of general formula (XII)

wherein Ri is C_1-4 alkyl, such as Me or Et;

R₂ is H or Hal;

R₃ is N or CH; and

R₄ is H or Hal;

2. A pharmaceutical composition comprising:

(A) a compound of formula (I):

R-L-CO-X (l) wherein R is a C 10-24 unsaturated hydrocarbon group optionally interrupted by one or more heteroatoms or groups of heteroatoms selected from S, O, N, SO, or SO₂, said hydrocarbon group comprising at least 4 non-conjugated double bonds;

X is an electron withdrawing group;

(B) a compound of formula (X)

a compound of formula (XI) pilaralisib); or a compound of general formula (XII)

wherein Ri is C_1-4 alkyl, such as Me or Et;

R₂ is H or Hal;

R₃ is N or CH; and

R₄ is H or Hal;

3. A pharmaceutical composition for simultaneous, sequential or separate use comprising a kit comprising a first composition comprising a compound (I) as defined in claim 1 and a pharmaceutically-acceptable diluent or carrier, and a second composition comprising a compound (X) to (XII) as defined in claim 1 and a pharmaceutically-acceptable diluent or carrier.

4. A composition or product as claimed in any preceding claim wherein in formula (I), the group X is CHa , preferably CF3.

5. A composition or product as claimed in any preceding claim wherein in formula (I), the group R is a linear unsubstituted C 10-24 unsaturated alkylene group comprising at least 4 non-conjugated double bonds.

6. A composition or product as claimed in any preceding claim wherein L is - SCH2-.

7. A composition or product as claimed in any preceding claim wherein said compound of formula (I) has the formula:

wherein X is as defined in claim 1 , e.g. CF3.

8. A composition or product as claimed in any preceding claim where the compound of formula (I) is Compound A1 or Compound A2:

A2

9. A composition or product as claimed in any preceding claim in which said compound B is of formula (X)

a compound of formula (XI)

acalisib). or a salt thereof.

10. A composition or product as claimed in any preceding claim in which said compound B is buparlisib, idelalisib or pilaralisib or a salt thereof.

11. A composition or product as claimed in any preceding claim in which said compound of formula (I) is

A2 or a salt thereof; and the compound (B) is of formula (X)

or a salt thereof.

12. A pharmaceutical composition or combination product as claimed in claim 1 to 11 for use in the treatment or prevention of a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer.

13. A method of treating, such as reducing symptoms of, or preventing a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer in a patient in need thereof comprising administering to said patient, preferably a human, an effective amount of a composition or combination product as claimed in claim 1 to 11.

14. A method of treating, such as reducing symptoms of, or preventing a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer in a patient in need thereof comprising administering to said patient, preferably a human, an effective amount of a compound of formula (I) as defined in claim 1 to 11 and simultaneously, separately or sequentially administering to said patient a compound of formula (X) to (XII) as defined in claim 1 to 11.

15. A method of treating such as, reducing symptoms of, or preventing a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer, in a patient in need thereof comprising: (i) identifying a patient who has received either a compound of formula (I) as defined in claim 1 to 11 or a compound of formula (X) to (XII) as defined in claim 1 to 11 respectively;

(ii) administering to said patient an effective amount of either a compound of formula (X) to (XII) or a compound of formula (I) as defined in claim 1 to 11 so that said patient is adminstered with both a compound of formula (I) and a compound of formula (X) to (XII).

16. Use of a composition or combination product as claimed in claim 1 to 11 in the manufacture of a medicament for treating or preventing a proliferative disorder such as cancer, especially breast carcinoma or lymphoid cancer.

17. The combination product of any of claims 1 to 11 wherein the product is a fixed combination or non-fixed combination.

18. The pharmaceutical composition or combination product of any of claims 1 to 11 in combination with one or more anti-proliferative compounds for use in cancer therapy.

19. The pharmaceutical composition or combination product for use as claimed in 18, wherein the anti-proliferative compound is selected from the group consisting of aromatase inhibitors, antiestrogens, topoisomerase I or II inhibitors, and microtubule active compounds, alkylating compounds, histone deacetylase inhibitors, and cyclooxygenase inhibitors.