WO2010130779A2 - COMBINATION OF A PBOSPBOINOSITKLE 3-KSπASE INHIBITOR AND AN ANTIDIABETIC COMPOUND - Google Patents

Abstract

The invention relates to a pharmaceutical combination which comprises (a) a phosphosnositide 3-kinase inhibitor compound and (b) an insulin sensitivity enhancer compound for the treatment of a proliferative disease, especially a solid tumor disease; a pharmaceutical composition comprising such a combination; the use of such a combination for the preparation of a medicament for the treatment of a proliferative disease; a commercial package or product comprising such a combination as a combined preparation for simultaneous, separate or sequential use; and to a method of treatment of a warm-blooded animal, especially a human.

Description

Combination of a phosphoinositide 3-kinase inhibitor and an antidiabetic compound

Field of the Invention

The invention relates to a pharmaceutical combination which comprises (a) a phosphoinositide 3-kmase (PI3K) inhibitor compound and (b) an antidiabetic compound and optionally at least one pharmaceutically acceptable earner for simultaneous, separate or sequential use, in particular for the treatment of a proliferative disease, especially a proliferative disease in whicn the PI3K/Akt and/or RAS/MAPK pathways are dysregulated , a pharmaceutical composition comprising such a combination, the use of such a combination for the preparation of a medicament for the treatment of a proliferative disease, a commercial package or product comprising such a combination as a combined preparation for simultaneous, separate or sequential use, and to a method of treatment of a warm-blooded animal, especially a human

Background of the Invention

Several inhibitors of the PI3K/AKT/mT0R pathway are currently present in early clinical trials for the treatment of cancer Some of them dramatically increase the blood glucose levels

Summary of the Invention

The present invention relates to pharmaceutical combinations comprising (a) a phosphoinositide 3-kιnase (PI3K) inhibitor compound and (b) an antidiabetic compound and optionally at least one pharmaceutically acceptable earner for simultaneous, separate or sequential use, for the treatment of a proliferative disease, especially a prolrferatrve disease in which the PI3K/Akt and/or RAS/MAPK pathways are dysregulated

The present invention also relates to pharmaceutical compositions comprising the combinations of (a) a phosphoinositide 3-kinase (PI3K) inhibitor compound and

(b) an antidiabetic compound and optionally at least one pharmaceutically acceptable earner for simultaneous, separate or sequential use, for the treatment of a prolrferative disease, especially a proliferative disease in which the PI3K/Akt and/or RAS/MAPK pathways are dysregulated

The present invention also relates to the use of such a combination for the preparation of a medicament for the treatment of a prolrferative disease

The present invention also relates to methods of treating a warm-blooded animal, especially a human, suffenng from a proliferative disease in which the Pi3K/Akt and/or RAS/MAPK pathways are dysregulated comprising admtnistenng a phosphoinositide 3-kιnase (PI3K) inhibitor compound and (b) an antidiabetic compound

The present invention also relates to a commercial package or product comprising the combination as a combined preparation for simultaneous, separate or sequential use, and to a method of treatment of a warm-blooded animal especially a human In one aspect of the invention the proliferative disease is a solid tumor, induding breast cancer, ovarian cancer, cancer of the colon such as e g colorectal cancer (CRC), and generally the Gl (gastro-intestmal) tract, cervix cancer, lung cancer such as e g non-small-cell lung cancer (NSCLC)₁ head and neck cancer bladder cancer, kidney cancer such as e g renal cell carcinoma (RCC), liver cancer, brain cancer endometrial cancer, neuroendocrine tumors thyroid cancer, pancreatic cancer, cancer of the prostate or Kaposi s sarcoma In another aspect of the invention, the proliferative disease Peutz-Jeghers synd rome which ts characterized by intestinal hamartomas and increased inctdence of epithelial cancers

Detailed Description of the Figures

Figure 1 illustrates metformin as an inhibitor of HER2 negative breast cancer cell proliferation

Figure 2 illustrates the combined treatment of COMPOUND A plus metformin results in an inhibitory effect on cell proliferation Figure 3 illustrates the biochemical effects of metformin and COMPOUND A where metformin reduces p-MAPK {via downregulation of HER2 and EGFR) and metformin activates p-AMPK inhfbrtmg mTOR function (pS6)

Detailed Description of the Invention

WO2006/122806 descnbes imidazoqutnoline derivatives, which have been described to inhibit the activity of lipid kinases, such as PI3-kιnases Specific imidazoquinoline derivatives which are suitable for the present invention, their preparation and suitable pharmaceutical formulations containing the same are described in WO2006/122806 and ind ude compounds of formula I

wherein

R₁ ts naphthyf or phenyl wherein said phenyl is substituted by one or two substituents independently selected from the group consisting of Halogen, lower alkyl unsubstituted or substituted by halogen, cyano, imidazolyl or triazoryl, cycloalkyl amino substituted by one or two substituents independently selected from the group consisting of lower alkyl, lower aikyl sulfonyl, lower alkoxy and lower alkoxy lower alkylamtno, piperazinyl unsubstituted or substituted by one or two substrtuents independently selected from the group consisting of lower alkyl and lower alkyl suffonyl, 2-oxo-pyrrolιdιnyl, lower alkoxy lower alkyl, imidazolyl pyrazolyl, and tnazolyi, R₂ ts O or S

R₃ is lower alkyl,

R₄ ts pyridyl unsubstituted or substituted by halogen, cyano, lower alkyl, lower alkoxy Of piperaziπyl unsubstituted or substituted by lower alkyl, pyrimtdinyl unsubstttuted or substituted by lower alkoxy, quinolinyl unsubstttuted or substituted by halogen, quinoxalinyl, or phenyl substituted with alkoxy

R₅ ts hydrogen or halogen,

n is 0 or 1,

R₆ is oxido;

with the proviso that rf n=1 , the N-atom bearing the radical R₆ has a positive charge,

R₇ is hydrogen or amino,

or a tautomer thereof, or a pharmaceutically acceptable salt, or a hydrate or solvate thereof

The radicals and symbols as used in the definition of a compound of formula I have the meanings as disclosed in WO2006/122806 which publication is hereby incorporated into the present application by reference

A preferred compound of the present invention is a compound which is specif ically described in WO2006/122806 A very preferred compound of the present invention is 2-methyl-2-[4-(3-methyl-2-oxo-8-quιnolιn-3-yl-2,3-dιhydro-ιmιdazo[4,5-c]quιnolιn-1-yl)- phenyl]-proptonitrile and its monotosylate salt (COMPOUND A) The synthesis of 2- methyl-2-[4-(3-methyl-2-oxo-8-quιnolιn-3-yl-2,3-dιhydro-ιmιdazo{4,5-c]quιnohn-1-yl)- phenylj-propionitrile is for instance described in WO2006/122806 as Example 1 Another very preferred compound of the present invention is 8-(6-methoxy-pyridin-3- yl)-3-methy[-1-(4-pιperazm-1-yl-3-trif]uoromethyl-phenyl)-1 ,3-d ιhydro-ιmιdazo[4,5- c]quinolιn-2-one (COMPOUND B) The synthesis of 8-(6-methoxy-pyridιn-3-yl)-3- methyl-1 -{4-piperazιn-1 -yl-3-trifluoromethyl-phenyl)-1 ,3-d ihydro-ιmιdazo[4,5- c]quιnoiιn-2-one is for instance described in WO2006/122806 as Example 86

WO07/084786 describes pyrimidine derivatives, which have been found the activity of lfpid kinases, such as PI3-kιnases Specific pyrirnidine derivatives which are suitable for the present invention, their preparation and suitable pharmaceutical form ulations containing the same are described in WO07/084786 and include compounds of formula il

or a stereoisomer tautomer, or pharmaceutically acceptable salt thereof, wherein,

W is CR_W or N, wherein R_w is selected from the group consisting of

(1 ) hydrogen,

(2) cyano,

(3) halogen,

(4) methyl,

(5) trifluoromethyl,

(6) sulfonamide

Ri is selected from the group consisting of (1) hydrogen, (2) cyano,

(3) nrtro,

(4) halogen,

(5) substituted and uπsubstituted alkyl,

(6) substituted and unsubstituted alkenyl,

(7) substituted and unsubstituted alkynyl,

(8) substituted and unsubstrtuted aryl,

(9) substituted and unsubstituted heteroaryl,

(10) substituted and unsubstituted heterocyclyl,

(11) substituted and unsubstituted cycloalkyl,

(12) -COR_1a,

(13) -CO₂R_1a,

(14) -CONR_1aR_1b,

(15) -NR_1aR,b,

(16) -NR_1aCOR_1b,

(17) -NR_1aSO₂R_1b,

(18) -OCOR_1a,

(19) -OR_1a,

(20) -SR_1a,

(21) -SOR_1a.

(22) -SO₂R_1a, and

(23) -SO₂NR_1aR_1b, wherein R_1a, and R_1b are independently selected from the group consisting of

(a) hydrogen,

(b) substituted or unsubstituted alkyl,

(c) substituted and unsubstituted aryl,

(d) substituted and unsubstituted heteroaryl,

(e) substituted and unsubstituted heterocycfyl, and

(f) substituted and unsubstituted cycloalkyl, R₂ is selected from the group consisting

(1) hydrogen,

(2) cyano, (3) nitro,

(4) halogen,

(5) hydroxy,

(6) amino,

(7) substituted and unsubstituted alkyl,

(8) -COR_2a, and

(9) -NR_2aCOR₂b, wherein R_2a, and R_2b are independently selected from the group consisting of

(a) hydrogen, and

(b) substituted or unsubstituted alkyl;

R₃ is selected from the group consisting of

(1 ) hydrogen,

(2) cyano,

(3) nitro,

(4) halogen,

(5) substituted and unsubstituted alkyl,

(6) substituted and unsubstituted alkenyl,

(7) substituted and unsubstituted alkynyl,

(8) substituted and unsubstituted aryl,

(9) substituted and unsubstituted heteroaryl,

(10) substituted and unsubstituted heterocyclyl,

(11) substrtuted and unsubstituted cycloalkyi,

(12) -COR_3a,

(13) -NR_3aR_3b.

(14) -NR_3aCOR_3b,

(15) -NR_3aSO₂R_3b,

(16) -OR_3a,

(17) -SR_3a,

(18) -SOR_3a,

(19) -SO₂R_3a, and

(20) -SO₂NR_3aR_3b, wherein R_3a, and R_3b are independently selected from the group consisting of (a) hydrogen,

(b) substituted or unsubstituted alkyl ,

(c) substituted and unsubstituted aryl,

(d) substituted and unsubstituted heteroaryi,

(e) substituted and unsubstituted heterocyclyl, and

(f) substituted and unsubstituted cyd oalkyl, and R₄ is selected from the group consisting of

(1) hydrogen, and

(2) halogen

The radicals and symbols as used in the definition of a compound of formula I have the meanings as disclosed in WO07/0&4786 which publication is hereby incorporated into the present application by reference

A preferred compound of the present invention is a compound which is specifically described in WO07/084786 A very preferred compound of the present invention is 5-(2₁6-dι-morpholιn-4-yl-pyrtmιdtn-4-yl)-4-trιfluoromethyl-pyridιn-2-ylamιne and its hydrochloride salt (COMPOUND C) The synthesis of 5-(2 6-di-morpholιn-4-yl- pyrimtdιn-4-yl)-4-trifluoromethyl-pyrιdιn-2-ylamιne is described in WO07/084786 as Example 10

In one aspect, the present invention pertains to a combination such as a combined preparation or a pharmaceutical composition which comprises (a) a phosphoinositide 3-kιnase (PI3K) inhibitor compound and (b) a antidiabetic which is an insulin sensitizer and is an activator of AMP-activated protein kinase (AMPK), such as e g a biguanide or a thiazohdinedione (glitazone)

Exemplary biguanide compounds include drugs that are insulin sensitivity enhancers and e g useful in controlling or managing non-insuhn-dependent diabetes mellitus

(NIDDM) Non-limiting examples of biguanides include metformin, phenformin or buformin and the like and pharmaceutically acceptable salts, or isomers thereof In a preferred embodiment, the biguanide is metformin The preparation of metformin (dimethyldiguanide) and its hydrochloride salt is state of the art and was disclosed first by Emil A Werner and James Bell, J Chem Soc 121, 1922, 1790-1794 Metformin, can be administered e g tn the form as marketed under the trademarks GLUCOPHAGE™

In another aspect, the present invention relates to a combination such as a combined preparation or a pharmaceutical composition which comprises (a) a phosphoinosrtide 3-kιnase (PI3K) inhibitor compound and (b) metformin

In another embodiment the antidiabetic is a thiazolidinedione (glitazone) Exemplary ghtazones include 5-{f4-(2-(5-ethyl-2-pyridyl)ethoxy)pheny[]-methyl}thιazolιdme-2 ,4- dione (piogiitazone, EP 0 193 256 A1) 5-{[4-(2-(methyl-2-pyridιnyl-amιno)- ethoxy)phenyl]methyl}-thiazolidιne-2₁4-dione (rosiglitazone, EP 0 306 228 A1), 5-{[4-

((3,4-dιhydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yi)methoxy)-phenyl]- methyl}thιazolιdιne-2,4-dιone (troglitazone, EP 0 139 421) (S)-((3 4-dthydro-2-

(phenyl-methyl)-2H-1-benzopyran-6-yl)methyl-thiazolidine-2,4-dione (englitazone, EP 0 207 605 B1), 5-(2,4-dιoxothιazolιdιn-5-ylmethyl)-2-methoxy-N-(4-trifluoromethyl- benzy1)benzamιde (KRP297, JP 10087641 -A), 5-[6-(2-fluoro-benzyloxγ)naphthalen- 2-ylmethylJthιazolιdιne-2,4-dιone (MCC555, EP 0 604 983 B1) 5-({4-(3-(5-methyl-2- phenyl-4-oxazoly1)-1-oxopropyl)-phenyl]-methy[}-thιazolιdιne-2,4-dιone (dargfrtazone, EP 0 332 332), 5-{2-naphthylsulfonyl)-thιazolιdιne-2,4-dιor>e (AY-31637, US 4,997,948), 5-{[4-(1-methyl-cyclohexyl)methoxy)-phenyl]methyl}-thiazolidine-2,4- dione (ciglitazone, US 4,287,200) 5-{4-[{6-methoxy-1-methyl-1 H-benzιmidazol-2-yl) methoxylbenzyi}-1,3-thιazofιdιne-2,4-dιone (nvoglitazone, CAS-No 185428-18-6) are in each case generically and specifically disclosed in the documents cited in brackets beyond each substance, m each case in particular in the compound claims and the final products of the working examples, the subject-matter of the final products, the pharmaceutical preparations and the claims are hereby incorporated into the present application by reference to these publications The preparation of DRF2189 and of 5-{[4-(2-(2,3-dιhydroιndol-1-yl)ethoxy)phenyl]methyl}-thiazolidιne- 2,4-dtone is described in B B Lohray et al , J Med Chem 1998, 41, 1619-1630, Examples 2d and 3g on pages 1627 and 1628 The preparation of 5-[3-(4- chlorophenyl])-2-propynyl]-5-phenylsurfonyl)-thιazolιdιne-2,4-dιone and the other compounds tn which A is phenylethynyl mentioned herein can be earned out according to the methods described in J Wrobel et al , J Med Chem 1998, 41 , 1084-1091

fn particular, MCC555 can be formulated as disclosed on page 49, lines 30 to 45, of EP 0 604 983 B1, englttazone as disclosed from page 6, line 52, to page 7, line 6, or analogous to Examples 27 or 28 on page 24 of EP 0 207 605 B1 , and darglrtazone and 5-{4-[2-{5-methyl-2-phenyl-4-oxazolyl)-ethoxy)]benzyt}-thιazolιdine-2₁4-dtone

(BM-13 1246) can be formulated as disclosed on page 8, line 42 to line 54 of EP 0 332 332 B1 AY-31637 can be administered as disclosed in column 4, lines 32 to 51 of US 4,997,948 and rosiglrtazone as disclosed on page 9, lines 32 to 40 of EP 0 306 228 A1 , the latter preferably as tts mateate salt Rosiglitazone can be administered in the form as it is marketed e g under the trademark AVANDIA™ Troglitazone can be administered in the form as it is marketed e g under the trademarks ReZu!tn^1Λ\ PRELA Y™, ROMOZIN'^ (in the United Kingdom) or NOSCAL ™ (tn Japan) Pioglrtazone can be administered as disclosed in Example 2 of EP 0 193 256 A1 , preferably in the form of the monohydrochloride salt Corresponding to the needs of the single patient it can be possible to administer pioglrtazone tn the form as it rs marketed e g under the trademark ACTOS^ΪM Ciglitazone can, for example, be formulated as disclosed in Example 13 of US 4,287,200 Other activators of AMP-activated protein kinase (AMPK) that are useful for the present invention include compounds described and cited in Zhou et al, Acta Physfologica 2009, 196, 175-190, including the compounds described in WO 2008/016278, US 2005/0038068, WO 2007/062568, WO 2008/006432, WO 2008^083124, WO 2007/005785, FR2846656, EP 1 754 483 A1, WO 2006/071095 A1 , which are herewith incorporated by reference

The term "a combined preparation", as used herein defines especially a "kit of parts" in the sense that the combination partners (a) and (b) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i e simultaneously or at different time points The parts of the kit of parts can then, e g , 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 ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can be varied , e g in order to cope with the needs of a patient sub-population to be treated or the needs of the single

In one embodiment of the invention, (a) the phosphoinosrtide 3-kinase (PI3K) inhibitor compound inhibitor is COMPOUND A, COMPOUND B or COMPOUND C

The term "treating" or "treatment" as used herein composes a treatment effecting a delay of progression of a disease The term "delay of progression" as used herein means administration of the combination to patients being in a pre-stage or in an early phase of the proliferative disease to be treated in which patients for example a pre-form of the corresponding disease is diagnosed or which patients are in a condition, e g during a medical treatment or a condition resulting from an accident, under which it is likely that a corresponding disease will develop

In one embodiment of the present invention, the proliferative disease is a solid tumor The term "solid tumor" especially means breast cancer, ovarian cancer, cancer of the colon such as e g colorectal cancer (CRC), and generally the Gl

(gastro-intestinal) tract, cervix cancer, lung cancer such as e g non-small-cell lung cancer (NSCLC), head and neck cancer, bladder cancer kidney cancer such as e g renal cell carcinoma (RCC)₁ liver cancer, brain cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, pancreatic cancer, cancer of the prostate or Kaposi's sarcoma

In a preferred embodiment the proliferative disease is lung cancer, in particular lung tumors carrying a germltne mutations in serine/threonine kinase 11 (STK11 , also called LKB1) Inactivating somatic mutations of LBK1 have been reported in primary human lung adenocarcinomas Thus, germline mutations in LKB1 have been found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamos cell carcinomas, respectively. A k>ss-of-function mutation of LKB1 may also strongly cooperate with a dysfunctional activation of the PI3K and/or RAS/MAPK pathways, which are also common alterations in lung tumors. It has now been found that lung tumors carrying a loss-of-function mutation of LKB1 can be effectively treated with the COMBINATION OF THE INVENTION.

In a preferred embodiment, the proliferative disease Peutz-Jeghers syndrome, which is characterized by intestinal hamartomas and increased incidence of epithelial cancers.

Proliferative diseases that may be treated with the COMBINATION OF THE INVENTION in accordance with another embodiment of the present invention, include Breast Cancer, Ovarian Cancer, Colon Cancer, Pancreas Cancer, Melanoma, Head and Neck Cancer. Endometrial Cancer and Brain Cancer.

The present combination inhibits the growth of solid tumors, but also liquid tumors. Furthermore, depending on the tumor type and the particular combination used a decrease of the tumor volume can be obtained . The combinations disclosed herein are also suited to prevent the metastatic spread of tumors and the growth or development of micrometastases. The combinations disclosed herein are in particular suitable for the treatment of poor prognosis patients, especially such poor prognosis patients having lung tumors.

The structure of the active agents identified by code nos., generic or trade names may be taken from the actual edition of the standard compendium 'The Merck Index" or from databases, e.g. Patents International (e.g. IMS World Publications). The corresponding content thereof is hereby incorporated by reference.

It will be understood that references to the combination partners (a) and (b) are meant to also include the pharmaceutically acceptable salts. If these combination partners (a) and (b) have, for example, at least one basic center, they can form acid addition saKs. Corresponding acid addition salts can also be formed having, if desired, an additionally present basic center The combination partners (a) and (b) having an acid group (for example CCOH) can also form salts with bases The combination partner (a) or (b) or a pharmaceutically acceptable salt thereof may also be used in form of a hydrate or include other solvents used for crystallization

A combination which comprises (a) a phosphotnosittde 3-kinase inhibitor compound and (b) a biguanide insulin sensitivity enhancer, in which the active ingredients are present in each case in free form or in the form of a pharmaceutically acceptable salt and optionally at least one pharmaceutically acceptable carrier, will be referred to hereinafter as a COMBINATION OF THE INVENTION

The COMBINATION OF THE INVENTION are both synergistic and additive advantages, both for efficacy and safety Therapeutic effects of combinations of a phosphoinosrtide 3-kιnase inhibitor compound wrth a compound which modulates the biguanide insulin sensitivity enhancer can result in lower safe dosages ranges of each component in the combination Moreover, an insulin sensitivity enhancer is useful in overcoming the potential increase in blood glucose caused by modulators of PI3K signaling

The pharmacological activity of a COMBINATION OF THE INVENTION may, for example, be demonstrated in a clinical study or in a test procedure as essentially descnbed hereinafter Suitable clinical studies are, for example, open label nonrandomized dose escalation studies in patients with advanced solid tumors Such studies can prove the additive or synergism of the active ingredients of the COMBINATIONS OF THE INVENTION The beneficial effects on proliferative diseases and/or glucose homeostasis can be determined directly through the results of these studies or by changes in the study design which are known as such to a person skilled in the art Such studies are, in particular, suitable to compare the effects of a monotherapy using the active ingredients and a COMBINATION OF THE INVENTION Preferably, the combination partner (a) is administered with a fixed dose and the dose of the combination partner (b) is escalated until the Maximum Tolerated Dosage is reached It is one objective of this invention to provide a pharmaceutical composition compnsing a quantity, which is therapeutically effective against a proliferative disease comprising the COMBINATION OF THE INVENTION In this composition, the combination partners (a) and (b) can be administered together, one after the other or separately in one combined unit dosage form or in two separate unit dosage forms The unit dosage form may also be a fixed combination

The pharmaceutical compositions according to the invention can be prepared in a manner known per se and are those suitable for enteral, such as oral or rectal, and parenteral administration to mammals (warm-blooded animals), including man Alternatively, when the agents are administered separately, one can be an enteral formulation and the other can be administered parenterally

The novel pharmaceutical composition contain, for example, from about 10 % to about 100 %, preferably from about 20 % to about 60 %, of the active ingredients Pharmaceutical preparations for the combination therapy for enteral or parenteral administration are, for example, those in unit dosage forms, such as sugar-coated tablets, tablets, capsules or suppositories, and furthermore ampoules If not indicated otherwise, these are prepared in a manner known per se, for example by means of conventional mixing, granulating, sugar-coating, dissolving or lyophilizing processes It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not in rtsetf constitute an effective amount since the necessary effective amount can be reached by ad ministration of a plurality of dosage units

In preparing the compositions for oral dosage form, any of the usual pharmaceutical med ia may be employed, such as, for example, water, glycols, oils alcohols, flavoring agents, preservatives, coloring agents, or earners such as starches, sugars, microcnstalline cellulose, diluents granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical earners are obviously employed

In particular, a therapeutically effective amount of each of the combination partner of the COMBINATION OF THE INVENTION may be administered simultaneously or sequentially and in any order, and the components may be administered separately or as a fixed combination For example, the method of delay of progression or treatment of a proliferative disease accordtng to the invention may compnse (ι) administration of the first combination partner in free or pharmaceutically acceptable salt form and (ιi) administration of the second combination partner in free or pharmaceutically acceptable salt form, simultaneously or sequentially in any order, in jointly therapeutically effective amounts, preferably in synergistically effective amounts The individual combination partners of the COMBINATION OF THE INVENTION can be adminfstered separately at different times during the course of therapy or concurrently in divided or single combination forms Furthermore, the term administenng also encompasses the use of a pro-drug of a combination partner that convert in vivo to the combination partner as such The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administenng" is to be interpreted accordingly

The COMBINATION OF THE INVENTION can be a combined preparation or a pharmaceutical composition

Moreover, the present invention relates to a method of treating a warm-blooded animal having a proliferative disease comprising admtnistenng to the animal a COMBINATION OF THE INVENTION in a quantity which is therapeutically effective against said proliferative disease

Furthermore, the present invention pertains to the use of a COMBINATION OF THE INVENTION for the treatment of a proliferative disease and for the preparation of a medicament for the treatment of a proliferative disease Moreover, the present invention provides a commercial package comprising as active ingredients COMBINATION OF THE INVENTION, together with instructions for simultaneous, separate or sequential use thereof in the delay of progression or treatment of a proliferative disease

Preferred embodiments of the invention are represented by combinations comprising

• COMPOUND A, COMPOUND B or COMPOUND C and metformin,

• COMPOUND A, COMPOUND B or COMPOUND C and phenformin,

• COMPOUND A, COMPOUND B or COMPOUND C and pioglitazone,

• COMPOUND A, COMPOUND B or COMPOUND C and rivogtttazone,

• COMPOUND A, COMPOUND B or COMPOUND C and rosiglitazone

• COMPOUND A, COMPOUND B or COMPOUND C and ciglrtazone

• COMPOUND A, COMPOUND B or COMPOUND C and darglitazone

• COMPOUND A, COMPOUND B or COMPOUND C and engiitazone

In further aspects, the present inventions provides

• a combination which comprises (a) a COMBINATION OF THE INVENTION, wherein the active ingredients are present in each case in free form or in the form of a pharmaceutically acceptable salt or any hydrate thereof, and optionally at least one pharmaceutically acceptable carrier, for simultaneous, separate or sequential use,

• a pharmaceutical composition comprising a quantity which is jointly therapeutically effective against a proliferative disease of a COMBINATION OF THE INVENTION and at least one pharmaceutically acceptable carrier,

• the use of a COMBINATION OF THE INVENTION for the treatment of a proliferative disease,

• the use of a COMBINATION OF THE INVENTION for the preparation of a medicament for the treatment of a proliferative disease, • the use of a combination COMBINATION OF THE INVENTION wherein the PI3K inhibitor is selected from COMPOUND A, COMPOUND B or COMPOUND C₁ and

• the use of a COMBINATION OF THE INVENTION wherein the biguanide insulin sensitivity enhancer compound is a biguanide, e g metformin or phenformin,

• the use of a COMBINATION OF THE INVENTION wherein the biguanide insulin sensitivity enhancer compound ts a glrtazone, e g pioglrtazone, rivoglrtazone, rosiglitazone, αglitazone, darglrtazone, englitazone

Moreover, in particular, the present invention relates to a combined preparation whtch comprises (a) one or more unit dosage forms of a phosphoinosrtide 3-kιnase inhibitor compound and (b) a biguanide insulin sensitivity enhancer compound

Furthermore, in particular, the present invention pertains to the use of a combination comprising (a) a phosphoinositide 3-kιnase inhibitor compound and (b) a biguanide insulin sensitivity enhancer compound for the preparation of a medicament for the treatment of a proliferative disease and/or overcoming the potential increase in blood glucose caused by inhibition of the PI3K/Akt pathway

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 , the seventy of the condition being treated Thus, the dosage regimen the COMBINATION OF THE INVENTION is selected in accordance with a variety of factors including the route of administration and the renal and hepatic function of the patient A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the single active ingredients required to prevent, counter or arrest the progress of the condition Optimal precision in achieving concentration of the active ingredients within the range that yields efficacy wrthout toxicity requires a regtmen based on the kinetics of the active ingredients' availability to target sites

When the combination partners employed in the COMBINATION OF THE INVENTION are applied in the form as marketed as single drugs, their dosage and mode of administration can take place in accordance with the information provided on the package insert of the respective marketed drug in order to result in the beneficial effect descnbed herein, if not mentioned herein otherwise

COMPOUND A may be administered to a human in a dosage range varying from about 25 to 1600 mg /day

COMPOUND B may be administered to a human in a dosage range varying from about 2 5 - 150 mg/3x/week or 2 5 to 75 mg/day

COMPOUND C may be administered to a human tn a dosage range varying from about 12 5 to 600 mg/day

Metformin may be administered to a human e g 850 mg bid

The beneficial effects of the COMBINATION OF THE INVENTION can also be determined by other test models known as such to the person skilled in the pertinent art

The following examples are offered by way of illustration and are not tntended to limit the scope of the invention Variations, modification, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and the essential characteristics of the present teachings The cell lines mentioned therein are not thougt to limit the scope of the invention as they are merely representatives and may be replaced with different ceil lines and tumor cells for which they are representatives Accordingly the scope of the invention is to be defined not by the preceding illustrative description but instead by the following claims, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein Example 1 Dual-targeting of AMPK and PI3K/mT0R in a panel of breast cancer celt lines

MCF-7(HER2), SK-BR-3, MDA-MB-231 and MDA-MB^68 breast cancer cells are treated with drfferent doses of 2-Methyl-2-[4-(3-methyl-2-oxo-8-quιnolin-3-yl-2₁3- dιhydro-ιmιdazo[4,5-c)quιnolin-1-yl)-phenyi]-propιonιtri[e, also known as COMPOUND A₁ metformin or both agents in combination Levels of phosphorylated and total AMPK, MAPK, EGFR, HER2 and S6 ribosomal protein are evaluated by western bfot. Cell proliferation analyses are performed in triplicates using the WST-1 and crystal violet colorimetric assays.

Metformin induces dose-dependent growth inhibition of MCF-7(HER2), SK-BR- 3, MDA-MB-231 and MDA-MB-468 breast cancer cell lines as illustrated in Figures 1 and 2. The combined treatment of COMPOUND A plus metformin results in an inhibitory effect on cell proliferation greater than with either treatment alone as illustrated in Figure 2 Metformin activates AMPK reducing mTORCI activity and decreasing the levels of p-S6 ribosomal protein Treatment with metformin is also associated with reduced receptor tyrosine kinase (EGFR and HER2) expression and decreased p-MAPK COMPOUND A potently decreases p-AKT and p-S6 However, as described for other mTOR inhibitors, COMPOUND A increases MAPK phosphorylation by transactivation of several receptors tyrosine kinase (RTKs) including EGFR and HER2 Metformin counteracts the MAPK pathway transactivatton induced by COMPOUND A likely by downregulating EGFR and/or HER2 as illustrated in Figure 3 This data provides the rationale of combining metformin with Pi3K/mT0R inhibitors in EGFR or HER2 over-expressing cells

The combination of metformin and COMPOUND A inhibits the growth of EGFR positive and HER2 positive breast cancer cell lines We provide the rationale for targeting both AMPK and PI3K/Akt/mTOR pathways to elicit strong anti-tumor effects m breast cancer.

Example 2 Combination effect of PI3K inhibitors and metformin on A549 non-small cell lung tumors m nude mice xenograft model A549 human non-small cell lung cancer (NSCLC) cells are treated wrth drfferent doses of the 2-Methyl-2-[4-(3-methyl-2-oxo-8-quιnolιn-3-yl-2,3-dιhydro-ιmιdazo[4₁5- c}quιnohn-1-y1)-phenyl]-proptonrtn!e monotosylate salt (also known as COMPOUND A) or 5-(2,6-di-morphotøn^-yi-pyrimιdtn-4-yl)-4-trifluoromethyl-pyrιdιn-2-ylarnιne hydrochloride salt (also known as COMPOUND C), a single dose of metformin or both agents in combination The A549 human NSCLC cells (ATCC-CCL-185, available from American Type Culture Collection, Rockvtlle, Md US) dtsplay characteristics of type Il alveolar epithelial cells (Lteber et al, lnt J Cancer 17(1) 62- 70 [1976]) The A549 NSCLC cells are homozygous for mutations in the tumor suppressor genes CDKN2A and STK11 (also called LKB1), and in KRAS

(Wellcome Trust Sanger Institute, Catalogue of Somatic Mutations in Cancer, Cosmic ID No 905949 avatlable at websrte http //www Sanger ac uk/peπ7genetιcs/CGP/coreJιne_ytewer? actro n=sample&name=A549 )

A549 tumor ceils are grown in Karghn's modified Ham's F12 med ium containing 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, 25 μg/mL gentamicin, 100% fetal bovine serum, 2 mM glutamme, and 1 mM sodium pyruvate The cells are cultured in tissue culture flasks in a humidified incubator at 37°C, tn an atmosphere of 5% CO2 and 95% air The cells are harvested for injection into 9- week old female nu/nu (nude) mice (Marian Laboratones Indianapolis IN) by detaching the monolayers with 2X trypsin and resuspending at 5 x 10⁷ cells/mL in cold phosphate-buffered saline containing 50% Matrigel

0 2 mL of A549 cell suspension (1 x 10⁷ cells) is injected subcutaneously in the rignt flank of 9-week old female nu/nu (nude) mice (Harlan Laboratories, Indianapolis, IN) having a body weight (BW) range of 19 9-27 3 g on Day 1 of the study Tumors are callipered in two dimensions to monitor their growth as their mean volume approached 150-220 mm³ Twenty-two days after implantation, the mice are sorted into 11 groups of eight or nine mice having individual tumor sizes of 108-221 mm ³ Tumor volume in mm³ is determined using the formula [(width)² x

(tength)]/2 where width = width of the tumor in mm and length = length of the tumor in mm Tumor weight can be estimated with the assumption that 1 mg is equivalent to 1 mm³ of tumor voiume

The 11 groups of nude mice are treated as follows One group of nine mice serves as Controls (C or Control) for all analyses and is ad ministered intraperitoneally (ι p ) 50 mM sodium acetate at pH 4 (Vehicle 1) and then administered by oral gavage (p o ) a composition comprising 10% N- methylpyrrolidone 90% polyethylene glycol 300 (PEG300) (Vehicle 2) All treatments with metformin (metformin hydrochloride, Glucophage®, Bristol-Myers Squibb Company) monotherapy are administered intraperitoneal (ι p ) once daily at a single dose of 192 3 mg/kg metformin until the end of the study as provided in the Results Table 1 Metformin is dissolved in 50 mM sodium acetate at pH 4 for dosing

All treatments with the Compound A or Compound C monotherapy are administered at varying doses by oral gavage (p o ) once daily until the end of the study as provided in the Results Table 1 Compound A and Compound C are stored at -2O°C protected from light Stock solutions (10X) in 100% N-methylpyrrohdone

(NMP) are prepared every five days, ahquotted, and stored in the dark at room temperature On each treatment day, stock solution aliquots are diluted with polyethylene glycol (PEG300) to provide the formulated drug (Compound A or Compound C) in 10% NMP 90% PEG300 Dosing solutions are protected from light, and the formulated drug is administered within 1 hour after preparation

For combination therapies, Compound A or Compound C are administered by oral gavage (p o ) wrthin 30 minutes after the intraperitoneal (ι p ) administration of metform in , except on Day 20 when Compound A or Compound C is given immediately after metformin Compound A, Compound C and metformin are prepared and administered as disclosed above for the monotherapy and in Results Table 1

Paclitaxel (Natural Pharmaceuticals, lnc Beverly, Massachusetts, USA) is administered by bolus tail-vein injections (ι v ) once daily on alternate days for five doses Paclrtaxel is dissolved in 50% ethanol and 50% Cremophor^® EL to prepare a 10X stock solution stored at room temperature On each day of dosing, an aliquot of the paclitaxel stock solution is diluted with 5% dextrose in water to yield a dosing solution containing 5% ethanol and 5% Cremophor*¹ EL In all groups, the dosing volume of 10 mL/ kg (0 2 mL/ 20 g mouse) is scaled to the weight of each animal as determined on the day of dosing, except on weekends when the previous BW is earned forward Acceptable toxicity for the maximum tolerated dose (MTD) is defined as a group mean BW loss of less than 15% during the test, and not more than 10% treatment-related mortality. Any animal wrth BW losses exceeding 15% for three consecutive measurements, or with a BW loss exceeding 20% for one measurement, is designated to be euthanized

Short-term efficacy for tumor growth inhibition in A549 cells is determined on Day 20, the day on which the Control mean tumor volume nearly attained the 500 mm³ endpoint By Day 20, no tumors had progressed to the endpoint; but 6 animals had died prior to Day 20 Statistical and graphical analyses was conducted by determining the difference in tumor volume between Day 1 (the start of dosing) and the endpoint day for each animal that remained on study on Day 20. Antitumor activity is expressed as % T/C (comparing the mean tumor volume change between the endpoint day and Day 1 for the treatment group to the Control), or % IfT₀

(comparing the mean tumor volume change between the endpoint day and Day 1 for the treatment group to its tumor volume at the beginning of the experiment (To) A T/C < 40% generally indicates potential therapeutic activity A partial regression indicates that the tumor volume was 50% or less of its initial volume on Day 1 for three consecutive measurements during the study, and equal to or greater than 13 5 mm³ for one or more of these three measurements A complete regression indicates that the tumor volume was less than 13.5 mm³ for three consecutive measurements dunng the course of the study

Results Table 1 summarizes results for A549 tumors, for the standard 20 day experiment The metformin monotherapy at 192 3 mg/kg did not appear active in the A549 human NSCLC xenograft model in a 20-day tumor growth inhibition assay The response to paclitaxel is consistent with prior results reported in this xenograft model

The combined treatment of 32 7 mg/kg of COMPOUND C plus metformin results in -17% TZT₀ and significant med ian tumor reduction (p < 0 01) as compared to the Control but improves non-sigmficantly as compared to the COMPOUND C monotherapy Combined treatment of 32 7 mg/kg of COMPOUND C plus metformin further results in significant median tumor reduction (p < 0 001) as compared to the metformin monotherapy. Combination therapy with 54 4 COMPOUND C and metformin was terminated earl y due to toxicity

The combined treatment of 41.1 mg/kg of COMPOUND A plus metformin results in -27% TyT₀ with stgmficant tumor reduction (p <0 001) as compared to Control but improves non-signtficantly as compared to the COMPOUND A monotherapy Combined treatment of 41 1 mg/kg of COMPOUND A plus metformin further results in significant median tumor reduction Combination therapy with 68.5 mg/kg of COMPOUND A plus metformin resulted tn a - 30% TzT₀ as compared to Control. This combination was not evaluable after one death among the group of eight mice exceeded 10% mortality limitations

The combination treatment of COMPOUND A plus metformin and COMPOUND C plus metformin inhibits the growth of human NSCLC cell lines It is demonstrated that the combination treatment of COMPOUND A plus metformin and COMPOUND C plus metformin in improved growth inhibrtion of human NSCLC cell lines as compared to Control and/or metformin monotherapy.

¹ Animals # 5, 6, 7 and 8 received 68 5 mg/kg Compound A on Day 17 instead of

32 7 mg of Compound C

* Animals # 7, 8 and 9 received metformin p o instead of i.p on Day 2.

^ Animals #7, 8 and 9 received metformin p o instead of t p on Day 2

T/C = 100 x (ΔT/ ΔC) = percent change between Day 1 and D 20 in the mean tumor volume of a treated group (ΔT) compared with change in Control (ΔC)

777 O - 100 x (ΔT/ To) = percent change between Day 1 and D 20 in the mean tumor volume of a treated group (ΔT) compared with its initial volume, when ΔT < 0

Statistical Significance (Kruskal-Wallis with post hoc Dunn^'s multiple companson test) vs indicated group of Control or To πe = not evaluable, ns = not significant at p

> 0 05

Mean BW Change = lowest group mean body weight, as change from Day 1 up to

Day 20, "-" indicates no decrease in mean body weight was observed.

Example 3. Combination effect of PI3K inhibitors and metformin on H520 non-smalt cell lung tumors m nude mice xenograft model

EGFR-null H520 human non-small cell lung cancer (NSCLC) cells are treated with drfferent doses of 2-Methyl-2-[4-(3-methyl-2-oxo-8-quιnofιn-3-yl-2,3-dιhydro- ιmιdazo{4,5-c]quιnolιn-1-yl)-pbenyl]-propιonttriie monotosylate salt (also known as COMPOUND A) or 5-(2,6-dι-morpholιn-4-yl-pyrimidιπ-4-yl>-4-trif^}uoromethyl-pyrrdιn-2- yiamme hydrochloride salt (also known as COMPOUND C), a single dose of metformin or both agents in combination The H520 human NSCLC cells (NCI- H520, ATCC-HTB-182, available from Amencan Type Culture Collection, Rockvtlle, Md US) are isolated from a sample of a (ung mass taken from a patient with squamous celt carcinoma of the lung (Banks-SchSegel et ai, Cancer Res, 45(3) 1187- 1197 (1985)

H520 tumor cells are grown in RPMI 1640 medium containing 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, and 25 μg/mL gentamicin The medium is supplemented with 10% fetal bovine serum, 2 mM glutamme, and 1 mM sodium pyruvate, and buffered with 10 mM HEPES and 0 075% sodium bicarbonate The cells are cultured in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO₂ and 95% air The cells are harvested for injection into 8- week old female nu/nu (nude) mice (Harian Laboratories, Indianapolis, IN) by detaching the monolayers with 1X trypsin and resuspending at 5 x 10⁷ cells/mL in phosphate-buffered saline containing 50% Matrigel

0 2 ml_ of H520 tumor cell suspension (1 x 10⁷ cells) is injected subcutaneously in the right flank of 8-week old female nu/nu (nude) mice (Harian Laboratones, Indianapolis, IN) having a body weight (BW) range of 18 1-26 9 g on Day 1 of the study Tumors are monitored twice weekly and then daily as their mean volume approaches 120-180 mm³ Eight days after implantation, the mice are sorted into 11 groups of eight mice having individual tumor sizes of 126-196 mm³ and a group mean tumor size of 151-153 mm³ Tumor volume in mm³ is determined using the formula [(wtdth)² x (length)]/2, where width = width of the tumor in mm and length = length of the tumor in mm Tumor weight can be estimated with the assumption that 1 mg is equivalent to 1 mm³ of tumor volume

The 11 groups of nude mice are treated as follows One group of eight mice serves as Controls (C or Control) for all analyses and is ad ministered mtraperitoneally (ι p ) 50 mM sodium acetate at pH 4 (Vehicle 1) and then administered by oral gavage (p o ) a composition comprising 10% N- methylpyrroOdone 9€% polyethylene glycol 300 (PEG300) (Vehicle 2) All treatments with metformin (metformin hydrochloride, Glucophage®, Bnstol-Myers Squibb Company) monotherapy are administered intraperitoneal^ (ι p ) once daily at a single dose of 192 3 mg/kg metformin until the end of the study as provided in the Results Table 2 Metformin is dissolved in 50 mM sodium acetate at pH 4 for dosing

All treatments with the Compound A or Compound C monotherapy are administered at varying doses by oral gavage (p o ) once daily until the end of the study as provided in the Results Table 2 Compound A is stored at -20°C Compound C is stored at -20°C protected from light Stock solutions (10X) in 100% N-methylpyrrolidone (NMP) are prepared every five days, aliquotted , and stored in the dark at room temperature On each treatment day, stock solution ahquots are diluted with polyethylene glycol (PEG300) to provide the formulated drug (Compound A or Compound C) in 10% NMP 90% PEG300 Dosing solutions are protected from light, and the formulated drug is administered wrthtn 1 hour after preparation

For combination therapies, Compound A or Compound C is administered by oral gavage (p o ) within 30 minutes after the intraperitoneal (ι p ) administration of metformin , except on Day 20 when Compound A or Compound C is given immediately after metformin Compound A, Compound C and metformin are each prepared and administered as disclosed above for the monotherapy and in Results Table 2

Paclitaxel (Natural Pharmaceuticals, lnc , Beverly, Massachusetts, USA) is administered by bolus tail-vein injections (ι v ) once daily on alternate days for five doses Paclitaxel is dissolved in 50% ethanol and 50% Cremophor^® EL to prepare a 10X stock solution stored at room temperature On each day of dostng, an aliquot of the padrtaxel stock solution is diluted with 5% dextrose in water to yield a dosing solution containing 5% ethanol and 5% Cremophor^® EL In all groups, the dosing volume of 10 mL/ kg (0 2 mL/ 20 g mouse) is scaled to the weight of each animal as determined on the day of dosing, except on weekends when the previous BW is carried forward Acceptable toxicity for the maximum tolerated dose (MTD) is defined as a group mean BW loss of less than 15% during the test, and not more than one treatment-related mortalrty among ten animals Any animal with BW losses exceeding 15% for three consecutive measurements, or wrth a BW loss exceeding 20% for one measurement, is designated to be euthanized

Short-term efficacy for tumor growth inhibition in H520 celts is determined on Day 20, the day on which the Control mean tumor volume nearly attained the 1000 mm³ endpoint By Day 20, no tumors had progressed to the endpoint but 16 animals had died or been euthanized prior to Day 20 Statistical and graphical analyses was conducted by determining the difference in tumor volume between Day 1 (the start of dosing) and the endpotnt day for each animal that remained on study on Day 20 Antitumor activity is expressed as % T/C (comparing the mean tumor volume change between the endpoint day and Day 1 for the treatment group to the Control) A T/C < 40% is classified as potential therapeutically active A partial regression indicates that the tumor volume was 50% or less of its inrtjal volume on Day 1 for three consecutive measurements during the study, and equal to or greater than 13.5 mm³ for one or more of these three measurements. A complete regression indicates that the tumor volume was less than 13 5 mm³ for three consecutive measurements during the course of the study.

Results Table 2 summarizes results for H520 tumors, for the standard 20 day experiment The metformin monotherapy at 192.3 mg/kg did not appear to modulate tumor growth in the H520 human NSCLC xenograft model in a 20-day tumor growth inhibition assay. The paclitaxel monotherapy at 30 mg/kg produced 5% T/C and statistically significant median tumor reduction (p<0 001) as compared to Control.

The combined treatment of 32.7 mg/kg of COMPOUND C plus metformin resulted in 62% T/C but was not statistically evaSuable due to two deaths The combined treatment of 54.4 mg/kg of COMPOUND C plus metformin resulted in 125% T/C but was not statistically evaluable due to five deaths

The combined treatment of 41 1 mg/kg of COMPOUND A plus metformin resulted in 60% T/C, which shows an improved growth inhibition that is not statistically significant as compared to Control The combined treatment of 68 5 mg/kg of COMPOUND A pius metformin resulted in 36% T/C which is an improvement over the corresponding COMPOUND A monotherapy. The results for the 68 5 mg/kg of COMPOUND A combination therapy and monotherapy were not statistically evaluable due to three deaths.

The combination of COMPOUND A plus metformin inhibits the growth of human NSCLC cell lines it is demonstrated that the combination treatment of COMPOUND A pius metformin improved inhibition of growth of human NSCLC cell lines as compared to Control and the COMPOUND A monotherapy Metformin may potentially increase the toxicity of COMPOUND A and/or COMPOUND C in this H520 xenograft model.

Table 2. Antitumor effect of P13K inhibitors and metformin, alone and in combination, on H520 non-small cell lung tumors in nude mice

Statistical Significance (ANOVA with post-hoc Dunnett's multiple comparison test vs. control, except for the group treated with paclitaxel, Kruskal-Wallis and post-hoc Dunn's multiple comparison test vs. control, including the group treated with paclitaxel). ne = not evaluable; ns = not significant * Treatment stopped at specified day due to toxicity

T/C = 100 x (ΔT/ ΔC) = percent change between Day 1 and D 20 in the mean tumor volume of a treated group (ΔT) compared with change in Control (ΔC). Mean BW Change = lowest group mean body weight, as change from Day 1 up to Day 20, "--" indicates no decrease in mean body weight was observed

Example 4: Combination Effect of PI3K inhibitors and metformin on H460 Non-Small Cell Lung Tumors in Nude Mice

H460 human non-small cell lung cancer (NSCLC) cells are treated with different doses of 2-Methyl-2-[4-(3-methyl-2-oxo-8-quιnolιn-3-y1-2,3-dihydro-imtdazo[4,5- c]quinolιn-1-yl)-phenyl]-propionrtrile monotosyiate salt (also known as COMPOUND A) or 5-(2_>6-d ι-morphoJin-4-yl-pyrimidin-4-yl)-4-trifiuoromethyl-pyridtn-2-ylamine hydrochloride salt (also known as COMPOUND C), a single dose of metformin or both agents in combination The H460 human NSCLC cells (NCI-H460, ATCC- HTB-177, available from American Type Culture Collection, Rockvtlle, Md US) are derived from the pleural fluid of a patient with large ceil cancer of the lung

H460 tumor cells are grown in RPMI 1640 medium containing 100 units/mL pemcillm G sodium, 100 μg/mL streptomycin sulfate, and 25 μg/mL gentamicin The medium is supplemented with 10% fetal bovine serum and 2 mM glutamine The cells are cultured in tissue culture flasks in a humidified incubator at 37°C, in an atmosphere of 5% CO₂ and 95% air The cells are harvested for injection into 9- week old female nu/nu (nude) mice (Harlan Laboratories, Indianapolis, IN) by detaching the monolayers with 2X trypsin and resυspending at 5 x 10⁷ cells/mL in phosphate-buffered salme

0 2 mL of H460 tumor ceil suspension (1 x 10⁷ cells) re injected subcutaneousfy in the right flank of 9-week old female nu/nu (nude) mice (Harlan Laboratories, Indianapolis, IN) having a body weight (BW) range of 18 1-26 9 g on Day 1 of the study Tumors are monitored twice weekly and then dairy as their mean volume approaches 100-150 mm³ Ten days after implantation, the mice are sorted into 11 groups of eight mice having individual tumor sizes of 75-196 mm³ and a group mean tumor size of 119-122 mm³ Tumor volume in mm³ is determined using the formula [(width)² x (!ength)j72, where width = width of the tumor in mm and length = length of the tumor in mm Tumor weight can be estimated with the assumption that 1 mg is equivalent to 1 mm³ of tumor volume

The 11 groups of nude mice are treated as follows One group of eight mice serves as Controls (C or Control) for all analyses and is administered intraperitoneal Iy (ι p ) 50 mM sodium acetate at pH 4 (Vehicle 1) and then administered by oral gavage (p o ) a composition comprising 10% N- methylpyrrolidone 90% polyethylene glycol 300 (PEG300) (Vehicle 2) All treatments with metformin (metformin hydrochloride, Glucophage<S> Bristoi-Myers Squibb Company) monotherapy are administered intraperitoneal^ (ι p ) once daily at a single dose of 192 3 mg/kg metformin until the end of the study as provided in the Results Table 3 Metformin is dissolved in 50 mM sodfurn acetate at pH 4 for dosing AII treatments with the Compound A or Compound C monotherapy are administered at varying doses by oral gavage (p o ) once daily until the end of the study as provided in the Results Table 3 Compound A is stored at -20°C Compound C is stored at -20°C protected from light Stock solutions (10X) in 100% N-methylpyrrolιdone (NMP) are prepared every five days, altquotted , and stored in the dark at room temperature On each treatment day, stock solution aliquots are diluted with polyethylene glycol (PEG300) to provide the formulated drug (Compound A of Compound C) in 10% NMP 90% PEG300 Dosing solutions are protected from light, and the formulated drug is administered within 1 hour after preparation

For combination therapies, Compound A or Compound C are administered by oral gavage (p o ) within 30 minutes after the intraperitoneal (ι p ) administration of metformin, except on Day 20 when Compound A or Compound C is given immediately after metformin Compound A, Compound C and metformin are each prepared and administered as disclosed above for the monotherapy and in Results Table 3

Paclrtaxel (Natural Pharmaceuticals, lnc , Beverly, Massachusetts, USA) is administered by bolus tail-vein injections (ι v ) once daily on alternate days for five doses Paclrtaxel is dissolved in 50% ethanol and 50% Cremophor^® EL to prepare a 1OX stock solution stored at room temperature On each day of dosing an aliquot of the paclitaxel stock solution is diluted with 5% dextrose in water to yield a dosing solution containing 5% ethanol and 5% Cremophor^® EL in all groups, the dosing volume of 10 mil kg (0 2 mL/ 20 g mouse) is scaled to the weight of each animal as determined on the day of dosing except on weekends when the previous BW is earned forward Acceptable toxicity for the maximum tolerated dose (MTD) is defined as a group mean BW loss of less than 15% during the test, and not more than one treatment-related mortality among ten animals Any animal with BW losses exceeding 15% for three consecutive measurements or with a BW loss exceeding 20% for one measurement, is designated to be euthanized

Short-term efficacy for tumor growth inhibition in H460 cells is determined on Day 12, the day on which the Control mean tumor volume nearly attained the 1000 mm³ endpoint Statistical and graphical analyses was conducted by determining the difference in tumor volume between Day 1 (the start of dosing) and the endpoint day for each animal that remained on study on Day 12 Antitumor activity is expressed as % T/C (comparing the mean tumor volume change between the endpoint day and Day 1 for the treatment group to the Control) A T/C < 40% is classified as potential therapeutically active A partial regression indicates that the tumor volume was 50% or less of its initial volume on Day 1 for three consecutive measurements during the study, and equal to or greater than 13 5 mm³ for one or more of these three measurements A complete regression indicates that the tumor volume was less than 13 5 mm³ for three consecutive measurements during the course of the study

Results Table 3 summanzes results for H460 tumors, for the standard 12 day experiment The metformin monotherapy at 192 3 mg/kg did not appear to impact tumor growth in the H460 human NSCLC xenograft model in a 12-day tumor growth inhibition assay The response to pacJitaxel ts consistent with prior results reported in this xenograft model

The combination treatment of 41 1 mg/kg of COMPOUND A plus metform in results in statistically significant improved inhibition of tumor growth at 19% T/C as compared to the Control (p < 0 05 when analyzed with Kruskal-Walhs and post-hoc Dunn's multiple comparison test, p < 0 01 when analyzed with ANOVA with post-hoc Dunnett's multiple comparison test) The combination treatment of 41 1 mg/kg of COMPOUND A plus metformin further results in statistically significant improved inhibition of tumor growth at 19% as compared to the metformin monotherapy (p < 0 01 when analyzed with ANOVA wrth post-hoc Dunnett^'s multiple comparison test) and non-statιstιca!iy significant improvement over the COMPOUND A monotherapy The combination treatment of 68 5 mgAg of COMPOUND A plus metformin results in improved inhibition of tumor growth at 31% T/C as compared to Control The combination treatment of 68 5 mg/kg of COMPOUND A plus metformin further results in statistically significant improved inhibition of tumor growth at 31% as compared to the metformin monotherapy (p < 0 01 when analyzed with ANOVA wrth post-hoc Dunnett's multiple comparison test) and no improvement over the COMPOUND A monotherapy

The combination treatment of 32 7 mg/kg of COMPOUND C and metformin did not improve inhibition of tumor growth at 59% T/C as compared to the corresponding COMPOUND C monotherapy Combination therapy with 54.4 COMPOUND C and metformin was terminated early due to toxicity.

The combination of COMPOUND A plus metformin inhibits the growth of human NSCLC cell lines It is demonstrated that the combination treatment of COMPOUND A plus metformin improved inhibition of growth of human NSCLC cell lines as compared to Control, the metformin monotherapy and the COMPOUND A monotherapy However, metformin does not appear to enhance efficacy or tolerability in combination with COMPOUND C in this H460 xenograft model

Treatment stopped at Day 10 due to toxicity

T/C = 100 x (ΔT/ ΔC) = percent change between Day 1 and D 20 in the mean tumor volume of a treated group (ΔT) compared with change in Control (ΔC) 551 = Statisttcal Significance (Kruskal-Wailis and post-hoc Dunn's multiple comparison test) as compared to Control ne = not evaluabte, ns = not significant

552 - Statistical Stgnrficance (ANOVA wrth post-hoc Dunnett's multipte comparison test, excluding the group treated with Paclitaxel) as compared to Control ne = not evaluable, ns = not significant

Mean BW Change = lowest group mean body weight, as change from Day 1 up to Day 12, "--" indicates no decrease in mean body weight was observed

Claims

What is claimed is 1 A combination which comprises (a) a phosphoinosrtide 3-ktnase inhibitor compound inhibitor and (b) a insulin sensitivity enhancer compound, wherein the active ingredients are present in each case in free form or in the form of a pharmaceutically acceptable saft or any hydrate thereof and optionally at least one pharmaceutically acceptable earner for simultaneous, separate or sequential use

2 A pharmaceutical composition comprising a quantity which is jointly therapeutically effective against a prolrferative disease of a combination according to claim 1 and at least one pharmaceutically acceptable earner

3 A pharmaceutical composrtion according to claim 1 or 2 wherein the sensitivity enhancer compound is activator of AMP-activated protein kinase (AMPK)

4 A combination as defined in claim 1 or a pharmaceutical composition according to claim 2 or 3 for use in the treatment of a proliferative disease and/or overcoming the potential increase in blood glucose caused by inhibition of the PI3K/Akt pathway

5 Use of a combination as defined in claim 1 or a pharmaceutical composition according to claim 2 or 3 for the preparation of a medicament for the treatment of a proliferative disease and/or overcoming the potential increase in blood glucose caused by inhibition of the PI3K/Akt pathway

6 Use according to claims 4 or 5 wherein the proliferative disease is a solid tumor disease

7 Use according to claims 4 or 5 wherein the proliferative disease ts lung tumors carrying a loss-of-function mutation of LKB1 Use according to claims 4 or 5 wherein the proliferative disease is Breast Cancer, Ovarian Cancer Colon Cancer, Lung Cancer, Pancreas Cancer, Melanoma, Head and Neck, Brain Cancer, Endometrial Cancer, Cancers in patients wrth Peutz Jeghers Syndrome

Use according to claims 4 or 5 wherein the phosphoinosrtide 3-kιnase inhibitor compound is selected from 2-methyl-2-[4-(3-methyl-2-oxo-8-quιnolιn-3-yl-2,3- dιhydro-imidazo[4,5-c]quιnolιn-1-yl)-phenyl]-propιonftrιle and its monotosylate salt, 8-(6-methoxy-pyridιn-3-yl)-3-methyl-1-(4-pιperazιn-1-yl-3-trtfluoromethyl-phenyl)- 1 ,3-dιhydro-imιdazo[4,5-c]quιnolιn-2-one or 5-(2,6-dι-morpholιn-4-yl-pyrimιdιn-4- yl)-4-trffluoromethyl-pyndin-2-yiamine and its hydrochloride salt

Use according to any of claim 6 wherein the insulin sensitivity enhancer compound is a biguanide or glrtzone

A combined preparation, which comprises (a) one or more unrt dosage forms of phosphoinosite-3 kinase inhibitor and (b) one or more unit dosage forms of a biguanide or glitazone insulin sensitivity enhancer compound

A method of treating a patient suffering from a proliferative disease comprising administenng an effective amount of a phosphotnositide 3-kιnase inhibitor compound inhibitor and an insulin sensitivity enhancer compound, wherein the active ingredients are present in each case in free form or in the form of a pharmaceutically acceptable salt or any hydrate thereof and optionally at least one pharmaceutically acceptable carrier, for simultaneous, separate or sequential use

The method according to claim 12 wherein the proliferative disease is a solid tumor disease

The method according to claim 12 wherein the proliferative disease is lung tumors carrying a loss-of -function mutation of LKB1 The method according to claim 12 wherein the proliferative disease is Breast Cancer Ovarian Cancer, Colon Cancer, Lung Cancer, Pancreas Cancer, Melanoma, Head and Neck, Brain Cancer, Endometrial Cancer, Cancers in patients with Peutz Jeghers Syndrome

The method according to claim 12 wherein the patient is overcoming the potential increase in blood glucose caused by inhibition of the PI3K/Akt pathway . The method according to claim 12, wherein the phosphoinositide 3-ktnase inhibitor compound is selected from 2-methyl-2-(4-(3-methvi-2-oxo-8-quιnoltn-3-yl- 2,3-dιhydro-imidazo[4,5-c}quinoltn-1-yl)-pheπyi]-propionrtrιle and its monotosylate salt, 8-(6-methoxy-pyndin-3-yl)-3-methyl-1-(4-piperazin-1-yi-3-trrfluoromethyl- phenyt)-1 ,3-dihydro-imιdazo[4,5-c)quιnolin-2-one or 5-(2,6-dι-morpholιn-4-yl- pyrimidιn-4-yl)-4-trifluofomethyl-pyridιn-2-ylamιne and rts hydrochloride salt