WO2016012982A1 - Traitement combiné pour le traitement de cancer du sein résistant - Google Patents

Traitement combiné pour le traitement de cancer du sein résistant Download PDF

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WO2016012982A1
WO2016012982A1 PCT/IB2015/055616 IB2015055616W WO2016012982A1 WO 2016012982 A1 WO2016012982 A1 WO 2016012982A1 IB 2015055616 W IB2015055616 W IB 2015055616W WO 2016012982 A1 WO2016012982 A1 WO 2016012982A1
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estrogen
formula
pharmaceutically acceptable
breast cancer
compound
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PCT/IB2015/055616
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English (en)
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Veena R Agarwal
Todd W MILLER
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Piramal Enterprises Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
    • C07J31/006—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring not covered by C07J31/003

Definitions

  • the present invention relates to a pharmaceutical combination comprising a PI3K/mTOR inhibitor of Formula (I) (as described herein) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agent selected from an estrogen antagonist and a cyclin dependent kinase inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof.
  • the present invention also relates to the use of the said combination in the treatment of anti-estrogen resistant endocrine receptor positive (ER+) breast cancer.
  • the invention also relates to a method of treating anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of a PI3K/mTOR inhibitor of Formula (I) (as described herein) or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of an anti-cancer agent selected from an estrogen antagonist, and a cyclin dependent kinase inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof; or a mixture thereof.
  • a PI3K/mTOR inhibitor of Formula (I) as described herein
  • an anti-cancer agent selected from an estrogen antagonist, and a cyclin dependent kinase inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof; or a mixture thereof.
  • Breast cancer based on the immunohistochemistry (IHC) expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) can be classified into four groups; namely ER/PR+, Her2+ or triple positive; ER/PR+, Her2- or endocrine receptor positive; ER/PR-, Her2+ or Her2 positive; and ER/PR-, Her2- or triple negative.
  • IHC immunohistochemistry
  • Breast cancer is the most common type of cancer essentially affecting female population and endocrine receptor positive (ER+) breast cancer is the most common subtype.
  • the steroid hormones, particularly estrogen is responsible for the growth and development of this subtype of breast cancer.
  • the estrogen circulating in the bloodstream binds to the estrogen receptors (ER) residing inside the cells of the target tissues such as breast and uterus, thereby stimulating cell division and tumor growth of the target tissue.
  • estrogen antagonist therapy also referred to as anti- estrogen therapy.
  • the anti-estrogen therapy is primarily based on targeting ER signaling pathway and acts by: (i) reducing estrogen levels; (ii) blocking estrogen production; (iii) blocking estrogen receptors with anti-estrogens; or (iv) downregulating estrogen receptors.
  • GnRH Gonadotropin-releasing hormone
  • goserelin act by reducing estrogen levels.
  • GnRH agonists downregulate pituitary GnRH receptors, thereby suppressing the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), thus reducing estrogen production.
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • aromatase inhibitors such as exemestane (irreversible inhibitor), letrozole or anastrozole (reversible inhibitors) inhibit the activity of aromatase enzyme, thereby blocking the production of estrogens.
  • the aromatase inhibitors are primarily used in postmenopausal women.
  • Estrogen production can also be blocked by removal of ovaries, termed as oophorectomy.
  • Anti-estrogen drugs such as tamoxifen act by blocking estrogen receptors. These agents competitively block the ER thereby inhibiting estrogen-dependent growth of the breast tumor.
  • Fulvestrant acts as estrogen receptor antagonists by downregulating ER. Fulvestrant is known as pure anti-estrogen because, on binding with ER it induces a conformational change leading to ER degradation thus achieving complete inhibition of estrogen signaling through the estrogen receptor.
  • Fulvestrant has no estrogen agonist activity and also does not exhibit cross- resistance with other estrogen antagonists, such as tamoxifen (William J. Gradishar, Community Oncology, 2007, 4, 220-232). It has been approved in many countries as a second-line treatment for postmenopausal women having hormone sensitive advanced breast cancer after progression or relapse on anti-estrogen therapy (John Robertson, The Oncologist, 2007, 12, 774-784). However, resistance to fulvestrant has given rise to limitation in its use in the treatment of breast cancer. Rui Huang et al. (PLOS one, 2014, 9(4), e94226) have reported a role of signal transducer and activator of transcription (STAT) signaling in endocrine resistance. STAT3 and STAT5 are known to have involvement in tumorigenicity, cell- cycle progression, cell survival, transformation and angiogenesis. They seemingly have pronounced role in the oncogenesis of breast cancer and in resistance to endocrine therapy.
  • STAT signal transducer and activator of transcription
  • Angelo Di Lo et al. (Journal of Clinical Oncology, 2012, 30, 16, 1897-1900), describe an early in-vivo study, wherein it was demonstrated that fulvestrant alone can be effective in low-estrogen environment.
  • a xenograft model of MCF-7 was developed by injecting the tumor cells in the mouse and the tumors were allowed to develop in an estrogenic environment which was provided by a subcutaneous estrogen pellet. After tumor development, estrogen pellet was removed and endocrine treatment was initiated. In this model, fulvestrant showed more potent antitumor effects compared to tamoxifen or estrogen deprivation.
  • TGI Tumor growth inhibition
  • the present invention relates to a pharmaceutical combination comprising a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agents selected from an estrogen antagonist and a cyclin dependent kinase inhibitor of Formula (I I) or a pharmaceutically acceptable salt or a solvate thereof.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agents selected from an estrogen antagonist and a cyclin dependent kinase inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof and at least one pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical combination of the present invention is provided for use in the treatment of anti-estrogen resistant endocrine receptor positive (ER+) breast cancer.
  • ER+ endocrine receptor positive
  • the present invention relates to a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof in combination with a therapeutically effective amount of one or more anti-cancer agents selected from an estrogen antagonist and a cyclin dependent kinase inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof.
  • the present invention relates to the use of a pharmaceutical combination comprising a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agents selected from estrogen antagonists and a cyclin dependent kinase inhibitor of Formula (II) in the manufacture of a medicament for the treatment of anti-estrogen resistant ER+ breast cancer.
  • a pharmaceutical combination comprising a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agents selected from estrogen antagonists and a cyclin dependent kinase inhibitor of Formula (II) in the manufacture of a medicament for the treatment of anti-estrogen resistant ER+ breast cancer.
  • the present invention relates to a method for inhibiting growth of anti-estrogen resistant ER+ breast cancer cells, comprising contacting the said cells with an effective amount of a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; in combination with an effective amount of one or more anti-cancer agents selected from an estrogen antagonist and a cyclin dependent kinase inhibitor of Formula (I I) or a pharmaceutically acceptable salt or a solvate thereof.
  • the present invention relates to a pharmaceutical kit comprising: (a) a pharmaceutical combination comprising a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agents selected from an estrogen antagonist and a cyclin dependent kinase inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof; and (b) optionally a package insert comprising instructions for using the said pharmaceutical combination.
  • FIG. 1 a is a western blot image showing mTORCI and mTORC2 inhibitory activity of Compound A (a representative example of the PI3K/mTOR inhibitor of Formula (I)) at different concentrations in anti-estrogen-sensitive and anti-estrogen-resistant endocrine receptor positive (ER+) breast cancer cells.
  • Compound A a representative example of the PI3K/mTOR inhibitor of Formula (I)
  • FIG. 1 b is a western blot image showing activin receptor-like kinase (ALK1 ) inhibitory activity of Compound A in anti-estrogen-sensitive endocrine receptor positive (ER+) breast cancer cells.
  • ALK1 activin receptor-like kinase
  • FIG. 2 is a western blot image showing cyclin dependent kinase (CDK) inhibitory activity of Compound B (a representative example of the CDK inhibitor of Formula (II)) at different concentrations in anti-estrogen-sensitive and anti-estrogen-resistant ER+ breast cancer cells.
  • CDK cyclin dependent kinase
  • FIG. 3a is a graphical representation showing decrease in proliferation and viability of ER+ breast cancer cells when treated with the Compound A.
  • FIG. 3b is a graphical representation showing decrease in proliferation and viability of ER+ breast cancer cells when treated with the Compound B.
  • FIG. 4 is a histogram showing the effects of the Compound A, the Compound B and Fulvestrant when used alone and in combination in anti-estrogen-sensitive and anti- estrogen-resistant ER+ breast cancer cells.
  • FIG. 5a and FIG. 5b are the graphical representations of tumor growth profile in anti- estrogen-sensitive ER+ breast cancer (MCF-7) xenograft model after administration of the Compound A, the Compound B and fulvestrant, each alone and in combination.
  • MCF-7 anti- estrogen-sensitive ER+ breast cancer
  • FIG. 6 is a western blot image showing mTORCI , mTORC2 and PI3K inhibitory activity of Compound A and fulvestrant alone and in combination at different concentrations in anti-estrogen-sensitive (MCF-7) endocrine receptor positive (ER+) breast cancer cells.
  • FIG. 7 is a graphical representation of tumor growth profile in anti-estrogen-resistant ER+ breast cancer (T47D-fulvestrant resistant) xenograft model after administration of fulvestrant alone and in combination with the Compound A.
  • FIG. 8 is western blot image showing mTORCI , mTORC2 and PI3K inhibitory activity of the Compound A in combination with fulvestrant in anti-estrogen-resistant (T47D-fulvestrant resistant) endocrine receptor positive (ER+) breast cancer cells.
  • FIG. 9a is an image showing Ki67 scores of the Compound A and fulvestrant in combination compared with vehicle and fulvestrant in anti-estrogen-resistant ER+ breast cancer (T47D-fulvestrant resistant) xenograft model.
  • FIG. 9b depicts photomicrographs of anti-estrogen-resistant (T47D-fulvestarnt resistant) tumors after treatment with vehicle+fulvestrant and the compound A+ fulvestrant; using hematoxylin-eosin (H&E) staining (i), immunohistochemically stained with antibodies against Ki67 (ii), and terminal deoxynucleotidyl transferase- deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) analysis (iii).
  • H&E hematoxylin-eosin
  • dUTP terminal deoxynucleotidyl transferase- deoxyuridine triphosphate
  • TUNEL nick-end labeling
  • FIG. 10 is western blot image showing effects of the compound A on PI3K and mTOR signaling in human breast tumors ex vivo.
  • substitution or “substituted by” or “substituted with” includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, as well as represents a stable compound, which does not readily undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • halo or halogen as used herein refers to an atom selected from fluorine, chlorine, bromine and iodine.
  • (Ci-C 4 )alkyl refers to the radical of saturated aliphatic groups, including straight or branched- chain containing from 1 to 4 carbon atoms.
  • alkyl groups include but are not limited to methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, ferf-butyl, and the like.
  • salt(s) as part of a term, includes reference to the term singly or in plurality, for example the term pharmaceutically acceptable salt(s) indicates a single salt or more than one salt of the PI3K/mTOR inhibitor of Formula (I).
  • solvate refers to a compound formed by the interaction of a solute (in respect of the present invention, PI3K/mTOR inhibitor of Formula (I) or CDK inhibitor Formula (II) or a salt thereof) and a solvent.
  • solvents for the purpose of the invention may not interfere with the biological activity of the solute.
  • suitable solvents include, but are not limited to, water, methanol, ethanol and acetic acid.
  • the solvent used is a pharmaceutically acceptable solvent.
  • suitable pharmaceutically acceptable solvents include, without limitation, water, ethanol and acetic acid. Most preferably the solvent used is water.
  • suitable solvates are the mono- or dihydrates or alcoholates of the compounds according to the invention.
  • pharmaceutically acceptable salt(s) refers to inorganic and organic salts of the PI3K/mTOR inhibitor of Formula (I) and CDK inhibitor of Formula (II) contained in the pharmaceutical combination or composition of the invention.
  • terapéuticaally effective amount refers to the amount of the PI3K/mTOR inhibitor of Formula (I) and that of the anti-cancer agent which is used in combination with the PI3K/mTOR inhibitor of Formula (I), when administered to a subject in need thereof, sufficient to: (i) prevent or delay the progression of anti-estrogen resistant ER+ breast cancer; (ii) prevent or delay the progression of hormone-sensitive breast cancer to hormone- resistant breast cancer; or (iii) treat the anti-estrogen resistant ER+ breast cancer.
  • treat and “treatment” as used herein refers to one or more of : (i) inhibition of anti-estrogen resistant ER+ breast cancer i.e., arresting the development of anti-estrogen resistant ER+ breast cancer; (ii) reduction in the regression of anti- estrogen resistant ER+ breast cancer or slowing down of the anti-estrogen resistant ER+ breast cancer; (iii) amelioration of anti-estrogen resistant ER+ breast cancer i.e., reducing the severity of the symptoms associated with the anti-estrogen resistant ER+ breast cancer; (iv) relief, to some extent, of one or more symptoms associated with the anti-estrogen resistant ER+ breast cancer; (v) achieving a stabilized state of the disease; and (vi) prolonging survival of the subject as compared to expected survival.
  • PI3K/mTOR inhibitor of Formula (I) refers to PI3K and/or mTOR inhibitor of Formula (I) or pharmaceutically acceptable salts or solvates thereof that inhibits expression or activity of phosphatidyl inositol 3 kinase (PI3K) or mammalian target of rapamycin kinase (mTOR) or of both.
  • PI3K phosphatidyl inositol 3 kinase
  • mTOR mammalian target of rapamycin kinase
  • anti-cancer agent refers to a compound that is capable of suppressing the functions of a cancer cell.
  • the term includes compounds that inhibit cell growth, cell proliferation and/or cell differentiation or cause cell death.
  • an anti-cancer agent is selectively toxic against certain cancer cells but is non-toxic or less toxic to the normal cells (non-cancerous cells).
  • the anti-cancer agents are the compounds selected from an estrogen antagonist and a cyclin dependent kinase inhibitor of Formula (I I) or a pharmaceutically acceptable salt or a solvate thereof
  • CDK inhibitor of Formula (II) refers to the compounds of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof, as disclosed herein which inhibits activity of cyclin dependent kinases (CDK).
  • estrogen antagonist(s) or "anti-estrogen(s)” as used herein refers to the compounds that antagonize the effect of estrogen at the estrogen receptor level.
  • pure anti-estrogen(s) refer to the compounds that bind the estrogen receptors and are devoid of any estrogen agonist effect.
  • the term includes a drug such as fulvestrant.
  • hormone-sensitive breast cancer refers to the ER+ breast cancers that depend on the hormones such as estrogen or progesterone for their growth and are sensitive to hormone therapy or endocrine therapy.
  • hormone-resistant state of breast cancer or “hormone-resistant breast cancer” as used herein refers to the ER+ breast cancers that are resistant to hormone therapy or endocrine therapy.
  • subject refers to an animal, preferably a mammal, and most preferably a human.
  • mammal refers to warmblooded vertebrate animals of the class 'mammalia', including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young.
  • mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.
  • a subject in need thereof means a subject (patient) in need for the treatment of anti-estrogen resistant ER+ breast cancer.
  • a subject in need thereof means a subject (patient) diagnosed having anti-estrogen resistant ER+ breast cancer.
  • synergistic or “synergistic effect” or “synergism” as used herein refers to the therapeutic effect of the combination of the compounds (e.g. PI3K/mTOR inhibitor, estrogen antagonists and/or the CDK inhibitor) which is greater than the additive effect of the said compounds used in combination.
  • the synergistic effect can be attained by administering the said compounds simultaneously through a unit dosage form or as separate formulations administered simultaneously or sequentially.
  • the present invention relates to a pharmaceutical combination comprising PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agents (as described herein).
  • PI3K/mTOR inhibitors of Formula (I) used in the pharmaceutical combination as per the present invention are imidazo[4,5-c]quinoline derivatives, which are disclosed in PCT Application Publication No. WO2012007926.
  • the PI3K/mTOR inhibitor contained and/or used in the pharmaceutical combination or composition of the present invention is selected from the compounds of Formula (I)
  • Xi is C, CH or N
  • R is pyridyl, wherein pyridyl is unsubstituted or substituted with one or more of Rb;
  • R a and R b at each occurrence are independently selected from the group consisting of halogen, -CN, -0-(Ci-C 4 )alkyl, -NR c R d and (Ci-C 4 )alkyl, wherein (d- C 4 )alkyl is unsubstituted or substituted with one or more of halogen or -CN; and R c and R d are hydrogen or (CrC 4 )alkyl; and
  • n is 0 or an integer from 1 to 5;
  • the PI3K/mTOR inhibitor is a compound of Formula (I), wherein X- ⁇ is N and R a at each occurrence is independently selected from the group consisting of CI, Br, F, -CN, -OCH 3 , CH 3 , CF 3 or -C(CH 3 ) 2 CN.
  • the PI3K/mTOR inhibitor is a compound of Formula (I), wherein R b is -NR c R d or (CrC 4 )alkyl, wherein (CrC 4 )alkyl is unsubstituted or substituted with one or more of halogen; and R c and R d are hydrogen or (C C 4 )alkyl.
  • the PI3K/mT0R inhibitor is a compound of Formula (I), wherein R is 3-pyridyl, which is unsubstituted or substituted with one or more of Rb and Rb at each occurrence is independently selected from -NR c R d and (CrC 4 )alkyl, wherein (CrC 4 )alkyl is unsubstituted or substituted with one or more halogens and R c and R d are hydrogen or (CrC 4 )alkyl.
  • R is 3-pyridyl, which is unsubstituted or substituted with one or more of Rb and Rb at each occurrence is independently selected from -NR c R d and (CrC 4 )alkyl, wherein (CrC 4 )alkyl is unsubstituted or substituted with one or more halogens and R c and R d are hydrogen or (CrC 4 )alkyl.
  • the PI3K/mTOR inhibitor is a compound of Formula (I), wherein R is pyridyl, which is unsubstituted or substituted with one or more of Rb and R b at each occurrence is independently selected from the group consisting of -NH 2 , - NH(Ci-C 4 )alkyl , -N(Ci-C 4 -alkyl) 2 and methyl, wherein methyl is unsubstituted or substituted with one to three halogen atoms.
  • R is pyridyl, which is unsubstituted or substituted with one or more of Rb and R b at each occurrence is independently selected from the group consisting of -NH 2 , - NH(Ci-C 4 )alkyl , -N(Ci-C 4 -alkyl) 2 and methyl, wherein methyl is unsubstituted or substituted with one to three halogen atoms.
  • the PI3K/mTOR inhibitor is a compound of Formula (I), wherein R is pyridyl, which is unsubstituted or substituted with one or more of Rb and R b at each occurrence is independently selected from the group consisting of - NH 2 , -NH-CH3, -N(CH 3 ) 2 and -CF 3 .
  • the PI3K/mTOR inhibitor is a compound of Formula (I), wherein R is 3-pyridyl, which is unsubstituted or substituted with one or more of Rb and R b at each occurrence is independently selected from the group consisting of - NH 2 , -NH-CH3, -N(CH 3 ) 2 and -CF 3 .
  • the PI3K/mTOR inhibitor is a compound of Formula (I), wherein n is 1 or 2.
  • the PI3K/mTOR inhibitor is the compound of Formula (I) selected from:
  • the PI3K/mTOR inhibitor of Formula (I) is N-(8-(6- amino-5-(trifluoromethyl)pyridin-3-yl)-1 -(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl- 1 H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamide, herein after referred to as Compound A (also referred to as panulisib).
  • the anti-cancer agents contained and/or used in the combination or composition according to the present invention are selected from cyclin dependent kinase (CDK) inhibitors or estrogen antagonists.
  • CDK cyclin dependent kinase
  • the anti-cancer agent is a cyclin dependent kinase (CDK) inhibitor.
  • CDK cyclin dependent kinase
  • the CDK inhibitor is a compound of Formula (II)
  • X is chlorine, bromine, fluorine or iodine
  • Ri is hydrogen, (Ci-C 4 )alkyl or trifluoromethyl
  • the CDK inhibitor is a compound of Formula (II), wherein X is chlorine and Ri is hydrogen, (Ci-C 4 )alkyl or trifluoromethyl; or a pharmaceutically acceptable salt or a solvate thereof.
  • the CDK inhibitor is a compound of Formula (II), wherein X is chlorine and Ri is hydrogen; or a pharmaceutically acceptable salt or a solvate thereof.
  • the CDK inhibitor is a compound of Formula (I I), wherein X is chlorine and is trifluoromethyl ; or a pharmaceutically acceptable salt or a solvate thereof.
  • the CDK inhibitor is a compound of Formula (I I), wherein X is chlorine and Ri is 4-trifluoromethyl; or a pharmaceutically acceptable salt or a solvate thereof.
  • the CDK inhibitor is a compound of Formula (I I) selected from:
  • the CDK inhibitor is (+)-trans-2-(2-Chloro-4- trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-3-yl)- chromen-4-one hydrochloride, herein after referred to as Compound B (also referred to as voruciclib).
  • the anti-cancer agent is an estrogen antagonist.
  • the anti-cancer agent is a pure anti-estrogen.
  • the estrogen antagonist is selected from RU 58668 (Sanofi), EM-652 (Endorecherche) or fulvestrant (AstraZeneca).
  • the estrogen antagonist is fulvestrant.
  • Fulvestrant can be synthesized by methods described in US 4,659,516 or can be obtained through commercial sources.
  • Fulvestrant (FASLODEX®, AstraZeneca, CAS Reg. No. 129453-61 -8) is used for the treatment of postmenopausal women with hormone receptor-positive metastatic breast cancer after progression or relapse on antiestrogen therapy (The Oncologist, 2007, 1 2, 774-784). It downregulates estrogen and progesterone receptors and degrades the estrogen receptor. It is a pure anti-estrogen and has no known agonist activity. Fulvestrant is a compound having the following structure (British Journal of Cancer, 2004, 90 (Suppl 1 ), S2 - S6).
  • the pharmaceutical combination comprises a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof and a CDK inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof.
  • the pharmaceutical combination comprises a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or solvate thereof and an estrogen antagonist.
  • the pharmaceutical combination comprises a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof, a CDK inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof and an estrogen antagonist.
  • CDK inhibitors of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof and an estrogen antagonist selected from CDK inhibitors of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof and an estrogen antagonist.
  • the pharmaceutical combination comprises Compound A and a CDK inhibitor of Formula (II); or a pharmaceutically acceptable salt or a solvate thereof.
  • the CDK inhibitor of Formula (II) is Compound B.
  • the pharmaceutical combination comprises Compound A or pharmaceutically acceptable salts or solvates thereof; and an estrogen antagonist.
  • the estrogen antagonist is a pure anti-estrogen.
  • the estrogen antagonist is fulvestrant.
  • the pharmaceutical combination comprises Compound A or a pharmaceutically acceptable salt or a solvate thereof; and fulvestrant.
  • the pharmaceutical combination comprises Compound A or a pharmaceutically acceptable salt or a solvate thereof; Compound B and fulvestrant.
  • the pharmaceutical combination as described above in one or more embodiments of the present invention are provided for use in the treatment of anti-estrogen resistant ER+ breast cancer.
  • PI3K/mTOR inhibitors of Formula (I) can be prepared by synthetic routes as described in PCT Application Publication No. WO2012007926, which is incorporated herein by reference.
  • the compounds may also be prepared by synthetic routes that include similar reaction steps or methods known in the art.
  • CDK inhibitors of Formula (II) can be prepared by synthetic routes as described in US Application Publication No. US20070015802, which is incorporated herein by reference.
  • PI3K/mTOR inhibitors of Formula (I) and CDK inhibitors of Formula (II) can be converted into their pharmaceutically acceptable salts by following procedures known to persons skilled in the art.
  • PI3K/mTOR inhibitors of Formula (I) and CDK inhibitors of Formula (II) which contain acidic groups may be converted into salts with pharmaceutically acceptable bases.
  • Such salts include, for example, alkali metal salts, like lithium, sodium and potassium salts; alkaline earth metal salts like calcium and magnesium salts, ammonium salts, for example, [tris(hydroxymethyl)aminomethane], trimethylamine salts and diethylamine salts; salts with amino acids such as lysine, arginine, guanidine and the like.
  • PI3K/mTOR inhibitors of Formula (I) and CDK inhibitors of Formula (II), which contain one or more basic groups, i.e. groups which can be protonated, can form an addition salt with an inorganic or organic acid.
  • suitable acid addition salts include: acetates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, cinnamates, citrates, ethanesulfonates, fumarates, glucuronates, glutamates, glycolates, hydrochlorides, hydrobromides, hydrofluorides, ketoglutarates, lactates, maleates, malonates, m ethanesulfonates, nitrates, oxalates, palmoates, perchlorates, phosphates, picrates, salicylates, succinates, sulfamate, sulfates, tartrates, toluene
  • PI3K/mTOR inhibitors of Formula (I) and CDK inhibitors of Formula (II) can also exist as hydrates or solvates.
  • PI3K/mTOR inhibitors of Formula (I) and CDK inhibitors of Formula (II) contained and/or used in the combination of the present invention can be used in their isotopically labeled forms, wherein one or more atoms of the compounds are replaced with their respective isotopes. All isotopes of any particular atom or element as specified are contemplated within the scope of the compounds disclosed in the invention.
  • isotopes of the atoms that can be incorporated into the compounds disclosed herein include, but are not limited to, isotopes of hydrogen such as 2 H and 3 H, carbon such as 11 C, 13 C and 14 C, nitrogen such as 13 N and 15 N, oxygen such as 15 0, 17 0 and 18 0, chlorine such as 36 CI, fluorine such as 18 F and sulfur such as 35 S.
  • isotopes of hydrogen such as 2 H and 3 H
  • carbon such as 11 C, 13 C and 14 C
  • nitrogen such as 13 N and 15 N
  • oxygen such as 15 0, 17 0 and 18 0, chlorine such as 36 CI
  • fluorine such as 18 F and sulfur such as 35 S.
  • Substitution with heavier isotopes for example, replacing one or more key carbon-hydrogen bonds with carbon-deuterium bond may show certain therapeutic advantages, resulting from longer metabolism cycles, (e.g., increased in- vivo half life or reduced dosage requirements), improved safety or greater effectiveness and hence may be preferred in certain circumstances.
  • the (+)-frans-enantiomers of the CDK inhibitor of Formula (II) can be obtained by methods disclosed in PCT Application Publication Nos. WO2004004632, WO2007148158 and WO2008007169 incorporated herein by reference or the enantiomers of the CDK inhibitor of Formula (II) can also be obtained by methods well known in the art, such as chiral HPLC and enzymatic resolution. Alternatively, the enantiomers can be synthesized by using optically active starting materials.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or solvate thereof; and a therapeutically effective amount of one or more anti-cancer agents selected from an estrogen antagonist and a CDK inhibitor of Formula (II) or a pharmaceutically acceptable salt or solvate thereof.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or solvate thereof; and a therapeutically effective amount of a CDK inhibitor of Formula (II); or a pharmaceutically acceptable salt or a solvate thereof.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof and a therapeutically effective amount of an estrogen antagonist.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; a therapeutically effective amount of a CDK inhibitor of Formula (II); or a pharmaceutically acceptable salt or a solvate thereof and a therapeutically effective amount of an estrogen antagonist.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of the Compound A or a pharmaceutically acceptable salt or a solvate thereof and a therapeutically effective amount of a CDK inhibitor of Formula (II); or a pharmaceutically acceptable salt or a solvate thereof.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of the Compound A or a pharmaceutically acceptable salt or a solvate thereof and a therapeutically effective amount of the Compound B.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of the Compound A or a pharmaceutically acceptable salt or a solvate thereof; and a therapeutically effective amount of an anti-estrogen antagonist.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of the Compound A or a pharmaceutically acceptable salt or a solvate thereof; and a therapeutically effective amount of fulvestrant.
  • a method for the treatment of anti-estrogen resistant ER+ breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of the Compound A or a pharmaceutically acceptable salt or solvate thereof; a therapeutically effective amount of the Compound B and a therapeutically effective amount of fulvestrant.
  • a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or solvate thereof and a therapeutically effective amount of one or more anti-cancer agents selected from an estrogen antagonist and a CDK inhibitor of Formula (II) or a pharmaceutically acceptable salt or solvate thereof for use in the treatment of anti- estrogen resistant ER+ breast cancer.
  • a pharmaceutical combination for use in the treatment of anti-estrogen resistant ER+ breast cancer wherein the anticancer agent is a pure anti-estrogen.
  • a pharmaceutical combination of the Compound A or a pharmaceutically acceptable salt or a solvate thereof and fulvestrant for use in the treatment of anti-estrogen resistant ER+ breast cancer is provided.
  • the present invention provides methods for the synergistic treatment of anti- estrogen resistant ER+ breast cancer.
  • the anti-estrogen resistant ER+ breast cancer is fulvestrant resistant breast cancer.
  • a pharmaceutical combination as described hereinbefore for use in the prevention of the progression of hormone-sensitive breast cancer to hormone-resistant state of the breast cancer.
  • a pharmaceutical combination as described hereinbefore for use in the delay of the progression of hormone-sensitive breast cancer to hormone-resistant state of the breast cancer.
  • a pharmaceutical combination as described hereinbefore for use in the treatment of anti-estrogen resistant ER+ breast cancer synergistically.
  • a method for inhibiting growth of anti-estrogen resistant ER+ breast cancer cells comprising contacting the cells with an effective amount of a pharmaceutical combination comprising a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agents selected from an estrogen antagonist and CDK inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof.
  • a method for inhibiting growth of anti-estrogen resistant ER+ breast cancer cells comprising contacting the cells with an effective amount of a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and an effective amount of one or more anti-cancer agents selected from an estrogen antagonist and a CDK inhibitor of Formula (II) or a pharmaceutically acceptable salt or a solvate thereof; wherein the anti-estrogen resistant ER+ breast cancer cells are MCF-7/fulvestrant resistant breast cancer cells or T47D/fulvestrant resistant breast cancer cells.
  • a method for inhibiting growth of anti-estrogen resistant ER+ breast cancer cells comprising contacting the cells with an effective amount of the Compound A or a pharmaceutically acceptable salt or a solvate thereof; and an effective amount of fulvestrant; wherein anti-estrogen resistant ER+ breast cancer cells are MCF- 7/fulvestrant resistant breast cancer cells or T47D/fulvestrant resistant breast cancer cells.
  • a method for inhibiting growth of anti-estrogen resistant ER+ breast cancer cells comprising contacting the cells with an effective amount of the Compound A or a pharmaceutically acceptable salt or solvate thereof and an effective amount of the Compound B; wherein anti-estrogen resistant ER+ breast cancer cells are MCF- 7/fulvestrant resistant breast cancer cells or T47D/fulvestrant resistant breast cancer cells.
  • the PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof and an anti-cancer agent (as described herein) contained and/or used in the pharmaceutical combination according to the present invention can be administered simultaneously or sequentially or spaced out over a period of time.
  • the PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof and the anti-cancer agent (as described herein) contained and/or used in the pharmaceutical combination according to the present invention can be administered simultaneously.
  • the compound A or a pharmaceutically acceptable salt, a solvate thereof and the anti-cancer agent (as described herein) contained and/or used in the pharmaceutical combination according to the present invention can be administered simultaneously.
  • the compound A or a pharmaceutically acceptable salt, or a solvate thereof; and CDK inhibitor of Formula (II) contained and/or used in the pharmaceutical combination according to the present invention can be administered simultaneously.
  • the compound A or a pharmaceutically acceptable salt or a solvate thereof and the Compound B contained and/or used in the pharmaceutical combination according to the present invention can be administered simultaneously.
  • the compound A or a pharmaceutically acceptable salt, or a solvate thereof and fulvestrant contained and/or used in the pharmaceutical combination can be administered simultaneously.
  • PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof and one or more anti-cancer agents (as described herein) contained and/or used in the pharmaceutical combination can be administered in a sequential manner.
  • Sequential administration means administration of one component of the combination before the administration of the other component of the combination such that the combination shows a synergistic effect. Accordingly, the PI3K/mTOR inhibitor of Formula (I) can be administered prior to or after the administration of the anti-cancer agent.
  • sequential administration of the PI3K/mTOR inhibitor of Formula (I) and the anti-cancer agents involves administration of the said compounds spaced out over a period of time i.e. after administration of one component of the combination; the other component is administered after a certain fixed period.
  • the compound A or a pharmaceutically acceptable salt, or a solvate thereof and the Compound B contained and/or used in the pharmaceutical combination or composition according to the present invention can be administered sequentially.
  • the compound A or a pharmaceutically acceptable salt or a solvate thereof and fulvestrant contained and/or used in the pharmaceutical combination can be administered sequentially.
  • administration of the PI3K/mTOR inhibitor of Formula (I) and/or anti-cancer agents can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal or intrarectal.
  • the present invention provides a pharmaceutical kit comprising PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof and one or more anti-cancer agents (as described herein).
  • the pharmaceutical kit can comprise two or more separate containers for the PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof; and one or more anticancer agents (as described herein).
  • the kit may further comprise a package insert, including information about the indication, usage, doses, direction for administration, contraindications, precautions and warnings.
  • the suitable container that can be used includes a bottle, a vial, an ampoule, a syringe or a blister pack.
  • the pharmaceutical kit may optionally comprise a further container comprising a pharmaceutically acceptable buffer, water for injection, phosphate-buffered saline, Ringer's solution and dextrose solution.
  • the pharmaceutical kit can comprise two or more separate containers for the compound A or a pharmaceutically acceptable salt or a solvate thereof; and one or more anti-cancer agents (as described herein).
  • the pharmaceutical kit can comprise two or more separate containers for the compound A or a pharmaceutically acceptable salt or a solvate thereof; and a CDK inhibitor of Formula (II).
  • the pharmaceutical kit can comprise two or more separate containers for the compound A or a pharmaceutically acceptable salt or a solvate thereof; and the compound B.
  • the pharmaceutical kit can comprise two or more separate containers for the compound A or a pharmaceutically acceptable salt or a solvate thereof; and a pure anti-estrogen.
  • the pharmaceutical kit can comprise two or more separate containers for the compound A or a pharmaceutically acceptable salt or a solvate thereof; and fulvestrant.
  • the pharmaceutical composition of the present invention comprises a PI3K/mTOR inhibitor of Formula (I) or pharmaceutically acceptable salts or solvates thereof; one or more anti-cancer agents selected from an estrogen antagonist and CDK inhibitor of Formula (II) and at least one pharmaceutically acceptable carrier or excipient.
  • the pharmaceutically active excipients include, but not limited to, lactose, corn starch or derivatives thereof, gum arabica, magnesia or glucose, etc.
  • the carriers that can be used include, but not limited to, fats, waxes, natural or hardened oils, etc.
  • Suitable carriers for the production of solutions are, for example injection solutions, or for emulsions or syrups are, for example, water, physiological sodium chloride solution or alcohols, for example, ethanol, propanol or glycerol, sugar solutions, such as glucose solutions or mannitol solutions, or a mixture of the various solvents which have been mentioned.
  • PI3K/mTOR inhibitor of Formula (I) and anti-cancer agents are formulated into pharmaceutical dosage forms using conventional pharmaceutical techniques familiar to one skilled in the art such as by means of conventional blending, granulating, dissolving or lyophilizing.
  • the pharmaceutical composition of the present invention includes suitable carriers, diluents or excipients such as, for example, filling agents, binding agents, buffering agents, lubricating agents, antioxidants, dispersants, disintegrants, emulsifiers, defoamers, flavors, preservatives, surfactants, wetting agents, stabilizing agents, solubilizers, coating agents or colorants.
  • suitable carriers diluents or excipients such as, for example, filling agents, binding agents, buffering agents, lubricating agents, antioxidants, dispersants, disintegrants, emulsifiers, defoamers, flavors, preservatives, surfactants, wetting agents, stabilizing agents, solubilizers, coating agents or colorants.
  • the pharmaceutical composition may be packaged in a suitable container depending upon the formulation and the method of administration of the composition.
  • suitable containers known to a person skilled in the art include bottles, vials, ampoules, infusion bag and blister pack.
  • the pharmaceutical composition can be administered orally, for example in the form of pills, tablets, coated tablets, lozenges, capsules, dispersible powders or granules, suspensions, emulsions, syrups or elixirs. Administration, however, can also be carried out rectally, for example in the form of suppositories, or parenterally, for example intravenously, intramuscularly or subcutaneously, in the form of injectable sterile solutions or suspensions, or topically, for example in the form of solutions or ointments or transdermal ⁇ , for example in the form of transdermal patches, or in other ways, for example in the form of aerosols, nasal sprays or nasal drops.
  • the effective doses of the therapeutic agents for administration will vary depending on the age, sex, body weight and sensitivity difference of the patient, the mode, time, interval and duration of administration, the nature, formulation and type of the preparation, etc.
  • the therapeutic agents are administered in a time frame where one or more of the therapeutic agents are still active.
  • One skilled in the art would be able to determine such a time frame by determining the half life of the administered therapeutic agents.
  • the therapeutic agents contained and/or used in the pharmaceutical combination can be administered simultaneously or sequentially. Those skilled in the art will recognize that several variations are possible within the scope and spirit of this invention.
  • the dose of the therapeutic agents (the PI3K/mTOR inhibitors of Formula (I) and the anti-cancer agent (as described herein)) to be administered daily is to be selected to produce the desired effect.
  • a suitable dosage is about 0.01 to 100 mg/kg of the PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof, for example, about 0.01 to 50 mg/kg of the PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof, about 0.01 to 20 mg/kg of a PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof.
  • the anti-cancer agent (as described herein) can administered at a dose from about 5 mg/day to about 500 mg/day. If required, higher or lower daily doses can also be administered.
  • Actual dosage levels of the active ingredients contained in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient.
  • the therapeutically effective amount of the compound A or a pharmaceutically acceptable salt or a solvate thereof can be about 5 mg/kg, about 10 mg/kg or about 20 mg/kg.
  • the therapeutically effective amount of the compound A or a pharmaceutically acceptable salt or a solvate thereof is about 5 mg/kg.
  • the therapeutically effective amount of compound A or a pharmaceutically acceptable salt or a solvate thereof is about 10 mg/kg.
  • the therapeutically effective amount of compound A or a pharmaceutically acceptable salt or a solvate thereof is about 20 mg/kg.
  • the carrier, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof and one or more anti-cancer agents (as described herein) contained and/or used in the pharmaceutical combination have been evaluated in certain assay systems, and in several different administration schedules in vitro. The experimental details are as provided herein below. The data presented herein clearly indicate that the anti-cancer agent when combined with a compound Formula (I) exhibits synergistic effect.
  • the representative PI3K/mTOR inhibitor of Formula (I), the compound A used in the pharmacological assays refers to N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)- 1 -(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl-1 H-imidazo[4,5-c]quinolin-2(3H)- ylidene)cyanamide.
  • Activity of the PI3K/mTOR inhibitor of Formula (I) or a pharmaceutically acceptable salt or a solvate thereof in combination with one or more anti-cancer agents (as described herein) (the pharmaceutical combination according to the present invention) can be determined according to any effective in vitro or in vivo method.
  • MCF-7/fulvestrant-resistant and T47D/fulvestrant-resistant cells were provided as gifts by Matthew Ellis (Washington University). Parental and fulvestrant-resistant cells were maintained in DMEM and 10% FBS (Hyclone) in the absence or presence of 1 ⁇ fulvestrant (Tocris Bioscience and Abmole Bioscience), respectively, (iii) Long-term estrogen-deprived (LTED) cells were cultured as described in, The Journal of Clinical Investigation, 2010,120, 2406-13 and were maintained in hormone-depleted medium [phenol red-free DMEM with 10% dextran/charcoal- treated FBS (DCC-FBS; Hyclone)].
  • Anti-estrogen sensitive cells (MCF-7, T47-D, HCC-1428, ZR75-1 and BT474), MCF-7/fulvestrant-resistant cells, T47D/ fulvestrant-resistant cells and LTED cells were treated with Compound A (0 to 400 nM) for 16-24 hours.
  • the cell were then lysed in RIPA buffer [50 mM Tris pH 7.4, 150 mM NaCI, 1 % NP-40, 0.5% deoxycholic acid, 0.1 % SDS, 1 mM EDTA, 1 mM EGTA, 5 mM NaPPi, 50 mM NaF, 10 mM ⁇ -glycerophosphate, 1 mM Na 3 V0 4 (New England Biolabs), protease inhibitor cocktail (Pierce)] on ice. Lysates were sonicated for 10 seconds and centrifuged at 18,000 x g for 10 min. Protein concentrations of supernatants were determined by Bicinchoninic acid (BCA) assay (Pierce).
  • BCA Bicinchoninic acid
  • the cell lysate were also analyzed by immunoblot using antibodies for P-
  • Antibody binding was detected using HRP-conjugated secondary antibodies against mouse or rabbit Ig (GE Healthcare), and ECL substrate (Pierce).
  • FIG. 1 a depicts the mTORCI and mTORC2 kinase inhibitory activity of Compound A.
  • FIG. 1 b depicts the ALK-1 inhibitory activity of Compound A.
  • Compound A inhibits mTORCI , mTORC2 kinase, and ALK1 activity in anti-estrogen sensitive and resistant ER positive breast cancer cells.
  • Anti-estrogen sensitive cells (MCF-7, T47-D, HCC-1428, ZR75-1 and BT474), MCF-7/fulvestrant-resistant cells, T47D/ fulvestrant-resistant cells and LTED cells were treated with Compound B (0 to 5 ⁇ ) for 16 to 24 hours.
  • the cells were then lysed in RIPA buffer [50 mM Tris pH 7.4, 150 mM NaCI, 1 % NP-40, 0.5% deoxycholic acid, 0.1 % SDS, 1 mM EDTA, 1 mM EGTA, 5 mM NaPPi, 50 mM NaF, 10 mM ⁇ -glycerophosphate, 1 mM Na 3 V0 4 (New England Biolabs), protease inhibitor cocktail (Pierce)] on ice. Lysates were sonicated for 10 seconds and centrifuged at 18,000 x g for 10 min. Protein concentrations of supernatants were determined by Bicinchoninic acid (BCA) assay (Pierce).
  • BCA Bicinchoninic acid
  • FIG. 2 depicts the CDK inhibitory activity of Compound B.
  • Compound B inhibits CDK activity in anti-estrogen sensitive and resistant ER positive breast cancer cells.
  • Anti-estrogen sensitive cells (MCF-7, T47-D, HCC-1428, ZR75-1 and BT474), MCF-7/fulvestrant-resistant cells, T47D/ fulvestrant-resistant cells and LTED cells were plated in triplicate in respective growth media at 5x10 3 cells/well in 96-well plates. The next day, cells were treated with Compound A (0 to 30 nM) or Compound B (0 to 5 ⁇ ). Five to eight days later, relative quantities of adherent cells were determined by sulforhodamine B (SRB) assay (as described in Nature Protocols 2006, 1 , 1 1 12-1 1 16). Relative cell numbers were used to calculate inhibitory effect.
  • SRB sulforhodamine B
  • Compound A and Compound B decrease the proliferation of anti- estrogen sensitive and resistant ER positive breast cancer cells.
  • Anti-estrogen sensitive cells MCF-7
  • MCF-7/fulvestrant-resistant cells T47D/ fulvestrant-resistant cells and MCF-7/LTED cells were seeded in triplicate in 6-well plates at 0.6-1 x10 6 cells/well and were treated with fulvestrant (1 ⁇ ), Compound A (200 nM) and Compound B (5 ⁇ ), alone and in combination for 3 days.
  • Floating and adherent cells were processed using ApoScreen Annexin Apoptosis kit (Southern Biotech), then analyzed by flow cytometry. Cells staining positively for Annexin-V and/or propidium iodide were considered apoptotic.
  • FIG. 4 shows the % apoptosis in anti-estrogen sensitive and resistant ER positive breast cancer cells when treated with Compound A and Compound B alone and in combination with fulvestrant.
  • mice Female NOD-scid I L2RY _/" (NSG; NOD.Cg-Prkdcscid N2rgtm 1 Wjl/SzJ) mice, 5 to 6 weeks old, (obtained from Norris Cotton Cancer Center Transgenics & Genetic Constructs Shared Resource). Animals were housed in animal isolator (Harlan Inc.) under specified pathogen-free conditions maintained at 22 to 25 °C and 55 to 75% humidity, with a 12- hour light/1 2- hour dark cycle. The mice were acclimatized for a period of at least 7 days before experimentation. Animals were handled in a laminar flow hood. Ail food and water was autoclaved. Mice had access to pelleted rodent diet and water ad libitum.
  • Compound A was suspended in (0.5%) methyl cellulose and dosed at 5 or 1 5 mg/kg/d in 100 ⁇ _.
  • Fulvestrant was procured as clinical formulation from AstraZeneca or dissolved in ethanol (50 mg/mL), then diluted 1 0-folds with castor oil to obtain the formulation of 5 mg/mL.
  • Treatment was initiated when tumor size volume of the MCF-7 tumors was about 200mm 3 .
  • Group 6 Tumor-bearing mice administered with a combination of Compound A
  • Tumors were harvested 3 days of treatment or at the end of the study (4 to 6 weeks) and cut in pieces for snap-freezing or formalin fixation followed by paraffin- embedding (FFPE).
  • FFPE paraffin- embedding
  • Tumor cells of mice from treatment groups 1 to 4 were isolated and then lysed in RIPA buffer [50 mM Tris pH 7.4, 150 mM NaCI, 1 % NP-40, 0.5% deoxycholic acid, 0.1 % SDS, 1 mM EDTA, 1 mM EGTA, 5 mM NaPPi, 50 mM NaF, 10 mM ⁇ - glycerophosphate, 1 mM Na 3 V0 4 (New England Biolabs), protease inhibitor cocktail (Pierce)] on ice. Lysates were sonicated for 10 seconds and centrifuged at 18,000 x g for 10 min. Protein concentrations of supernatants were determined by BCA assay (Pierce).
  • FIG. 5a shows tumor growth inhibition results of Compound A alone and in combination with fulvestrant in anti-estrogen-sensitive ER+ breast cancer (MCF- 7) xenograft model.
  • FIG. 5b shows tumor growth inhibition results of Compound B alone and in combination with Compound A in anti-estrogen-sensitive ER+ breast cancer (MCF-7) xenograft model.
  • FIG. 6 shows mTORCI , mTORC2 and PI3K inhibitory activity of Compound A and fulvestrant in ER+ breast cancer (MCF-7) xenograft model.
  • Compound A alone, and in combination with fulvestrant shows significant tumor growth inhibition in anti-estrogen-sensitive ER+ breast cancer (MCF-7) xenograft model.
  • Treatment was initiated when tumor size volume of the T47D-fulvestrant resistant tumors were about 200 mm 3 .
  • Tumors were harvested after 3 days of treatment for immunoblotting, or at the end of 6 weeks for immunohistochemistry. Tumors were cut in pieces for snap-freezing or formalin fixation followed by paraffin-embedding (FFPE). Immunoblotting:
  • Tumor cells of mice from treatment groups 1 and 2 were isolated and then lysed in RIPA buffer [50 mM Tris pH 7.4, 150 mM NaCI, 1 % NP-40, 0.5% deoxycholic acid, 0.1 % SDS, 1 mM EDTA, 1 mM EGTA, 5 mM NaPPi, 50 mM NaF, 10 mM ⁇ -glycerophosphate, 1 mM Na 3 V0 4 (New England Biolabs), protease inhibitor cocktail (Pierce)] on ice. Lysates were sonicated for 10 seconds and centrifuged at 18,000 x g for 10 min. Protein concentrations of supernatants were determined by BCA assay (Pierce).
  • Samples were reduced and denatured by addition of 1 .25% ⁇ - mercaptoethanol in NuPage sample buffer (Invitrogen). Samples were heated for 1 min. at 95 °C before SDS-PAGE. Proteins were transferred to nitrocellulose membranes, which were blocked with 5% BSA/TBS-T and probed using antibodies against P-AKT T 308, P-AKT S4 73, P-S6 and Actin. Antibody binding was detected using HRP-conjugated secondary antibodies against mouse or rabbit Ig (GE Healthcare), and ECL substrate (Pierce).
  • FFPE tumor tissue Five-micron sections of FFPE tumor tissue were used for H&E staining, IHC with Ki67 antibody (Biocare Medical), and TUNEL (Promega). For Ki67 IHC and TUNEL, 4-5 high-power (400x magnification) microscopic fields were used to count the numbers of positively-stained and total cells. Percentages of positively stained cells/field were used to calculate a single score for each tumor.
  • FIG. 7 Compound A and fulvestrant in combination shows significant tumor growth inhibition in anti-estrogen-resistant ER+ breast cancer (T47D- fulvestrant resistant) xenograft model.
  • FIG. 8 shows that Compound A and fulvestrant in combination inhibits mTORCI , mTORC2 and PI3K activity in anti-estrogen-resistant ER+ breast cancer (T47D- fulvestrant resistant) xenograft model.
  • FIG. 9a and FIG. 9b shows significant decrease in Ki67 scores in the treatment group 2 compared with the treatment group 1 .
  • FIG. 9b shows significant increase in TUNEL positivity in treatment group 2
  • Compound A and fulvestrant in combination shows significant tumor growth inhibition in anti-estrogen-resistant ER+ breast cancer (T47D- fulvestrant resistant) xenograft model.
  • Compound A in combination with fulvestrant suppresses T47D-fulvestarnt resistant tumor growth by inhibiting proliferation, and increasing apoptosis.
  • FIG. 10 depicts mTORCI , mTORC2 and PI3 kinase inhibitory activity of the Compound A.
  • Compound A inhibits mTORCI , mTORC2 and PI3 kinase activity in ER+/HER2- human breast tumors.

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Abstract

La présente invention concerne une combinaison pharmaceutique comprenant un inhibiteur de PI3K/mTOR de Formule (I) (telle que décrite ici) ou un sel ou un solvate pharmaceutiquement acceptable de celui-ci ; et un ou plusieurs agents anticancéreux choisis parmi un antagoniste d'œstrogène et un inhibiteur de kinase dépendant des cyclines de Formule (II) ou un sel ou un solvat pharmaceutiquement acceptable de ceux-ci. La présente invention concerne également l'utilisation de ladite combinaison dans le traitement d'un cancer du sein positif pour les récepteurs endocriniens (ER+) résistant aux antiœstrogènes. L'invention concerne également un procédé de traitement d'un cancer du sein ER+ résistant aux antiœstrogènes, comprenant l'administration à un sujet le nécessitant d'une quantité thérapeutiquement efficace d'un inhibiteur de PI3K/mTOR de Formule (I) (telle que décrite ici) ou d'un sel pharmaceutiquement acceptable de celui-ci et une quantité thérapeutiquement efficace d'un agent anticancéreux choisi parmi un antagoniste d'œstrogène et un inhibiteur de kinase dépendante des cyclines de Formule (II) ou un sel ou un solvat pharmaceutiquement acceptable de ceux-ci ; ou un mélange de ceux-ci.
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