WO2016134486A1 - Utilisation de canagliflozine et de ses dérivés dans le traitement du cancer - Google Patents

Utilisation de canagliflozine et de ses dérivés dans le traitement du cancer Download PDF

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WO2016134486A1
WO2016134486A1 PCT/CA2016/050211 CA2016050211W WO2016134486A1 WO 2016134486 A1 WO2016134486 A1 WO 2016134486A1 CA 2016050211 W CA2016050211 W CA 2016050211W WO 2016134486 A1 WO2016134486 A1 WO 2016134486A1
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cancer
canagliflozin
alkyl
halo
substituted
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PCT/CA2016/050211
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Gregory STEINBERG
Theos TSAKIRIDIS
Linda VILLANI
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Steinberg Gregory
Tsakiridis Theos
Villani Linda
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present application relates to methods, uses, compositions and kits for treating cancer, including cancer prevention.
  • the present application relates to the use of Canagliflozin, optionally in combination with an adjunct cancer treatment, for the treatment of cancer.
  • Cancer is the leading cause of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer related deaths in 2012. 1 Deaths from cancer are projected to continue rising, with an estimated 13.1 million deaths in 2030. An age-related increase in cancer incidence exists since somatic mutations occur at a constant rate over time. The prospect of targeting cancerous cells between the time intervals in which these mutations appear provides an opportunity to prevent cancers from transitioning to successive stages of malignancy. 2 Early interventions have therefore been associated with improved patient outcomes. However, the apparent failure to control cancer related deaths highlights the need for discovering biological agents that will better protect against neoplastic events.
  • chemoprevention can be defined as the use of natural, synthetic or biological agents to protect against the progression of premalignant cells to invasive disease or the use of chemicals, bioactive plant compounds or dietary components to block or reverse cancer progression.
  • AMPK 5'-Adenosine monophosphate activated protein kinase
  • This heterotrimeric enzyme is composed of a catalytic a subunit and regulatory ⁇ and Y subunits.
  • 3,4 AMPK is activated >100-fold by phosphorylation of Thr172 in the a subunit by upstream kinases liver kinase B1 (LKB1 ) or the Ca 2+ - dependent kinase CaMKK.
  • AMPK suppresses protein synthesis through inhibition of the mammalian target of rapamycin (mTOR), a protein kinase that regulates cell growth, proliferation, protein synthesis, and has been implicated in malignant transformation and cancer.
  • mTORCI mammalian target of rapamycin complex 1
  • AMPK can target and block the mammalian target of rapamycin complex 1 (mTORCI ) complex through multiple mechanisms to conserve energy expenditure.
  • AMPK can inhibit mTOR through phosphorylation of the tuberous sclerosis complex (TSC) proteins (TSC1 :TSC2).
  • TSC1 :TSC2 have GTPase activity towards the small G-protein Rheb, which activates mTORCI when it is GTP-bound.
  • AMPK triggers TSC2 activity via direct phosphorylation on its Thr1227 and Ser1345 residues, which in turn inactivates Rheb by converting it to a GDP-bound confirmation. 12 AMPK also inhibits mTORCI activity through phosphorylation and inhibition of its binding partner Raptor. 13
  • AMPK In addition to inhibiting mTOR, AMPK also regulates cell survival and growth by phosphorylating a wide range of transcription factors, their co- activators, and histones. In response to metabolic stress AMPK can phosphorylate the transcription factor p53 on its Ser15 residue and cause G1 -S phase cell cycle arrest. 14 Alternatively, AMPK was also shown to phosphorylate the cyclin-dependent kinase inhibitor (CDKI) p27 on Thr198 to sequester it in the cytoplasm and promote survival in response to nutrient or growth-factor withdrawal. 15 Furthermore, AMPK can provoke transcriptional regulation of genes in response to bioenergenic strain through direct phosphorylation of histone H2B on Ser36.
  • CDKI cyclin-dependent kinase inhibitor
  • AMPK induces a metabolic switch that inhibits pathways that are important for cell growth and proliferation. 17
  • AMPK can target multiple signaling pathways by acute phosphorylation or alterations in gene transcription to attenuate stress and modulate cell survival; effects that may limit cancer cell proliferation and survival.
  • the homeostatic mechanisms regulating blood glucose involve an intricate balance between multiple organ systems.
  • the inhibition of sodium glucose transporter 2 (SGLT2) prevents reabsorption of glucose in the distal kidney; thus resulting in the excretion of glucose into the urine.
  • Phlorizin is a phenolic-glucoside that was first isolated from the bark of apple trees in 1835. Phlorizin is known to induce glucosuria in animals and humans as is it able to non-selectively inhibit the sodium-dependent glucose transporters (SGLT1 -2) at low nanomolar concentrations. 19 However, it is not orally bioavailable and causes gastrointestinal distress because it inhibits SGLT1 (the glucose transporter found mainly in the small intestine) as well as SGLT2 (which is expressed almost exclusively in the kidney). 18 These features unfortunately limit the compound's therapeutic use. 20
  • C-glucosides have been used as an alternative to ⁇ - glucosides as they are resistant to degradation by ⁇ -glucosidase enzymes. 21 Furthermore, they increase glucosuria and reduce hyperglycemia in an insulin- independent manner.
  • Two such drugs include Canagliflozin (trade name InvokanaTM) and Dapagliflozin (trade name ForxigaTM) which have recently received approval by the FDA, Health Canada and the European Commissions for the treatment of type 2 diabetes.
  • Canagliflozin and Dapagliflozin display similar half-maximal inhibitory concentrations (IC 50 ) to the SGLT2 transporter (approximately 1 -2 nM). They exhibit a 400 and 1200-fold selectively towards SGLT2 respectively, which to date, is their only described target.
  • Canagliflozin has been shown to lower blood sugar in patients with type 2 diabetes. Its primary mechanism of action is reported to involve the inhibition of the sodium glucose transporter 2 (SGLT2) protein which in turn blocks glucose resorption in the distal kidney resulting in increased glucose excretion. Studies have established that these drugs do not induce carcinogenesis or increase the risk of bladder cancer. 18 However, whether or not Canagliflozin inhibits cancer cell proliferation and cancer cell clonogenic survival has not previously been reported.
  • SGLT2 sodium glucose transporter 2
  • Type 2 diabetics on SGLT2 inhibitors have increased insulin- sensitivity, reduced body weight and increased lipid metabolism 22 , effects which are similar to other anti-diabetic agents such as the biguanide metformin.
  • Metformin has been shown to inhibit cancer cell growth and survival 24 and is currently the subject of over 600 cancer clinical trials.
  • AMPK Acetyl-CoA carboxylase
  • Cytotoxic therapy with chemotherapy or radiotherapy is used to treat inoperable cancers including metastatic disease or to prevent tumor recurrence. Cytotoxic therapy combinations are frequently used for tumors showing resistance to chemotherapy or radiation, which develops secondary to activation of cell survival mechanisms and due to genomic instability that leads to development of more aggressive tumor cell sub-populations. 25 Single treatments often will not provide identical response in tumour cells.
  • combination therapies will employ drugs that are effective as single agents and have different mechanisms of action.
  • Docetaxel an inhibitor of microtubule disassembly
  • CRPC castrate- resistant prostate cancer
  • the prognosis for CRPC remains poor. It is designated as a spectrum disorder where patients can present as asymptomatic or have incurred significant metastases and morbidities while on androgen deprivation therapies.
  • few second-line chemotherapies have been shown to improve survival beyond docetaxel treatments. It is therefore desirable to discover novel treatments that can potentiate the effects of current therapies with minimal toxicity.
  • cisplatin a drug that produces cross-links within DNA
  • NSCLC stage IB-IV non-small cell lung cancer
  • Radiation is also a key cancer treatment.
  • it is used as a curative therapy for unresected lung and prostate cancers both of these diseases show high degree of radio-resistance, leading to poor outcomes and increased toxicity due to dose escalation.
  • it is desirable to develop radiation sensitizers to enhance cancer killing.
  • Canagliflozin is a medication that lowers blood sugar in type 2 diabetics. It is a sodium glucose transporter 2 (SGLT2) inhibitor; a medication that lowers blood sugar in type 2 diabetes by increasing glucose excretion from the kidney. It is disclosed herein that clinical concentrations of Canagliflozin (10 ⁇ ) activate AMP-activated protein kinase (AMPK) and inhibits the clonogenic survival and proliferation of lung (A549, H1299), prostate (22RV1 , PC3), colon (MC38, HCT1 16), liver (HepG2), breast (MCF7) and ovarian (SKOV-3) cancer cells.
  • AMPK AMP-activated protein kinase
  • the present application includes a method for the treatment or prevention of cancer progression in a subject, said method comprising administering to the subject an effective amount of Canagliflozin, or an active analog thereof.
  • the Canagliflozin, or the active analog thereof is administered in combination with an adjunct cancer treatment.
  • the present application also includes a use of Canagliflozin, or an active analog thereof, for treating cancer in a subject in need thereof.
  • the Canagliflozin, or the active analog thereof is for use in combination with an adjunct cancer treatment.
  • the present application also includes a method of treating cancer comprising administering an effective amount of Canagliflozin, or an active analog thereof, in combination with an adjunct cancer treatment to a subject in need thereof.
  • the present application further includes a use of Canagliflozin, or an active analog thereof, in combination with an adjunct cancer treatment for treating cancer in a subject.
  • the present application also includes a method of improving the efficacy of an adjunct cancer treatment comprising administering an effective amount of Canagliflozin, or an active analog thereof, in combination with the adjunct cancer treatment to a subject in need thereof.
  • the present application further includes a use of Canagliflozin, or an active analog thereof, for improving the efficacy of an adjunct cancer treatment.
  • the present application also includes a pharmaceutical composition comprising Canagliflozin, or an active analog thereof, in combination with one or more other anti-cancer agents.
  • the present application also includes a kit for the treatment of cancer, the kit comprising:
  • the present application also includes a kit for improving the efficacy of an anti-cancer agent for the treatment of cancer, the kit comprising:
  • the adjunct cancer treatment is one or more other anti-cancer agents and/or radiation therapy.
  • the other anti-cancer agent is a biguanide derivative, such as Metformin and/or Phenformin.
  • the other anticancer agent is a salsalate/salicylate derivative.
  • the other anti-cancer agent is selected from Metformin, Phenformin, Doxorubicin, Docetaxel, Duanorubicin, Epirubicin, Paclitaxel, Cyclophosphamide, Methotrexate, Cisplatin, 5-Fluorouracil, Etoposide, carboplatin, Gemcitabine, Vinorelbine and combinations thereof.
  • the other anti-cancer agent is Cisplatin or Docetaxel.
  • the other anti-cancer agent is Metformin.
  • the other anti-cancer agent is a biological agent.
  • the cancer is selected from prostate cancer, pancreatic cancer, ovarian cancer, lung cancer, breast cancer, bladder cancer, colon cancer, brain cancer, head and neck cancer, endometrial cancer, leukemia, lymphoma and sarcoma.
  • the cancer is of the lung, prostate, colon, breast or ovary.
  • the present application also includes a method for treating or preventing cancer progression comprising administration to the subject a combination of therapeutically effective amounts of Canagliflozin, or an active analog thereof, and an additional anti-neoplastic agent.
  • the present application also includes a method for treating or preventing cancer progression comprising administration to the subject therapeutically effective amounts of Canagliflozin or an active analog thereof, in combination with an anti-neoplastic agent or radiation.
  • the active analog of Canagliflozin is a compound of the Formula I:
  • R A is selected from halo and Ci -4 alkyl; and Ring C is phenyl unsubstituted or substituted with 1 to 3 substituents selected from halo, cyano, Ci -4 alkyl, halo-substituted Ci -4 alkyl, OCi -4 alkyl, halo-substituted OC-i- alkyl, methylenedioxy, ethyleneoxy, mono-Ci -4 alkylamino, di-Ci -4 alkylamino, carbamoyl, mono-Ci -4 alkylcarbamoyl and di-Ci -4 alkylcarbamoyl.
  • Figure 1 shows plots demonstrating the proliferation of cancers of the lung (A549 and H1299; Figures 1A and B, respectively) prostate (PC3 and 22RV-1 ; Figures 1 C and D, respectively) colon (HCT1 16; Figure 1 E), liver (HepG2; Figure 1 F), breast (MCF7; Figure 1 G) and ovarian (SKOV-3; Figure 1 H) cancer cells treated with Canagliflozin, or Dapagliflozin at the indicated concentrations relative to vehicle treated controls for 72 h.
  • Figure 2 shows the clonogenic survival of cancers of the lung (A549: Figure 2A, left panel; Figure 2B, top panel; and H1299: Figure 2A, right panel; Figure 2B, bottom panel), prostate (PC3: Figure 2C, left panel; Figure 2D, top panel; and 22RV-1 : Figure 2C, right panel, Figure 2D; bottom panel) and colon (MC38; Figure 2E) treated with Canagliflozin or Dapagliflozin at the indicated concentrations relative to the vehicle treated controls for 5-10 days.
  • Figure 3 shows that Canagliflozin rapidly induces the activation of AMPK in cancer cells.
  • Canagliflozin quickly increases the phosphorylation of AMPK and ACC while potently inhibiting mTORCI activity in PC3 and H1299 cells.
  • Canagliflozin (30 ⁇ ) increased the expression of phosphorylated AMPK and ACC within 0.5 hours in PC3 (A, B) and H1299 (C, D) cells, which was sustained up to 24 h over three independent experiments.
  • the phosphorylation of S6 kinase and its substrate, the ribosomal protein S6, decreased time dependently in PC3 and H1299 cells.
  • Dapagliflozin treatment did not influence the phosphorylation status of AMPK or ACC in these cells.
  • Figure 4 shows that Canagliflozin dose-dependently activates AMPK in cancer cells.
  • Canagliflozin also dose dependently increases the phosphorylation of AMPK and ACC in PC3 and H1299 cells at clinically effective concentrations.
  • Canagliflozin and Dapagliflozin significantly increased the expression of phosphorylated AMPK and ACC at doses >10 ⁇ and >100 ⁇ respectively in PC3 cells.
  • a similar response was observed in H1299 cells at doses >30 ⁇ and >100 ⁇ respectively.
  • FIG. 5 shows that Dapagliflozin is a weak AMPK activator. Dapagliflozin dose response is shown for PC3 (A) and H1299 (B) cells for the numeric data presented in Figures 4B and 4D.
  • Figure 6 shows the 14 C-acetate synthesis into PC3 cancer cells treated with indicated concentrations of vehicle or Canagliflozin for 2 hours.
  • Figure 7 shows Oxygen consumption of H1299 cancer cells treated with indicated concentrations of vehicle or Canagliflozin for 2 hours.
  • Figure 8 shows that Canagliflozin (30 ⁇ ) decreased complex-l supported respiration in PC3 and H1299 cancer cells (A). Percent inhibition of complex-l facilitated respiration in PC3 and H1299 cells is shown in (B). Cell proliferation as a function of Canagliflozin dose in the presence of 1 1 mM glucose, no galactose or in the presence of 10 mM galactose, no glucose is shown for PC3 (left panel) and H1299 (right panel) cells (C).
  • FIG. 9 shows that Canagliflozin inhibits the activity of Akt in PC3 cells.
  • Canagliflozin (30 ⁇ ) decreases the phosphorylation of Akt at residues Thr308 and Ser473 within 0.5 h in PC3 cells (A-B). Under these same conditions, the phosphorylation status of Akt in H1299 cells remained generally unchanged (C-D).
  • Figure 10 shows that Canagliflozin but not Dapagliflozin inhibits H 3 -2DG uptake in PC3 (A) and H1299 (B) cells treated at the indicated concentrations relative to the vehicle treated controls.
  • Figure 1 1 demonstrates graphically that Canagliflozin potentiates Docetaxel's anti-clonogenic (A) and anti-proliferative (B) effects in PC3 cells.
  • Figure 1 1 A shows clonogenic survival as a function of Canagliflozin dose for PC3 cells treated either with Canagliflozin or Canagliflozin and 0.5 nM Docetaxel.
  • Figure 1 1 B shows cell proliferation as a function of Canagliflozin dose for PC3 cells treated either with Canagliflozin or Canagliflozin and 2 nM Docetaxel.
  • Figure 12 shows graphically that Canagliflozin potentiates Cisplatin's anti-proliferative effects in H 1299 cells.
  • Figure 12A shows cell proliferation as a function of Canagliflozin dose for H1299 cells treated either with Canagliflozin or Canagliflozin and 5 ⁇ Cisplatin.
  • Figure 12B shows cell proliferation as a function of Cisplatin dose for H1299 cells treated either with Cisplatin or Cisplatin and 30 ⁇ Canagliflozin.
  • Figure 13 shows graphically that Canagliflozin potentiates the anti- clonogenic effects of radiation in PC3 cells. Clonogenic survival is shown as a function of Canagliflozin dose for PC3 cells treated with 0, 2 or 4 Gy radiation.
  • the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • the foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.
  • the term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • an anti-cancer agent should be understood to present certain aspects with one anti-cancer agent or two or more additional anti-cancer agents.
  • an additional or second component such as an additional or second anticancer agent
  • the second component as used herein is chemically different from the other components or first component.
  • a “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
  • the compounds described herein have at least one asymmetric center. Where compounds possess more than one asymmetric center, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (e.g. less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the application having alternate stereochemistry.
  • radiation therapy includes any form of ionizing radiation therapy for cancer, including external beam radiotherapy such as conventional fraction radiotherapy, hypofractionated radiotherapy (treatment delivered in a shorter period of time), 3D-conformal, intensity modulated and volumetric arc therapy radiation, stereotactic radiotherapy and radiosurgery; brachytherapy and injectable radio-active therapies.
  • external beam radiotherapy such as conventional fraction radiotherapy, hypofractionated radiotherapy (treatment delivered in a shorter period of time), 3D-conformal, intensity modulated and volumetric arc therapy radiation, stereotactic radiotherapy and radiosurgery; brachytherapy and injectable radio-active therapies.
  • subject includes all members of the animal kingdom including mammals, and suitably refers to humans.
  • composition refers to a composition of matter for pharmaceutical use.
  • an effective amount of the Canagliflozin, or the active analog thereof is an amount that, for example, reduces the cancer compared to the cancer without administration or use of the Canagliflozin, or the active analog thereof.
  • reducing the cancer can refer to reducing the tumor burden compared to the tumor burden without administration or use of the Canagliflozin, or the active analog thereof.
  • reducing the cancer also includes preventing tumor progression or reducing the rate of progression of cancer compared to tumor progression or rate of progression of cancer without administration or use of the Canagliflozin, or the active analog thereof.
  • Effective amounts may vary according to factors such as the disease state, age, sex, weight and/or species of the subject.
  • the amount of Canagliflozin, the active analog thereof, or another anti-cancer agent that will correspond to such an amount will also vary depending upon various factors, such as the given anti-cancer agent, the pharmaceutical formulation, the route of administration or use, the type of cancer being treated, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • to treat means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of a disease, and remission (whether partial or total, whether detectable or undetectable).
  • to treat mean prolonging survival as compared to expected survival if not receiving treatment.
  • to treat include prophylactic treatment.
  • a subject with early cancer is treated to prevent progression.
  • a subject in remission is treated to prevent recurrence.
  • Treatment methods comprise administering to a subject or use of a therapeutically effective amount of Canagliflozin, or the active analog thereof, and optionally consist of a single administration or use, or alternatively comprise a series of administrations or uses.
  • the Canagliflozin, or the active analog thereof is administered or used at least once a week.
  • the Canagliflozin, or the active analog thereof is administered to the subject or used from about one time per 2, 3 or 4 weeks, or less, or about one time per week to about once daily for a given treatment.
  • the Canagliflozin, or the active analog thereof is administered or used 2, 3, 4, 5 or 6 times daily.
  • the length of the treatment period depends on a variety of factors, such as the severity of the cancer, the age of the subject, the concentration and/or the activity of a Canagliflozin, or an active analog thereof formulation, and/or a combination thereof. It will also be appreciated that the effective dosage of the Canagliflozin, or the active analog thereof, used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage result and become apparent by standard diagnostic assays known in the art. In some embodiments, chronic administration or use is required. For example, in an embodiment, the Canagliflozin, or the active analog thereof, is administered to the subject or used in an amount and for duration sufficient to treat the subject.
  • "Palliating" a cancer means that the extent and/or undesirable clinical manifestations of a cancer are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the cancer.
  • prevention or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with cancer or manifesting a symptom associated with cancer.
  • administered means administration of a therapeutically effective amount of the Canagliflozin, or the active analog thereof, optionally in combination with another anti-cancer agent to a cell either in cell culture or in a subject.
  • Canagliflozin refers to a compound having the following structure:
  • Canagliflozin is available commercially. Its synthesis is described, for example, in U.S. Patent No. 7,943,788. A crystalline form of the hemihydrate of Canagliflozin is described in U.S. Patent No. 7,943,582 and is within the scope of the present application.
  • active analog of Canagliflozin refers to compounds having a structure that is analogous to Canagliflozin, with structural variations that do not substantially impact the compound's activity as an activator of AMPK and/or inhibitor of the proliferation of cancer cells. Whether an analog of Canagliflozin is an activator of AMPK and/or inhibitor of the proliferation of cancer cells can be determined by a person skilled in the art using standard assays, for example as described in the Examples herein.
  • Dapagliflozin refers to a compound having the following structure:
  • the selective sodium-dependent glucose transporter 2 (SGLT2) inhibitors are a new class of anti-diabetic medications that lower blood glucose. It was tested whether the SGLT2 inhibitors Canagliflozin and Dapagliflozin inhibit the growth and survival of cancer cells. It was found that Canagliflozin inhibited lung, prostate, colon, liver, breast and ovarian cancer cell proliferation at concentrations that can be clinically achieved (20-65 ⁇ ). Low dose treatment also inhibited colony formation of lung and prostate cancer cells (IC50s of 8-13 ⁇ ). These effects were potentiated when combined, for example, with gamma radiation or the cytotoxic drugs Cisplatin and Docetaxel.
  • Dapagliflozin was 5-10-fold less effective in all of these same assays. Reductions in growth and survival with Canagliflozin were accompanied by activation of the AMP-activated protein kinase (AMPK) and reductions in Akt and the mTORCI substrate S6 kinase. Consistent with the activation of AMPK, Canagliflozin reduced mitochondrial respiration through the inhibition of complex-l. These data suggest that in addition to being a medication for the treatment of diabetes, Canagliflozin is also useful in limiting, for example, the growth and survival of adenocarcinomas.
  • AMPK AMP-activated protein kinase
  • the present application includes a method of treating cancer comprising administering Canagliflozin, or an active analog thereof, to a subject in need thereof.
  • the present application also includes a use of Canagliflozin, or an active analog thereof, for treating cancer in a subject; a use of Canagliflozin, or an active analog thereof, for preparation of a medicament for treating cancer in a subject; and Canagliflozin, or an active analog thereof, for use to treat cancer in a subject.
  • the present application further includes a method for the treatment or prevention of cancer progression in a subject, said method comprising administering to the subject an effective amount of Canagliflozin, or an active analog thereof.
  • the present application also includes a use of Canagliflozin, or an active analog thereof, for treatment or prevention of cancer progression in a subject; a use of Canagliflozin, or an active analog thereof, for preparation of a medicament for treating or preventing cancer progression in a subject; and Canagliflozin, or an active analog thereof, for use to treat or prevent cancer progression in a subject.
  • the cancer is selected from the group consisting of prostate cancer, pancreatic cancer, ovarian cancer, lung cancer, breast cancer, bladder cancer, colon cancer, brain cancer, head and neck cancer, endometrial cancer, leukemia, lymphoma and sarcoma.
  • the cancer is of the lung, prostate, colon, liver, breast or ovary.
  • the cancer is of the lung, prostate, colon, breast or ovary. It is an embodiment that the cancer is of the lung, prostate or colon.
  • the subject is a human.
  • the Canagliflozin, or the active analog thereof is useful as an adjunct therapy with other cancer treatments such as radiation and/or other anti-cancer agents. Accordingly, the present application also includes a method of treating cancer comprising administering, to a subject in need thereof, Canagliflozin, or an active analog thereof, in combination with an adjunct cancer treatment.
  • the present application also includes a use of Canagliflozin, or an active analog thereof, in combination with an adjunct cancer treatment for treating cancer in a subject; a use of Canagliflozin, or an active analog thereof, in combination with one or more other anti-cancer agents for preparation of a medicament for treating cancer in a subject; and Canagliflozin, or an active analog thereof, in combination with an adjunct cancer treatment for use to treat cancer in a subject.
  • the adjunct cancer treatment is radiation therapy.
  • the adjunct cancer treatment is one or more other anti-cancer agents. It is an embodiment that the adjunct cancer treatment is a combination of radiation and one or more other anti-cancer agents.
  • the administration or use of Canagliflozin, or the active analog thereof, in combination with the adjunct cancer treatment enhances the effectiveness of the adjunct cancer treatment in the treatment of cancer.
  • the present application also includes a method of improving the efficacy of an adjunct cancer treatment for treating cancer comprising administering Canagliflozin, or an active analog thereof, in combination with the adjunct cancer treatment, to a subject in need thereof.
  • the present application also includes a use of Canagliflozin, or an active analog thereof, for improving the efficacy of an adjunct cancer treatment; a use of Canagliflozin, or an active analog thereof, for preparation of a medicament for improving the efficacy of an adjunct cancer treatment; and Canagliflozin, or an active analog thereof, for use to improve the efficacy of an adjunct cancer treatment.
  • the other anti-cancer agent can be any suitable other anticancer agent, the selection of which can be made by a person skilled in the art.
  • the anti-cancer agent is a drug targeting cancer mitochondrial metabolism, glucose uptake, isocitrate dehydrogenase or lipid metabolism.
  • the anti-cancer agent is a direct small molecule activator of AMPK.
  • the other anti-cancer agent is a biguanide derivative, such as Metformin and/or Phenformin.
  • the other anticancer agent is a salsalate/salicylate derivative.
  • the one or more other anti-cancer agents are selected from the group consisting of Phenformin, Metformin, Doxorubicin, Docetaxel, Duanorubicin, Epirubicin, Paclitaxel, Cyclophosphamide, Methotrexate, Cisplatin, 5-Fluorouracil, Etoposide, carboplatin, Gemcitabine, Vinorelbine and combinations thereof.
  • the other anti-cancer agent is Cisplatin or Docetaxel. It is an embodiment that the other anti-cancer agent is Cisplatin. In another embodiment of the present application, the other anti-cancer agent is Docetaxel.
  • the other anti-cancer agent is Metformin. It is an embodiment that the other anti-cancer agent is a chemotherapeutic agent used for the treatment of lung cancer such as Etoposide, carboplatin, Gemcitabine or Vinorelbine.
  • the other anti-cancer agent is a biological agent.
  • the biological agent can be any suitable biological agent, the selection of which can be made by a person skilled in the art.
  • the biological agent targets tumor growth mechanisms such as epidermal growth factor receptor (EGFR) and associated pathway-related, anaplastic lymphoma kinase (Alk)-related, K-Ras-related, p53-related therapy (for lung cancer), androgen receptor-related therapy (for prostate cancer) and immune-system modulating therapies (for all cancers).
  • EGFR epidermal growth factor receptor
  • Alk anaplastic lymphoma kinase
  • K-Ras-related therapy for lung cancer
  • p53-related therapy for prostate cancer
  • immune-system modulating therapies for all cancers.
  • the biological agent is selected from the group consisting of: monoclonal antibodies, cytokines, vaccines, oncolytic viruses and combinations thereof.
  • the active analog of Canagliflozin is a compound of the Formula I:
  • R A is selected from halo and Ci -4 alkyl; and Ring C is phenyl unsubstituted or substituted with 1 to 3 substituents selected from halo, cyano, Ci- alkyl, halo-substituted Ci- alkyl, OCi- alkyl, halo-substituted OC-
  • alkyl methylenedioxy, ethyleneoxy, mono-Ci -4 alkylamino, di-Ci -4 alkylamino, carbamoyl, mono-Ci -4 alkylcarbamoyl and di-Ci -4 alkylcarbamoyl.
  • Ring C is a phenyl group substituted with 1 -2 substituents selected from halo, cyano, Ci -4 alkyl, halo-substituted Ci -4 alkyl, OCi -4 alkyl, halo-substituted OCi -4 alkyl, mono-C-i -4 alkylamino and di-C-i- alkylamino.
  • Ring C is a phenyl group substituted with 1 substituent selected from F, CI, CH 3 , OCH 3 , cyano, CF 3 , OCF 3 and N(CH 3 ) 2 .
  • the substituent on Ring C is located at the para position.
  • Ring C is para-fluorophenyl.
  • R A is selected from F, CI and CH 3 . In a further embodiment, R A is CH 3 .
  • the methods and uses of the application comprise the use or administration of Canagliflozin, a hydrate, solvate and/or crystalline polymorph thereof, including a crystalline hemihydrate form of Canagliflozin.
  • the Canagliflozin, or the active analog thereof, and optionally the one or more other anti-cancer agents can be administered to a subject or used in a variety of forms depending on the selected route of administration or use, as will be understood by those skilled in the art.
  • the Canagliflozin, or the active analog thereof, and/or optionally the one or more other anti-cancer agents are administered to the subject, or used, by oral (including sublingual and buccal) or parenteral (including intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, topical, patch, pump and transdermal) administration or use and the Canagliflozin, or the active analog thereof, and/or optionally the other anti-cancer agent(s) formulated accordingly.
  • the Canagliflozin, the active analog thereof, and/or optionally the other anticancer agent(s) are administered or used by injection, in a spray, in a tab let/cap let, in a powder, topically, in a gel, in drops, by a patch, by an implant, by a slow release pump or by any other suitable method of administration or use, the selection of which can be made by a person skilled in the art.
  • the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are orally administered or used, for example, with an inert diluent or with an assimilable edible carrier, or enclosed in hard or soft shell gelatin capsules, or compressed into tablets, or incorporated directly with the food of the diet.
  • the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are incorporated with excipient and administered or used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Oral dosage forms also include modified release, for example immediate release and timed-release, formulations.
  • modified- release formulations include, for example, sustained-release (SR), extended- release (ER, XR, or XL), time-release or timed-release, controlled-release (CR), or continuous-release (CR or Contin), employed, for example, in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g., as of molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet.
  • SR sustained-release
  • ER extended- release
  • CR controlled-release
  • Contin continuous-release
  • timed-release compositions are formulated, e.g. liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc.
  • Liposome delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • liposomes are formed from a variety of phospholipids, such as cholesterol, stearylamine and/or phosphatidylcholines.
  • the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are freeze dried and the lyophilizates obtained, are used for example, for the preparation of products for injection.
  • Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are administered or used parenterally.
  • solutions of the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • dispersions of the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and administration or use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations.
  • the pharmaceutical form is suitable for injectable administration or use and includes sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists.
  • compositions for nasal administration or use are formulated as aerosols, drops, gels or powders.
  • the aerosol formulation comprises a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and is presented in single or multidose quantities in sterile form in a sealed container, which, for example, take the form of a cartridge or refill for use with an atomising device.
  • the sealed container is a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after administration or use.
  • the dosage form comprises an aerosol dispenser
  • a propellant which is, for example, a compressed gas, such as compressed air or an organic propellant such as a fluorochlorohydrocarbon.
  • the aerosol dosage forms take the form of a pump-atomizer.
  • the composition is suitable for buccal or sublingual administration or use such as in tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, gelatin and/or glycerine.
  • the composition is suitable for rectal administration or use such as in suppositories containing a conventional suppository base such as cocoa butter.
  • the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are coupled with soluble polymers as targetable drug carriers.
  • soluble polymers include, for example, polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide- phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
  • a drug for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
  • the Canagliflozin, or active analog thereof is administered or used alone or, as noted above, in combination with other known anti-cancer agents.
  • the Canagliflozin, or active analog thereof is administered or used contemporaneously with those anti-cancer agents.
  • "contemporaneous" administration or use of two substances to a subject means providing each of the two substances so that they are both biologically active in the individual at the same time.
  • the exact details of the administration or use will depend on the pharmacokinetics of the two substances in the presence of each other, and include, for example, administering or using the two substances at the same time, within a few hours of each other, or administering or using one substance within 24 hours of administration or use of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens is routine for one skilled in the art.
  • two substances will be administered or used substantially simultaneously, i.e., within minutes of each other, or in a single composition that contains both substances.
  • Canagliflozin, or active analog thereof, and the other anti-cancer agent(s) are administered to a subject or used in a non- contemporaneous fashion.
  • the dosage of the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration or use, the age, health and weight of the recipient, the nature and extent of the symptoms of the cancer, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the subject to be treated.
  • One of skill in the art can determine the appropriate dosage based on the above factors.
  • the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are administered or used initially in a suitable dosage that is optionally adjusted as required, depending on the clinical response.
  • oral dosages of the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents will range between about 1 mg per day to about 1000 mg per day for a human adult.
  • the pharmaceutical compositions are formulated for oral administration or use and the Canagliflozin, the active analog thereof, and/or optionally the one or more other anti-cancer agents are, for example in the form of tablets containing 0.25, 0.5, 0.75, 1 .0, 5.0, 10.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 75.0, 80.0, 90.0, 100.0, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg of active ingredient per tablet.
  • the Canagliflozin, the active analog thereof and/or optionally the one or more other anti-cancer agents are administered or used in a single daily dose.
  • the total daily dose is divided into two, three or four, or more, daily doses.
  • the dosage of the Canagliflozin, or active analog thereof is less than the dosage of the Canagliflozin, or active analog thereof, when administered or used alone.
  • the dosage of the other anti-cancer agent is less than the dosage of the other anti-cancer agent when administered or used alone.
  • the present application also includes a composition comprising Canagliflozin, or an active analog thereof, one or more other anti-cancer agents and optionally a carrier.
  • the Canagliflozin, or active analog thereof, and the one or more other anti-cancer agents are suitably formulated into pharmaceutical compositions for administration to subjects or use in a biologically compatible form suitable for administration or use in vivo. Accordingly, the present application further includes a pharmaceutical composition comprising Canagliflozin, or active analog thereof, one or more other anti-cancer agents and optionally a pharmaceutically acceptable carrier.
  • the one or more other anti-cancer agents are selected from the group consisting of Phenformin, Doxorubicin, Docetaxel, Duanorubicin, Epirubicin, Paclitaxel, Cyclophosphamide, Methotrexate, Cisplatin, 5-Fluorouracil, Etoposide, carboplatin, Gemcitabine, Vinorelbine and combinations thereof.
  • the other anti-cancer agent is Cisplatin or Docetaxel. It is an embodiment that the other anti-cancer agent is Cisplatin.
  • the other anticancer agent is Docetaxel. It is an embodiment that the other anti-cancer agent is selected from Etoposide, carboplatin, Gemcitabine and Vinorelbine.
  • the other anti-cancer agent is a biological agent.
  • the biological agent can be any suitable biological agent, the selection of which can be made by a person skilled in the art.
  • the biological agent targets tumor growth mechanisms such as epidermal growth factor receptor (EGFR) and associated pathway-related, anaplastic lymphoma kinase (Alk)-related, K-Ras-related, p53-related therapy (for lung cancer), androgen receptor-related therapy (for prostate cancer) and immune-system modulating therapies (for all cancers).
  • the biological agent is selected from the group consisting of: monoclonal antibodies, cytokines, vaccines, oncolytic viruses and combinations thereof.
  • the active analog of Canagliflozin is a compound of the Formula I:
  • R A is selected from halo and Ci -4 alkyl; and Ring C is phenyl unsubstituted or substituted with 1 to 3 substituents selected from halo, cyano, Ci -4 alkyl, halo-substituted Ci -4 alkyl, OCi -4 alkyl, halo-substituted OC-i- alkyl, methylenedioxy, ethyleneoxy, mono-Ci -4 alkylamino, di-Ci -4 alkylamino, carbamoyl, mono-Ci -4 alkylcarbamoyl and di-Ci -4 alkylcarbamoyl.
  • Ring C is a phenyl group substituted with 1 -2 substituents selected from halo, cyano, Ci- alkyl, halo-substituted Ci- alkyl, OCi -4 alkyl, halo-substituted OCi -4 alkyl, mono-Ci -4 alkylamino and di-C-i- alkylamino.
  • Ring C is a phenyl group substituted with 1 substituent selected from F, CI, CH 3 , OCH 3 , cyano, CF 3 , OCF 3 and N(CH 3 ) 2 .
  • the substituent on Ring C is located at the para position.
  • Ring C is para-fluorophenyl.
  • R A is selected from F, CI and CH 3 . In a further embodiment, R A is CH 3 .
  • compositions of the application comprise Canagliflozin, a hydrate, solvate and/or crystalline polymorph thereof, including a crystalline hemihydrate form of Canagliflozin.
  • the present application also includes a kit for the treatment of cancer, the kit comprising:
  • the present application also includes a kit for the treatment of cancer, the kit comprising:
  • the present application also includes a kit for improving the efficacy of an anti-cancer agent for the treatment of cancer, the kit comprising:
  • the present application also includes a kit for improving the efficacy of an anti-cancer agent for the treatment of cancer, the kit comprising:
  • kits of the present application can be varied as discussed herein for the methods and uses of the present application.
  • the active analog of Canagliflozin is a compound of the Formula I:
  • R A is selected from halo and Ci -4 alkyl; and Ring C is phenyl unsubstituted or substituted with 1 to 3 substituents selected from halo, cyano, Ci -4 alkyl, halo-substituted Ci -4 alkyl, OCi -4 alkyl, halo-substituted OC-i- alkyl, methylenedioxy, ethyleneoxy, mono-Ci -4 alkylamino, di-Ci -4 alkylamino, carbamoyl, mono-Ci -4 alkylcarbamoyl and di-Ci -4 alkylcarbamoyl.
  • Ring C is a phenyl group substituted with 1 -2 substituents selected from halo, cyano, Ci- alkyl, halo-substituted Ci- alkyl, OCi -4 alkyl, halo-substituted OCi -4 alkyl, mono-Ci -4 alkylamino and di-C-i- alkylamino.
  • Ring C is a phenyl group substituted with 1 substituent selected from F, CI, CH 3 , OCH 3 , cyano, CF 3 , OCF 3 and N(CH 3 ) 2 .
  • the substituent on Ring C is located at the para position.
  • Ring C is para-fluorophenyl.
  • R A is selected from F, CI and CH 3 . In a further embodiment, R A is CH 3 .
  • kits of the application comprise Canagliflozin, a hydrate, solvate and/or crystalline polymorph thereof, including a crystalline hemihydrate form of Canagliflozin.
  • the following non-limiting examples are illustrative of the present application:
  • Example 1 The SGLT2 inhibitor Canagliflozin activates AMPK and Inhibits the Growth and Survival of Cancer Cells
  • H1299, PC3, 22RV-1 , HCT1 16 and SKOV-3 cells were cultured in RPMI 1640 media (Gibco: Mississauga, ON) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1 % (v/v) antibiotic-antimycotic (100x) solution (Gibco: Mississauga, ON).
  • HepG2 and MCF-7 cells were cultured in MEM media (Gibco: Mississauga, ON) supplemented with 10% (v/v) FBS and 1 % (v/v) antibiotic-antimycotic (100x).
  • MCF-7 media was also supplemented with 1 % (v/v) non-essential amino acids (100X) solution (Gibco: Mississauga, ON) and 1 % sodium-pyruvate (100X) solution (Gibco: Mississauga, ON). All cells were maintained at 37°C in 5% C0 2 and were treated with the concentrations of Canagliflozin and Dapagliflozin (Selleck Chemicals LLC), Phenformin, Metformin, Galactose (Sigma: Toronto, ON), Cisplatin and Docetaxel (Cayman: Ann Arbor, Ml) indicated herein. Working solutions were prepared so that the vehicle comprised less than 0.1 % of the media.
  • Clonogenic Survival Assay A549, H1299, PC3 and 22RV-1 cells were seeded at a density of 500-1000 cells per well. The following day, cells were treated in triplicate with the indicated drug for 5-10 days. Cells were fixed with 0.5% crystal violet DNA stain (1 g crystal violet (Sigma: Toronto, ON) in 50% methanol) and viable colonies (> 50 cells) were counted. [00117] Proliferation Assay: A549, H1299, PC3, 22RV-1 , HepG2, HCT1 16, MCF-7 and SKOV-3 cells were seeded at a density of 1000-2000 cells per well into 96-well plates. The following day, cells were treated in quadruplicate with the indicated drug for 72 hours.
  • lysis buffer (1 M DTT, 200 mM Na 3 V0 4 , 20% triton-X, protease inhibitor cocktail tablet (Roche), 50 mM HEPES, 150 mM NaCI, 100 mM NaF, 10 mM Na pyrophosphate, 5 mM EDTA, 250 mM Sucrose). Cells were scraped and measured for radioactive counts.
  • Mitochondrial Respiration Assay Cells were treated in duplicate with the indicated treatment for 30-90 minutes. Cells were subsequently washed and collected in MiR05 buffer (1 10 mM sucrose, 60 mM potassium lactobionate, 20 mM HEPES, 10 mM KH 2 P0 4 , 3 mM MgCI 2 , 0.5 mM EGTA, 1 g/L BSA, pH 7.1 ) supplemented with an equivalent dose of the indicated treatment. Respiratory measurements were conducted using the Oxygraph- 2K OROBOROS® apparatus at 30 °C with stirring.
  • Cellular ATP Assay PC3 and H1299 cells were seeded at a density of 20,000 cells per well into white-walled 96-well plates. The following day, cells were treated in duplicate with the indicated treatment for 30 minutes.
  • the Abeam Luminescent ATP Detection Assay Kit (ab1 1 3849) was used to detect levels of cellular ATP according to the manufacturer's protocol.
  • Membranes were blocked (5% BSA solution in TBST (50 mM Tris, 150 mM NaCI, 1 M HCI, pH 7.4, 0.1 % Tween-20)) and incubated with the indicated primary and HRP-conjugated secondary antibodies. Densitometry values were quantified using Image J software and are expressed as percent of control.
  • Canagliflozin blocks the cellular proliferation and clonogenic survival of cancer cells.
  • Lung cancer cells appeared to be wholly resistant to the same concentrations of Dapagliflozin, while prostate, colon, breast and ovarian cancer cell proliferation was only significantly reduced at concentrations of 100 ⁇ . Additionally, the estimated IC50s for Dapagliflozin are well above the tolerable clinical exposure level. Liver cancer cells (HepG2) were the only cell line whose proliferation was inhibited by Dapagliflozin at lower doses (IC50 ⁇ 55 ⁇ ).
  • Activating phosphorylation of AMPK at T172 and the phosphorylation of its downstream substrate ACC at Ser 79/221 was measured following treatment of prostate, lung and colon cancer cells with 30 ⁇ Canagliflozin or Dapagliflozin for varying times. Within 30 minutes of treatment, Canagliflozin increased the phosphorylation of AMPK (Thr172) and ACC (Ser79) by 7 and 4-fold respectively in PC3 prostate cancer cells, a response that was sustained for up to 24 hours (Figure 3A-B). A smaller response was detected in the H1299 cells (2-fold increase, Figure 3C-D).
  • Canagliflozin also decreased the phosphorylation of S6K (Thr389) and its substrate S6 (Ser 240/244) by approximately 50% at the 0.5 and 1 hour time point respectively, in the PC3 cells. A similar response by S6 was not observed until the 24 hour time point in the H1299 cells. Dapagliflozin did not significantly alter the phosphorylation status of any of the markers measured in the PC3 or H1299 cells over time.
  • Canagliflozin inhibits cell growth and proliferation through AMPK regulation of protein and lipid synthesis.
  • AMPK may prevent cancer cell growth and proliferation due to the inhibition of protein synthesis and lipogenesis; effects mediated through mTOR and ACC respectively.
  • mTOR is a useful pathway controlling cancer cell growth and proliferation whose activity can be inferred through the phosphorylation of downstream effectors such as p70S6.
  • Canagliflozin (30 ⁇ ) totally eliminated the phosphorylation of p70S6 within 1 hr of treatment without altering total protein expression indicating the potent ability of this compound to supress mTOR activity ( Figure 3).
  • Dapagliflozin had no effect on the phosphorylation of p70S6 ( Figure 3).
  • Canagliflozin inhibits de novo lipogenesis in PC3 prostate cancer cells
  • Canagliflozin reduces oxygen consumption; SGLT2 inhibitors block mitochondrial respiration through the inhibition of complex-l.
  • AMPK can be activated by reductions in cellular energy charge, increases in cellular calcium or through a direct allosteric mechanism involving Ser108 of the AMPK betal subunit. Most xenobiotics and drugs that activate AMPK reduce mitochondrial oxidative phosphorylation leading to increases in cellular AMP/ADP therefore the effects of Canagliflozin on oxygen consumption were measured.
  • cancer cells which were cultured in growth media containing galactose, were subjected to a greater drop in cellular ATP with a 30 minute treatment of Canagliflozin, in comparison to cells cultured in growth media containing glucose (Table 4). This response was also observed when the cells were treated with the known complex-l inhibitor phenformin.
  • Canagliflozin inhibits Akt activity in PC3 prostate cancer cells
  • Canagliflozin inhibits glucose uptake independently of SGLT2
  • Canagliflozin but not Dapagliflozin decreased H 3 -2DG uptake in PC3 and H1299 cells ( Figure 10A-B).
  • a 10 and 30 ⁇ dose of Canagliflozin reduced 2-DG uptake by over 30 and 60%, respectively.
  • These same doses reduced 2-DG uptake by 50 and 60% in H1299 cells.
  • Canagliflozin potentiates Docetaxel's anti-proliferative and anticlonogenic effects in PC3 cells.
  • Figure 1 1A shows the clonogenic survival of prostate cancer cells treated with Canagliflozin at the indicated concentrations relative to the vehicle treated control as a single agent (grey) or in combination with 0.5 nM Docetaxel (black). 5 ⁇ and 10 ⁇ doses of Canagliflozin combined with 0.5 nM docetaxel inhibited PC3 clonogenic survival by an additional 50% and 90%, respectively, compared to docetaxel treatment alone. [00139] The effect of a similar combination regimen towards these cells' proliferative ability was also tested.
  • Figure 1 1 B shows the proliferation of prostate cancer cells treated with Canagliflozin at the indicated concentrations relative to the vehicle treated control as a single agent (grey) or in combination with 2 nM Docetaxel (black). Consistently, a 30 ⁇ dose of Canagliflozin combined with a 2 nM dose of Docetaxel inhibited PC3 cell proliferation by an additional 50% compared to docetaxel treatment alone.
  • Canagliflozin potentiates Cisplatin's anti-proliferative effects in H1299 cells.
  • Figure 12A shows the proliferation of lung cancer cells treated with Canagliflozin at the indicated concentrations relative to the vehicle treated control as a single agent (grey) or in combination with 5 ⁇ Cisplatin (black).
  • a 30 ⁇ dose of Canagliflozin combined with a 5 ⁇ dose of Cisplatin inhibited H1299 cell proliferation by an additional 12% compared to Cisplatin treatment alone.
  • Figure 12B shows the proliferation of lung cancer cells treated with Cisplatin at the indicated concentrations relative to the vehicle treated control as a single agent (grey) or in combination with 30 ⁇ Canagliflozin (black).
  • a 30 ⁇ dose of Canagliflozin combined with a 10 ⁇ dose of Cisplatin inhibited H1299 cell proliferation by an additional 25% compared to Canagliflozin treatment alone.
  • Canagliflozin potentiates radiation's anti-clonogenic effects in PC3 cells.
  • Figure 13 shows the clonogenic survival of prostate cancer cells treated with a 5 or 10 ⁇ dose of Canagliflozin combined with different doses of radiation (0, 2 or 4 Gy). As can be seen from the data in Figure 13, when combined with 2 Gy of radiation, a 5 ⁇ and 10 ⁇ dose of Canagliflozin inhibited PC3 clonogenic survival by an additional 60 and 80% respectively when compared to radiation treatment alone.
  • Cancers of the lung and prostate are among the 5 most common sites of cancer diagnoses across genders. 1 In view of these striking cancer risks, it is an object to discover novel chemotherapeutic agents that will improve the clinical outcomes of patients currently living with cancer.
  • Canagliflozin at concentrations within the therapeutic window of exposure used for treating type 2 diabetes 22 , displays significant anti-cancer effects across a variety of cell lines; effects not observed with the other approved SGLT2 inhibitor Dapagliflozin. Furthermore, it has been observed that suppression of cancer growth and survival are associated with the activation of AMPK, and the inhibition of de novo lipogenesis, glucose uptake and AKT/mTOR signalling.
  • Canagliflozin is an AMPK Activator: Consistently, a 10-30 ⁇ dose of Canagliflozin increased the phosphorylation of AMPK (Thr172) and its downstream substrate ACC (Ser79) within 30 minutes in PC3 and H1299 cells ( Figure 3). Other AMPK activators such as metformin, salicylate and AICAR have only been able to induce similar patterns of activation at millimolar concentrations in these same cell lines.
  • complex-l inhibitors such as metformin 36 and berberine
  • indirectly activate AMPK 37 while not wishing to be limited by theory, this may be the mechanism by which Canagliflozin increases AMPK activity. Therefore, while not wishing to be limited by theory, even small reductions in complex-l activity would be expected to compromise the ability of these cells to maintain energy homeostasis.
  • Canagliflozin is toxic to mitochondrial function
  • the cancer cells were cultured in growth media containing galactose.
  • the oxidation of galactose to pyruvate generates no net ATP.
  • the cells are therefore compelled to oxidize pyruvate in order to sustain their energy demands.
  • the PC3 and H1299 cells proved to be more sensitive to the anti-proliferative effects of Canagliflozin ( Figure 8C).
  • Canagliflozin's effects on mitogen signaling were also investigated.
  • Canagliflozin was observed to suppress the activating phosphorylation of Akt by more than 50% in PTEN negative PC3 cells but had minimal inhibitory activity in H1299 cells which express the PTEN protein 39 (Figure 9).
  • PTEN suppresses tumour cell growth by inhibiting PI3K- Akt-mTOR pathway by dephosphorylating phosphatidylinositol-(3,4,5)- triphosphate (PIP 3 ), an activator of 3-phosphoinositide-dependent kinase (PDK) and Akt.
  • PIP 3 phosphatidylinositol-(3,4,5)- triphosphate
  • PDK 3-phosphoinositide-dependent kinase
  • PC3 cells may have elevated Akt activity to which Canagliflozin can suppress, as opposed to the H1299 cells whose Akt activity is within the normal homeostatic range.
  • Canagliflozin's ability to suppress this pathway suggests that it may be targeting proteins that reside upstream.
  • Akt has recently been discovered to directly phosphorylate the a1 -subunit of AMPK at residue Ser487 and prevent the activating phosphorylation at Thr172 in HEK293 cells. 41 Canagliflozin's ability to inhibit Akt activity, in combination with complex-l inhibition and a subsequent reduction in the cellular adenylate charge, may therefore further promote AMPK activation in the PC3 cancer cells.
  • Canagliflozin dose dependently decreased 2-DG uptake While not wishing to be limited by theory, it is possible that Canagliflozin inhibits other glucose transporters expressed on cancer cell surfaces. However, thus far, it has only been noted that Canagliflozin is a weak inhibitor of GLUT1 42 This is noted because acute glucose deprivation induces an energetic challenge within the cell to which AMPK will respond. 43 However, this activity is likely not sustainable for 24 hours suggesting, while not limited by theory, that there is an alternative mechanism of AMPK activation. Still, by inhibiting glucose uptake, a considerable source of energy on which cancer cell growth may depend is reduced. This effect may become useful when, as discussed above, mitochondrial function is also impaired.
  • ACC considered a sensitive measure of cellular AMPK activity; it is also the rate-limiting enzyme in the fatty acid biosynthetic pathway. Since an increase in the phosphorylation of ACC (Ser79) in response to Canagliflozin treatment was detected, while not wishing to be limited by theory, a proportional decrease in de novo lipogenesis may be observed. Furthermore, it has previously been demonstrated that the inhibition of lipogenesis is strongly correlated with salicylate activated AMPK and inhibition of clonogenic survival in lung and prostate carcinomas 44 , a result which has been replicated by other groups using other AMPK activators. 45 Consistently, a dose-dependent decrease in fatty acid synthesis by Canagliflozin was noted at concentrations >15 ⁇ in PC3 cells ( Figure 6).
  • Canagliflozin's use in concurrent chemotherapy and radiation therapy Canagliflozin is an effective single agent in terms of its ability to inhibit PC3 and H1299 cell viability. It was therefore of interest to determine whether this drug could also be useful in combination with the chemotherapy drugs Docetaxel and Cisplatin as well as radiation; well established therapies for the treatment of castrate resistant prostate cancer (CRPC) and non-small cell lung cancer (NSCLC) respectively.
  • Docetaxel is the first-line therapy for the treatment of metastatic CRPC as it increases the overall survival of patients when used in combination with prednisone. 46 It is reported herein that low dose Canagliflozin (5-30 ⁇ ) in combination with clinically relevant concentrations of Docetaxel inhibited PC3 colony formation and cell proliferation by 50-90% more in comparison to Canagliflozin treatment alone.
  • Salicylate activates AMPK and synergizes with metformin to reduce the survival of prostate and lung cancer cells ex vivo through inhibition of de novo lipogenesis. Biochem. J. (2015).
  • IC50 Half maximal inhibitory concentrations (IC50) of Canagliflozin and Dapagliflozin on the proliferation of lung (A549, H1299), prostate (22RV1, PC3) colon (MC38, HCT116), liver (HepG2), breast (MCF7) and ovarian (SKOV-3) cells.

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Abstract

La présente invention concerne l'utilisation de canangliflozine et de ses dérivés dans le traitement et la prévention du cancer. Il a été établi précédemment que lesdits composés sont des inhibiteurs sélectifs du transporteur 2 de glucose et de sodium (SGLT2) utiles dans le traitement du diabète ayant des effets qui sont similaires à ceux de la metformine. Il a été établi aujourd'hui que la canagliflozine active la protéine kinase activée par l'AMP (AMPK) et inhibe la croissance d'une gamme de cancers. En outre, il a été établi aujourd'hui que l'utilisation de canagliflozine en association avec d'autres médicaments chimiothérapeutiques déclenche une activité anti-cancéreuse accrue.
PCT/CA2016/050211 2015-02-27 2016-02-29 Utilisation de canagliflozine et de ses dérivés dans le traitement du cancer WO2016134486A1 (fr)

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US201562121539P 2015-02-27 2015-02-27
US62/121,539 2015-02-27
US201562169156P 2015-06-01 2015-06-01
US62/169,156 2015-06-01

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Cited By (6)

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WO2018064405A1 (fr) * 2016-09-28 2018-04-05 Abraxis Bioscience, Llc Méthodes de traitement de troubles mitochondriaux et métaboliques
CN111617075A (zh) * 2020-07-23 2020-09-04 中国药科大学 卡格列净在制备治疗胰腺癌药物中的应用
CN112439067A (zh) * 2019-09-03 2021-03-05 清华大学深圳研究生院 Sglt2抑制剂在制备改善抗肿瘤药物敏感性的产品中的应用
EP3735969A4 (fr) * 2019-05-13 2021-05-19 Zhejiang University Utilisation de canagliflozine dans la préparation d'un médicament anti-tumoral
CN114727624A (zh) * 2019-11-28 2022-07-08 勃林格殷格翰动物保健有限公司 Sglt-2抑制剂在非人哺乳动物的停奶中的用途
WO2022161510A1 (fr) * 2021-01-29 2022-08-04 南京医科大学附属逸夫医院 Utilisation de canagliflozine dans la préparation d'un médicament pour le traitement d'une maladie médiée par la scléronodulaire

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WO2009117367A1 (fr) * 2008-03-18 2009-09-24 Bristol-Myers Squibb Company Procédé de traitement de cancers ayant de hautes exigences en glucose employant un inhibiteur de sglt2 et leurs compositions
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WO2009117367A1 (fr) * 2008-03-18 2009-09-24 Bristol-Myers Squibb Company Procédé de traitement de cancers ayant de hautes exigences en glucose employant un inhibiteur de sglt2 et leurs compositions
WO2013158143A1 (fr) * 2012-04-19 2013-10-24 Eleison Pharmaceuticals LLC Polythérapies par glufosfamide pour le cancer
WO2014182955A2 (fr) * 2013-05-08 2014-11-13 The University Of Houston System Ciblage de l'interaction entre un egfr et un sglt1 pour la cancérothérapie

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018064405A1 (fr) * 2016-09-28 2018-04-05 Abraxis Bioscience, Llc Méthodes de traitement de troubles mitochondriaux et métaboliques
KR20230003239A (ko) 2016-09-28 2023-01-05 아브락시스 바이오사이언스, 엘엘씨 미토콘드리아 및 대사 장애를 치료하는 방법
EP3735969A4 (fr) * 2019-05-13 2021-05-19 Zhejiang University Utilisation de canagliflozine dans la préparation d'un médicament anti-tumoral
US11534448B2 (en) 2019-05-13 2022-12-27 Zhejiang University Use of canagliflozin in preparation of antitumor drug
CN112439067A (zh) * 2019-09-03 2021-03-05 清华大学深圳研究生院 Sglt2抑制剂在制备改善抗肿瘤药物敏感性的产品中的应用
CN114727624A (zh) * 2019-11-28 2022-07-08 勃林格殷格翰动物保健有限公司 Sglt-2抑制剂在非人哺乳动物的停奶中的用途
CN111617075A (zh) * 2020-07-23 2020-09-04 中国药科大学 卡格列净在制备治疗胰腺癌药物中的应用
WO2022161510A1 (fr) * 2021-01-29 2022-08-04 南京医科大学附属逸夫医院 Utilisation de canagliflozine dans la préparation d'un médicament pour le traitement d'une maladie médiée par la scléronodulaire

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