WO2011017809A1 - Autophagy inhibitor compounds, compositions and methods for the use thereof in the treatment of cancer - Google Patents

Autophagy inhibitor compounds, compositions and methods for the use thereof in the treatment of cancer Download PDF

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WO2011017809A1
WO2011017809A1 PCT/CA2010/001243 CA2010001243W WO2011017809A1 WO 2011017809 A1 WO2011017809 A1 WO 2011017809A1 CA 2010001243 W CA2010001243 W CA 2010001243W WO 2011017809 A1 WO2011017809 A1 WO 2011017809A1
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och
cooh
cells
cancer
verteporfin
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PCT/CA2010/001243
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French (fr)
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Michel Roberge
Aruna Balgi
Elizabeth Donohue
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The University Of British Columbia
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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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to therapeutics, their uses and methods for the modulation of autophagy.
  • the invention relates to compounds, compositions, therapies, and methods of treatment of cancer cells.
  • Autophagy is a catabolic process responsible for the bulk degradation of cytoplasmic components (1 ). It is characterized by formation of sequestering membranes called phagophores that expand, engulf cytoplasmic material and then fuse to form double- membraned autophago somes. The autophagosomes, undergo maturation via fusion with endosomal vesicles and ultimately fuse with lysosomes which deliver hydrolytic enzymes that breakdown sequestered molecules and release of the products into the cytoplasm for macromolecular synthesis. Constitutive autophagy functions as a housekeeping mechanism by controlling the turnover of long-lived proteins and organelles (2).
  • Autophagy also functions as an adaptive response to maintain cellular homeostasis; it is stimulated in response to cellular stresses such as nutrient depletion, oxidative stress, protein aggregation, and several cancer drugs (3, 4). By removing aberrant organelles and limiting the production of reactive oxygen species, autophagy is believed to protect cells from genotoxic stress and allow for cancer pathogenesis (5). This is supported by the observation that Beclin 1 and ultraviolet irradiation resistance-associated gene (UVRAG), which are involved in autophagosome assembly, also have tumour suppressor activity (6, 7). Furthermore, autophagy-deficient cells are susceptible to enhanced gene amplification and chromosomal instability, both of which may be considered tumourigenic
  • RNA interference (RNAi) technology to decrease autophagy while exposing cells to autophagy-stimulating cancer agents have been tried.
  • RNAi-mediated knockdown of the autophagy gene Atg5 resulted in decreased cell survival in tamoxi fen-treated MCF-7 cells (19) and in glioma cells treated with a DNA-damaging agent (20) or the tyrosine kinase inhibitor imatinib (14). Inhibition of autophagy also sensitizes tamoxifen-resistant T47D cells to treatment (19), further supporting a prosurvival role.
  • lysosomotropic agents chloroquine
  • V-ATPase inhibitors bafilomycin Al
  • pepstatin A lysosomal protease inhibitors
  • Known early stage inhibitors of autophagy are compounds that inhibit
  • phosphatidylinosital 3 (PB)-kinases such as 3 -MA, wortmannin, and LY294002.
  • PB phosphatidylinosital 3
  • these compounds affect a number of cellular processes and are toxic after prolonged exposure (1, 21).
  • Verteporfin (CAS # 129497-78-5) is a benzoporphyrin derivative which has been used clinically for photodynamic therapy of age related macular degeneration (23).
  • Verteporfin is photoactivated for photodynamic therapy to eliminate the abnormal blood vessels in the eye associated with conditions such as the wet form of macular
  • Verteporfin accumulates in these abnormal blood vessels and, when stimulated by nonthermal red light with a wavelength of 693 ran in the presence of oxygen, the photoactivated verteporfin produces highly reactive short-lived singlet oxygen and other reactive oxygen radicals, resulting in local damage to the endothelium and blockage of the vessels.
  • Benzoporphoryrins are described for example, in US patents 5,095,030, 5,214,036, and 6,008,241.
  • BPDs benzoporphyrin derivatives
  • compounds identified herein show inhibition of early stage autophagy, which may be useful for the treatment of cancer.
  • chemotherapeutic treatments or cells that are metabolically stressed cells For example, nutrient deprived cancer cells.
  • the activity of the compounds identified herein do not require any photo activation and the methods and uses do not require a photoactivation step. Accordingly, the compounds may be administered systemically or locally and may have the desired activity without the need to photoactivate in situ after administration.
  • the compounds described herein may be used for in vivo or in vitro research uses (i.e. non-clinical) to investigate the mechanisms of autophagy inhibition and the response of nutrient deprived cancer cells to various cancer therapeutic treatments. Furthermore, these compounds may be used individually or as part of a kit for in vivo or in vitro research to investigate autophagy inhibition, the response of metabolically stressed cancer cells maintained in culture, combination treatments of cancer cells maintained in culture, and/or animal models. Alternatively, the compounds described herein may be combined with commercial packaging and/or instructions for use.
  • This invention is also based in part on the discovery that the compounds described herein, may also be used to modulate autophagy activity either in vivo or in vitro for both research and therapeutic uses.
  • the compounds may be used in an effective amount so that autophagy activity may be modulated.
  • the compounds may be used to inhibit early stage autophagy activity.
  • the compounds modulatory activity may be used in either an in vivo or an in vitro model for the study of cancer. For example, in early stage autophagy and in cells that are metabolically stressed (for example, nutrient deprived cancer cells or cells that have been or are about to be given a cancer chemotherapeutic agent.
  • the compounds modulatory activity may be used for the treatment of cancer to sensitize cells to other cancer therapeutics or to cells that are already undergoing autophagy in response to a metabolic stress or cancer therapeutic.
  • the cancer cells may be nutrient deprived cancer cells, hypoxic cancer cells or otherwise prone to enter autophagy in response to a cancer therapy.
  • the autophagy may be early stage autophagy.
  • benzoporphyrin derivatives, as described herein may be autophagy inhibitors, as described herein.
  • Compounds identified herein show inhibition of autophagy, which may be useful for the treatment of cancer in the absence of photoactivation.
  • Compounds identified herein may be useful as chemosensitizers for the treatment of cancer.
  • the chemosensitization activity may occur in the absence of photoactivation of the compounds and in the absence of a photoactivation step.
  • the treatment may be of nutrient deprived cancer cells or otherwise metabolically stressed cells.
  • the treatment may be of cancer cells undergoing early stage autophagy.
  • a method of modulating autophagy including administering a compound of formula IA or IB to a cell to modulate autophagy, the compound of Formula IA or IB having the structure:
  • Formula IA Formula IB wherein R 1 is selected from the group: CH 2 OH, CO 2 G 1 , CO 2 G 1 OG 1 ,
  • R 2 is selected from the group: CH 2 OH, CO 2 G 2 , CO 2 G 2 OG 2 , CO 2 G 2 OG 2 OG 2 , and CO 2 G 2 OG 2 OG 2 OG 2 ;
  • R 3 is selected from the group: CH 2 OH, CO 2 G 3 , CO 2 G 3 OG 3 , CO 2 G 3 OG 3 OG 3 , and CO 2 G 3 OG 3 OG 3 ; wherein G 1 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group;
  • G" is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
  • G 3 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and wherein only one of R 1 , R ,
  • the method modulation may occur in the absence of photoactivation.
  • the modulation may be of early stage autophagy.
  • the modulation may of a cancer cell.
  • the cancer cell may be in an animal.
  • the animal may be a human.
  • the cancer therapeutic regimen may be selected from one or more of the agents set out in TABLE 3.
  • the modulation may be for the treatment of cancer.
  • a method of sensitizing a cancerous cell to a cancer therapeutic agent comprising: administering to a subject in need thereof, a compound of formula IA or IB.
  • the sensitization occurs in the absence of photoactivation.
  • composition including a compound of Formula IA or IB and a pharmaceutically acceptable carrier, for treating cancer.
  • a commercial package comprising (a) the compound a compound of Formula IA or IB ; and (b) instructions for the use thereof for treating cancer.
  • compositions which may include one or more compounds having Formula IA and IB and a cancer therapeutic agent in TABLE 3. Furthermore, the pharmaceutical compositions may further include a pharmaceutically acceptable excipient.
  • the treatment of cancer may be in the absence of photoactivation.
  • the compounds and compositions described herein may be used in the absence of photoactivation.
  • the treatment may be carried out in the absence of activating light to prevent photoactivation.
  • the sensitization may be of cancerous cells in early stage autophagy.
  • the cancerous cell may be in an animal.
  • the animal may be a human.
  • the sensitization may precede the administration of a cancer therapeutic regimen.
  • the sensitization may follow the administration of a cancer therapeutic regimen.
  • the sensitization may be simultaneous with the administration of a cancer therapeutic regimen.
  • the cancer therapeutic regimen may be selected from one or more of the agents set out in TABLE 3.
  • a pharmaceutical composition including one or more of the agents set out in TABLE 3 and a compound of Formula IA or IB.
  • the compounds of Formula IA or IB may be active in the absence of photoactivation.
  • the compounds of Formula IA or IB may be selected from any one or more of the compounds of TABLE 2.
  • R 1 may be selected from the group: CH 2 OH, CO 2 G 1 , CO 2 G 1 OG 1 , and
  • R 2 may be selected from the group: CH 2 OH, CO 2 G 2 , CO 2 G 2 OG 2 , and CO 2 G 2 OG 2 OG 2 .
  • R 3 may be selected from the group: CH 2 OH, CO 2 G 3 , CO 2 G 3 OG 3 , and CO 2 G 3 OG 3 OG 3 .
  • G 1 may be hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
  • G may be hydrogen or a linear, or branched, saturated or unsaturated one to ten carbon alkyl group.
  • G 1 may be hydrogen or a linear saturated or unsaturated one to ten carbon alkyl group.
  • G 1 may be hydrogen or a linear saturated one to ten carbon alkyl group.
  • G 1 may be hydrogen or a one to 9 carbon alkyl group.
  • G 1 may be hydrogen or a one to 8 carbon alkyl group.
  • G 1 may be hydrogen or a one to 7 carbon alkyl group.
  • G 1 may be hydrogen or a one to 6 carbon alkyl group.
  • G 1 may be hydrogen or a one to 5 carbon alkyl group.
  • G 1 may be hydrogen or a one to 4 carbon alkyl group.
  • G may be hydrogen or a one to 3 carbon alkyl group.
  • G 1 may be hydrogen or
  • G 2 may be hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
  • G may be hydrogen or a linear, or branched, saturated or unsaturated one to ten carbon alkyl group.
  • G 2 may be hydrogen or a linear saturated or unsaturated one to ten carbon alkyl group.
  • G may be hydrogen or a linear saturated one to ten carbon alkyl group.
  • G may be hydrogen or a one to 9 carbon alkyl group.
  • G may be hydrogen or a one to 8 carbon alkyl group.
  • G 2 may be hydrogen or a one to 7 carbon alkyl group.
  • G 2 may be hydrogen or a one to 6 carbon alkyl group.
  • G 2 may be hydrogen or a one to 5 carbon alkyl group.
  • G may be hydrogen or a one to 4 carbon alkyl group.
  • G 2 may be hydrogen or a one to 3 carbon alkyl group.
  • G 2 may be hydrogen or a one to
  • G 3 may be hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group.
  • G 3 may be hydrogen or a linear, or branched, saturated or unsaturated one to ten carbon alkyl group.
  • G 3 may be hydrogen or a linear saturated or unsaturated one to ten carbon alkyl group.
  • G may be hydrogen or a linear saturated one to ten carbon alkyl group.
  • G 3 may be hydrogen or a one to 9 carbon alkyl group.
  • G 3 may be hydrogen or a one to 8 carbon alkyl group.
  • G 3 may be hydrogen or a one to 7 carbon alkyl group.
  • G 3 may be hydrogen or a one to 6 carbon alkyl group.
  • G 3 may be hydrogen or a one to 5 carbon alkyl group.
  • G 3 may be hydrogen or a one to 4 carbon alkyl group.
  • G may be hydrogen or a one to 3 carbon alkyl group.
  • G 3 may be hydrogen or
  • R , R " , and R may be independently selected from one of more of the following: COOH; CH 2 OH; CO 2 CH 2 OH; CO 2 CH 2 OCH 2 OH; CO 2 CH 2 OCH 2 OCH 2 OH;
  • R 1 , R 2 , and R 3 may be independently selected from one of more of the following: COOH; CH 2 OH; CO 2 CH 3 ; CO 2 CH 2 OH; CO 2 (CH 2 ) 2 OH; CO 2 (CH 2 ) 2 O(CH 2 ) 2 OH;
  • R 1 , R ⁇ , and R 3 may be independently selected from one of more of the following: COOH; CH 2 OH; CO 2 CH 3 ; CO 2 CH 2 OH; CO 2 CH 2 OCH 3 ; CO 2 (CH 2 ) 2 OH;
  • R 1 , R 2 , and R 3 may be independently selected from one of more of the following: COOH; CH 2 OH; CO 2 CH 3 ; CO 2 CH 2 OH; CO 2 (CH 2 ) 2 OH; CO 2 (CH 2 ) 2 O(CH 2 ) 2 OH; and CO 2 CH 2 OCH 2 OH; provided only one of R 1 , R 2 , and R 3 is COOH.
  • the compound may be selected from any one or more of the following compounds:
  • Figure IA shows a schematic model of Autophagy.
  • Figure IB shows a schematic early and late stage (punctuate accumulation of
  • Figure 2 A shows a cell-based assay for the identification of inhibitors of
  • MCF-7 EGFP-LC3 cells were exposed to 0.1 % DMSO (a, b), 10 mM 3-MA (c, d), or 10 ⁇ M verteporfin (e, f) without (a, c, e) or with 30 ⁇ M chloroquine (b, d, f) for 4 h in complete cell culture medium.
  • the images were acquired by confocal microscopy. Scale bar, 10 ⁇ m.
  • Figure 3 shows that verteporfin inhibts autophagosome formation in serum starved cells.
  • FIG. 4 shows that verteporfin inhibits autophagosome accumulation stimulated by rapamycin or serum starvation.
  • MCF-7 EGFP-LC3 cells were exposed for 4 h to 30 nM rapamcycin without (a) or with 10 ⁇ M verteporfin (b) in complete cell culture medium or to medium lacking serum without (c) or with 10 ⁇ M verteporfin (d). Images were acquired by confocal microscopy. Scale bar, 10 ⁇ m.
  • Figure 5. shows an ultrastructural examination of inhibition of CQ-induced
  • MCF-7 EGFP-LC3 cells were exposed for 4 h to 0.1% DMSO (a, b) or 75 ⁇ M CQ (c, d) without (a, c) or with 10 ⁇ M verteporfin (b, d) and then images were acquired by transmission electron microscopy, with arrowheads pointing to autophagic vesicles (Scale bar, 0.5 ⁇ m).
  • Figure 6. shows inhibition of EGFP-LC3 degradation and long-lived protein degradation by verteporfin.
  • A-C MCF-7 EGFP-LC3 cells were treated for 4 h with (A) 30 nM rapamycin without or with 10 niM 3 -MA or 100 nM bafilomycin Al in complete medium; (B) different concentrations of verteporfin without or with 30 nM rapamycin in complete medium; (C) 10 ⁇ M verteporfin in complete medium or in serum-free medium. (A-C), cells were exposed to 0.1% DMSO as a vehicle control and EGFP-LC3 processing and degradation was monitored by western blotting with anti- GFP antibody.
  • Figure 7 shows inhibition of the sequestration of cytosolic FITC-dextran into
  • FITC-dextran was scrape-loaded into MCF-7 cells and its distribution was analyzed by confocal microscopy, wherein DMSO- treated MCF-7 cells were fixed 2 h (a) or 24 h (b, c) after FITC-dextran loading and MCF-7 cells loaded with FITC-dextran were incubated in 10 mM 3 -MA (d) or 10 ⁇ M verteporfin (e) for 24 h in complete medium, following which cells were fixed, DNA was stained and images were acquired by confocal microscopy (Scale bar, 10 ⁇ m).
  • Figure 8. shows verteporfin does not inhibit LC3 processing or membrane
  • Figure 10 shows the effect of selected verteporfin analogues on chloroquine-induced autophagosome accumulation, wherein (A) shows the structures of protoporphyrin IX, verteporfin, and benzopophyrin derivatives tested in (B).
  • A shows the structures of protoporphyrin IX, verteporfin, and benzopophyrin derivatives tested in (B).
  • Figure 11 shows the effect of additional selected verteporfin analogues B BPD hexyl ester dimethyl ester, B BPD hexyl ester diacid, B BPD diethylene glycol ester, A BPD diethylene glycol ester, and verteporfin (A) and A BPD tri(ethylene glycol) ester, A BPD tri(diethylene glycol) ester, and verteporfin (B) on chloroquine-induced autophagosome accumulation, wherein autophagosome accumulation is plotted against varying concentrations of different porphyrins, including verteporfin.
  • Figure 12 shows a summary of autophagy inhibitors tested for autophagosome
  • FIG. 13 shows PK values for VisudyneTM (verteporfin) in HT-29 tumours in
  • Figure 14 shows normalized efficacy of verteporfin dosed qdxl i.v. in combination with HTI-286 dosed i.v. (administered 30 minutes post verteporfin at tumour CMAX) compared to saline and HTI-286 controls.
  • Figure 15 shows a decrease in tumor volume when verteporfin (10 mg/kg) is
  • gefitinib i.e. Iressa 50 mg/kg
  • “early stage autophagy” is the process of autophagosome formation and the sequestration of cytoplasmic material into autophagosomes.
  • nutrient deprived cancer cells may be cancer cells that are undergoing autophagy, wherein autophagy may occur due to a metabolic stress such as nutrient deprivation or hypoxia. Nutrient deprivation may be induced by a metabolic stress promoting agent. Cancer cells may be nutrient deprived due to a lack of blood flow or access to sufficient nutrients, wherein nutrient deprived cells may be deficient in oxygen, serum, amino acids, sugar (for example, glucose) or any combination thereof.
  • MCF7 cells are human breast cancer cells
  • HeLa cells are human cervical cancer cells
  • HT29 cells are human colorectal cancer cells
  • H460 cells are human large cell lung cancer cells.
  • Cancer refers to a proliferative or neoplastic disorder caused or characterized by the proliferation of cells which have lost susceptibility to normal growth control.
  • the term cancer includes tumors and any other proliferative disorders. Cancers of the same tissue type usually originate in the same tissue, and may be divided into different subtypes based on their biological characteristics. Four general categories of cancers are carcinoma (epithelial tissue derived), sarcoma (connective tissue or mesodermal derived), leukemia (blood-forming tissue derived) and lymphoma (lymph tissue derived). Over 200 different types of cancers are known, and every organ and tissue of the body may be affected.
  • organs and tissues that may be affected by various cancers include pancreas, breast, brain, thyroid, ovary, uterus, testis, prostate, thyroid, pituitary gland, adrenal gland, kidney, stomach, esophagus or rectum, head and neck, bone, nervous system, skin, blood, nasopharyngeal tissue, lung, urinary tract, cervix, vagina, exocrine glands and endocrine glands.
  • a cancer may be multicentric or of unknown primary site (CUPS).
  • a "cancerous cell” refers to a cell that has undergone a
  • transformation event and whose growth is no longer regulated to the same extent as before said transformation event.
  • a “tumor” refers to a collection of cancerous cells, often found as a solid or semi-solid lump in or on the tissue or a patient or test subject.
  • a "chemosensitizer” or “sensitizer” is a medicament that may enhance the therapeutic effect of a cancer therapeutic agent, and therefore improve efficacy of such treatment or agent.
  • the sensitivity or resistance of a tumor or cancerous cell to treatment may also be measured in an animal, such as a human or rodent, by, e.g., measuring the tumor size, tumor burden or incidence of metastases over a period of time. For example, about 2, about 3, about 4 or about 6 months for a human and about 2-4, about 3-5, or about 4-6 weeks for a mouse.
  • a composition or a method of treatment may sensitize a tumor or cancerous cell's response to a therapeutic treatment if there is an increase in sensitivity to the treatment or where there is a reduction in the amount of therapeutic treatment needed to achieve the same result.
  • An increase in sensitivity may be about 10% or more, for example, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more compared to the absence of such composition or method.
  • a reduction in the amount of therapeutic treatment needed to achieve the same result may be about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, compared to the absence of such composition or method.
  • the determination of sensitivity to a therapeutic treatment is routine in the art and within the skill of a person versed in the art.
  • Verteporfin (CAS # 129497-78-5) as used herein may include the two regioisomers as shown below:
  • the verteporfin as disclosed herein contains at least one chiral center and thus may exist in various stereoisomeric forms. If desired, such stereoisomers, including enantiomers, may be separated using techniques standard in the art (for example, chiral columns). However, racemic mixtures or mixtures containing more than one diastereomer may also be used and are contenplated herein. However, the compounds tested herein were in either of the trans entantiomers shown above.
  • the compounds shown in Formulas IA, IB, Tables 1, 2 and Figure 10 are representative of the individual optical isomers, enantiomers or diastereomers as the case may be, as well as mixtures of these individual chiral isomers.
  • VisudyneTM is the liposomal formulation of verteporfin used in humans for photodynamic therapy. VisudyneTM is given intravenously, usually within 15 minutes prior to laser treatment to eliminate the abnormal blood vessels in the eye in the treatment of wet macular degeneration. The verteporfin compound accumulates in these abnormal blood vessels and, when stimulated by a nonthermal red light laser with a wavelength of 693 nm in the presence of oxygen, produces highly reactive short-lived singlet oxygen and other reactive oxygen radicals, resulting in local damage to the endothelium and blockage of the vessels. Patients given VisudyneTM experience photosensitivity and are advised to avoid exposure to sunlight and unscreened lighting for at least 48 hours after the injection of verteporfin.
  • subjects administered the BPDs described herein, in accordance with the methods and uses described herein do not require photoactivation of the BPD via nonthermal red light laser with a wavelength of 693 nm or otherwise.
  • the activity of the BPDs to inhibit early stage autophagy is independent of the activity associated with photoactivation and would likely be hindered by photoactivation. Accordingly, a person of skill in the art would appreciate that the precautions associated with photosensitivity should also apply to the present methods and uses (i.e. avoid exposure to sunlight and unscreened lighting for at least 48 hours after the injection of of the BPD).
  • C x-y alkyl or "C x -C y alkyl” is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that has a carbon skeleton or main carbon chain comprising a number from x to y (with all individual integers within the range included, including integers x and y) of carbon atoms.
  • a "C 1-1O alkyl” is a chemical entity that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atom(s) in its carbon skeleton or main chain.
  • branched is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises a skeleton or main chain that splits off into more than one contiguous chain.
  • the portions of the skeleton or main chain that split off in more than one direction may be linear, cyclic or any combination thereof.
  • Non-limiting examples of a branched alkyl are tert-butyl and isopropyl.
  • unbranched is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises a skeleton or main chain that does not split off into more that one contiguous chain.
  • Non-limiting examples of unbranched alkyls are methyl, ethyl, n-propyl, and n-butyl.
  • substituted is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that has one chemical group replaced with a different chemical group that contains one or more heteroatoms.
  • a substituted alkyl is an alkyl in which one or more hydrogen atom(s) is/are replaced with one or more atom(s) that is/are not hydrogen(s).
  • chloromethyl is a non-limiting example of a substituted alkyl, more particularly an example of a substituted methyl.
  • Aminoethyl is another non-limiting example of a substituted alkyl, more particularly it is a substituted ethyl.
  • the functional groups described herein may be substituted with, for example, and without limitation, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents.
  • unsubstituted is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that is a hydrocarbon and/or does not contain a heteroatom.
  • unsubstituted alkyls include methyl, ethyl, tert-butyl, and pentyl.
  • saturated when referring to a chemical entity is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises only single bonds.
  • saturated chemical entities include ethane, tert-butyl, and N + H 3 .
  • Non-limiting examples of saturated C 1 -Ci O alkyl may include methyl, ethyl, n- propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, i-pentyl, sec- pentyl, t-pentyl, n-hexyl, i-hexyl, 1,2-dimethylpropyl, 2-ethylpro ⁇ yl, l-methyl-2- ethylpropyl, 1 -ethyl-2-methylpropyl, 1 , 1 ,2-trimethylpropyl, 1,1,2-triethylpropyl, 1,1- dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, 1,3-dimethylbutyl, 2-methylpentyl, 3- methylpentyl, sec-hexyl,
  • Non-limiting examples Of C 2 -CiO alkenyl may include vinyl, allyl, isopropenyl, 1- propene-2-yl, 1-butene-l-yl, l-butene-2-yl, l-butene-3-yl, 2-butene-l-yl, 2-butene-2-yl, octenyl and decenyl.
  • Non-limiting examples OfC 2 -Ci 0 alkynyl may include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl.
  • Saturated Ci-Cio alkyl, C 2 -C) O alkenyl or C 2 -Ci O alkynyl may be, for example, and without limitation, interrupted by one or more heteroatoms which are independently nitrogen, sulfur or
  • the compounds as described herein or acceptable salts thereof above may be used for systemic treatment of a cancer.
  • the compounds as described herein or acceptable salts thereof above may be used in the preparation of a medicament or a composition for systemic treatment of a cancer.
  • methods of systemically treating any of the cancers described herein are also provided. Some embodiments, make use of compositions comprising a compound described herein and a pharmaceutically acceptable excipient or carrier. Methods of treating any of the indications described herein are also provided. Such methods may include administering a compound as described herein or a composition of a compound as described herein, or an effective amount of a compound as described herein or composition of a compound as described herein to a subject in need thereof.
  • Compounds as described herein may be in the free form or in the form of a salt thereof.
  • compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (Berge et al., J. Pharm. Sd. 1977, 66, 1 ).
  • Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable).
  • compounds and all different forms thereof may be in the solvent addition form, for example, solvates.
  • Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent in physical association with the compound or salt thereof.
  • the solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent.
  • hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.
  • compounds and all different forms thereof may include crystalline and/or amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof.
  • Polymorphs include different crystal packing arrangements of the same elemental composition of a compound.
  • Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and/or solubility. Those skilled in the art will appreciate that various factors including
  • recrystallization solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate.
  • compounds and all different forms thereof include isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and
  • compositions in accordance with this invention may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt.
  • Pharmaceutical preparations will typically comprise one or more carriers, excipients or diluents acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers, excipients or diluents include those known in the art for use in such modes of administration.
  • Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner.
  • a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K.
  • the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release.
  • formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogels, or solutions which can be used topically or locally to administer a compound.
  • a sustained release patch or implant may be employed to provide release over a prolonged period of time.
  • Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for modulatory compounds include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • the formulations may be specifically prepared for intranasal delivery. For example, nasal inhalation.
  • Compounds or pharmaceutical compositions in accordance with this invention or for use in this invention may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc.
  • a medical device or appliance such as an implant, graft, prosthesis, stent, etc.
  • implants may be devised which are intended to contain and release such compounds or compositions.
  • An example would be an implant made of a polymeric material adapted to release the compound over a period of time.
  • an “effective amount” of a pharmaceutical composition as described herein includes a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduced cancer burden, increased life span or increased life expectancy.
  • a therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as less severe infection or delayed or no onset, increased life span, increased life expectancy or prevention of the progression of infection.
  • a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
  • dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.
  • the amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • compounds and all different forms thereof as described herein may be used, for example, and without limitation, in combination with other treatment methods.
  • Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LDlOO (the dose lethal to 100% of the population). In some circumstances, however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions. Some compounds described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Toxicity may be evaluated by examining a particular compound's or composition's specificity across cell lines. Animal studies may be used to provide an indication if the compound has any effects on other tissues.
  • a "subject” may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.
  • the subject may be suspected of having or having cancer. Diagnostic methods for cancer, and the clinical delineation of cancer, are known to those of ordinary skill in the art. TABLE 1 shows compounds made and tested for early stage autophagy modulatory activity.
  • a “cancer therapeutic regimen” may be selected from a cancer therapeutic agent or a combination of cancer therapeutic agents.
  • a “cancer therapeutic agent” may be a metabolic stress promoting agent or an autophagy inducing agent, which may include a nutrient deprivation promoting agent or hypoxia inducing agent, which may include a vascular targeting agent or an anti- angiogenic agent, wherein the cancer therapeutic agent may be, for example, one or more of the agents set inTABLE 3.
  • the vascular targeting agent may be selected from one or more of combretastatin A-4-phosphate, combretastatin analogues, HTI-286, ZD6126, or OXI 4503, and wherein an anti-angiogenic agent may be selected from one or more of, endostatin, TNP-470, or VEGF trap (see also, Table 1 in Roy and Debnath Semin Immunopathol (2010) Published online 30 June 2010 or one or more of the treatments set out in Livesey K. M. et al. Current Opinion in Investigational Drugs (2009) 10(12):1269-1279).
  • Gefitinib (CAS # 184475- 35-2 sold by AstraZenecaTM under the trade name IressaTM) was tested in combination with verteporfin.
  • Gefitinib inhibits the epidermal growth factor receptor (EGFR) tyrosine kinase by binding to the adenosine triphosphate (ATP)-bmding site of EGFR, which leads to inhibition of EGFR tyrosine kinase activation of the Ras signal transduction cascade, which m turn results in malignant cell inhibition
  • EGFR tyrosine kinase a selective inhibitor of the EGFR tyrosine kinase (i e Herl or ErbB-1)
  • EGFR is known to be overexpressed in human carcinomas such as lung and breast cancers, which may lead to inappropriate activation of the apoptotic Ras signal transduction cascade and to uncontrolled cell proliferation
  • Bevacizumab Humanized anti-VEGF antibody
  • CAS # 216974-75-3 A recombinant humanized monoclonal IgGl antibody that binds to and inhibits the biologic activity of human vascular endothelial growth factor (VEGF) approved for the treatment of metastatic colorectal cancer
  • VEGF vascular endothelial growth factor
  • Cetuximab Humanized anti-EGF receptor (EGFr) antibody
  • CAS # 205923-56-4 A chimeric IgGl monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR) preventing hgand binding and activation of the receptor This blocks downstream signaling of EGFR, inhibiting cell proliferation and angiogenesis, among other effects Metastatic colorectal cancer, head and neck cancer
  • PIGF vascular endothelial growth factor
  • Sorafenib (4-[4-[[4-chloro-3-(t ⁇ fluoromethyl)phenyl]carbamoylammo]phenoxy]-N-methylpy ⁇ dme-2- carboxamide) CAS # 284461-73-0 is approved for the treatment of advanced renal cell carcinoma (primary kidney cancer) and has received "Fast Track” designation by the FDA for the treatment of advanced hepatocellular carcinoma (primary liver cancer) ⁇ _ ⁇ __
  • Vandetanib / ZD6474 (4-(4-Bromo-2-fluoroanilmo)-6-methoxy-7-[(l-methylpipendin-4- yl)methoxy]quinazolme) CAS # 443913-73-3
  • An orally bioavailable 4-anilinoquinazolme Vandetanib selectively inhibits the tyrosine kinase activity of vascular endothelial growth factor receptor 2 (VEGF2), thereby blocking VEGF-stimulated endothelial cell proliferation and migration and reducing tumor vessel permeability
  • This agent also blocks the tyrosine kinase activity of epidermal growth factor receptor (EGFR) a receptor tyrosine kinase that mediates tumor cell proliferation and migration and angiogenesis
  • EGFR epidermal growth factor receptor
  • Vatalanib / PTK787 (N-(4-Chlorophenyl)-4-(4-pyridinylmethyl)-l-phthalazinamine dihydrochlo ⁇ de) CAS # 212141-51-0 is a small molecule protein kinase inhibitor that inhibits angiogenesis It is being studied as a possible treatment for several types of cancer, particularly cancer that is at an advanced stage or has not responded to chemotherapy Vatalanib is orally active
  • Axitinib / AG-013736 N-Methyl-2 ((3-((lE)-2-( y ⁇ dine-2-yl)ethenyl)-lH-indazol-6- yl)sulfanyl)benzamide) CAS # 319460-85-0.
  • Axitmib is an oral selective inhibitor of vascular endothelial growth factor receptors (VEGFR) 1 , 2 and 3
  • Axitinib is being studied as both a single agent and in combination across many tumor types, including as a treatment for renal cell carcinoma and non-small cell lung cancer
  • Tyrosine kinase inhibitor that inhibits three growth factor receptors simultaneously vascular endothelial Temozolamide (3-methyl-4-oxoimidazo[5,l-d][l,2,3,5]tetrazine-8-carboxamide) CAS# 85622-93-1
  • trasstuzumab Humanized anti-HER2 antibody
  • Arginine deiminase (a hydrolase enzyme that catalyzes the chemical reaction L-argmine + H 2 O to L- citrulline + NH, ) CAS# 9027-98-9
  • Temozolomide (3-methyl-4-oxoimidazo[5.1-d][l,2,3,5]tetrazine-8-carboxamide) CAS# 85622-93-1
  • Camptothecin / CPT (4-ethyl-4-hydroxy-l,12-dihydro-4h-2-oxa-6,12a-diaza- Dibenzo[b,h]fluorene-3,13- dione) CAS# 7689-03-4
  • Polyinosine-polycytidylic acid / PIC poly[(2R,3S.4R,5R)-5-(4-ammo-2-oxopy ⁇ midm-l-yl)-3,4- dihydroxyoxolan-2-yl]methyl dihydrogen phosphate, or poly[(2R,3S,4R,5R)-3,4-dihydroxy-5-(6-oxo-3H- punn-9-yl)oxolan-2-yl]methyl dihydrogen phosphate) CAS# 24939-03-5
  • Carboplatin (azanide, cyclobutane-l,l-dicarboxylic acid, platinum) CAS# 41575-94-4
  • Paclitaxel (5 beta,20-Epoxy-l,2a,4,7 beta, 10 beta, 13 alpha-hexahydroxytax-1 l-en-9-one 4,10-diacetate 2- benzoate 13-ester with (2 R,3S)-N-benzoyl-3-phenylisose ⁇ ne) CAS# 33069-62-4
  • Histone deacetylase inhibitors HDAC
  • Vitamin D3 analogs are currently in clinical trials for the treatment of various malignancies
  • Seocalcitol / EB-1089 ((lr,3s,5z)-5-((2e)-((lr,3as,7ar)-l-((lr,2e,4e)-6-ethyl-6-hydroxy-l-methyl-2,4- octadienyl)octahydro-7a-methyl-4h-mden-4-ylidene)ethylidene)-4-methylene- 1 ,3-cyclohexanediol) CAS # 134404-52-7
  • the vitamin D3 analog Seocalcitol is effective in the growth arrest and induction of cell death in various malignant cells
  • Cell death following Seocalcitol administration in breast cancer cells is associated with autophagy
  • Rapamycin (23,27-Epoxy-3H- ⁇ y ⁇ do[2,l-c]fl,4]oxaazacyclohent ⁇ acontine) CAS # 53123-88-9
  • This macrolide antibiotic inhibits the mTORCl complex and is in advanced clinical t ⁇ als for the treatment of va ⁇ ous malignancies
  • Temsirolimus / CCI-779 (Rapamycm 42-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate) CAS # 162635-04-3 This rapamycm derivative is showing promising results m advanced clinical t ⁇ als
  • Everolimus / RADOOl (dihydroxy-12-[(2i?)-l-[(lS",3 ⁇ ,4 ⁇ )-4-(2-hydroxyethoxy)-3- methoxycyclohexyl]propan-2-yl]- 19,30-dimethoxy- 15,17.21 ,23 ,29,35-hexamethyl- 11 ,36-dioxa-4- azat ⁇ cyclo[30 3 1 0 4 9 ]hexat ⁇ aconta-16,24,26,28-tetraene-2,3,10,14,20-pentone) CAS # 159351-69-6 Approved for advanced renal cell carcinoma after failure of treatment with sumtinib or sorafenib
  • Amiodarone ((2- ⁇ 4-[(2-butyl-l-benzofuran-3-yl)carbonyl]-2,6-diiodophenoxy ⁇ ethyl)diethylamme) CAS # 1951-25-3
  • Resveratrol (5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-l,3-diol) CAS # 9010-10-0
  • AICAR [(2R,3 S ,4R,5R)-5-(5-amino-4-carbamoylimidazol- 1 -yl)-3 ,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate
  • Metformin (3-(diaminomethyhdene)-l ,l-dimethylguanid ⁇ ne hydrochloride) CAS # 56258-19-6
  • Triciribine (l ,5-Dihydro-5-methyl-l-beta-D- ⁇ bofuranosyl-1.4,5,6,8-pentaazaacenaphthylen-3-amme) CAS
  • SB 202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-lH-imidazole) CAS # 152121-30-7
  • any of the above agents may fit into one or more of the above subcatego ⁇ es of activity, whereby the above TABLE is not meant to be limiting as to the mechanism of activity.
  • Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.
  • Autophagy entails the recruitment of cytosolic LC3/Atg8 protein to the membrane of nascent autophagosomes, a criterion often used to monitor the formation and accumulation of autophagosomes.
  • human breast cancer MCF-7 or HeLa, HT29, H460 cells stably expressing LC3 linked at its N-terminus to GFP were established.
  • GFP-LC3 fluorescence was largely diffuse throughout the cytoplasm, with a few dots denoting autophagosomes generated at a basal level.
  • the number of GFP-LC3 dots increased during serum and amino acid withdrawal, conditions that induce autophagy.
  • the lysosomotropic agent chloroquine is known to cause the accumulation of autophagosomes through inhibiting their fusion to lysosomes and preventing their degradation. Chloroquine treatment produced extensive
  • MCF-7 cells were transfected with GFP-LC3 plasmid using EffecteneTM.
  • the transfected cells were maintained in 400 ⁇ g/ml G418 for a period of 2 months.
  • the GFP labeled cells were sorted by FACS and further maintained in medium containing 400 ⁇ g/ml G418 as pooled stable transfectants.
  • Cells were thereafter maintained in RPMI- 1640 supplemented with 100 units/ml penicillin/streptomycin, ImM Hepes, 10% fetal bovine serum in humidified 5% CO2 at 37°C.
  • MCF-7 cells stably expressing GFP-LC3 were added to wells of 96 well plates and allowed to adhere.
  • Test drugs were added to a final concentration of 17 ⁇ M using a Biorobotics Biogrid IITM robot equipped with a 0.7mm dia 96-pin tool. The cells were incubated in the presence of test drug for 4 hours. The medium was removed and cells were fixed with 3% paraformaldehyde containing 500ng/ml of Hoechst 33342 for 15 min at room temperature. Wells were washed once with warm PBS and stored in PBS at 4°C. Plates were read using a Cellomics Arrayscan VTITM high automated fluorescence imager.
  • GFP-LC3 processing was assayed by Western blotting.
  • Cell extracts were prepared in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 raM EDTA, 1 mM EGTA, 1% Triton Xl 00TM, 25 mM sodium pyrophosphate, 1 mM ⁇ -glycerophosphate, 1 mM Na3VO4, and RocheTM protease inhibitor cocktail) and lO ⁇ g protein was subjected to 12% SDS-PAGE. Proteins were transferred to nitrocellulose and incubated with anti-GFP stabilized antibody preparation (1 :7000, RocheTM) followed by HRP-conjugated anti- Mouse antibody (1 : 10000, PierceTM).
  • Immunostained proteins were revealed using Immobilon Western Chemiluminescent HRP substrate (MilliporeTM) and X-ray film. Western blotting with anti ⁇ -tubulin rabbit polyclonal antibody (1 :20000, Santa CruzTM) and HRP-conjugated goat anti -rabbit antibody (1 : 10000, PierceTM) was used for loading controls.
  • Cells were seeded in 96 well plate and grown for 18 h in complete medium. The cells were washed three times with warm RPMI medium (serum deprivation) or HBSS medium (amino acid and serum deprivation) and immediately treated with drugs in the same medium. The cells were fixed and analysed for punctuate staining (as above) using the Arrayscan VTI.
  • Cells were seeded in 96 well plates and grown for 18 h. They were then exposed to drugs diluted in appropriate medium for 4 h. Media included DPBS, DPBS containing 11 mM glucose, DPBS containing 1 1 mM glucose and 10% FBS, DPBS containing 11 mM glucose and 10% dialysed FBS, DPBS containing 10% FBS, and complete medium. The medium was removed carefully and replaced with complete medium without drug. After 48h, cell numbers were assessed using the MTT assay.
  • MCF-7 cells and MCF-7 cells stably expressing EGFPLC3 were maintained in RPMI 1640 supplemented with 100 units/ml penicillin/streptomycin, 1 mM Hepes, and 10% (v/v) FBS.
  • EGFP-LC3 expressing cells were supplemented with 400 ⁇ g/ml G418 (Sigma, G8168). For starvation experiments, cells were washed twice with DPBS
  • Serum starvation was carried out by incubating cells in the previously described growth medium lacking FBS.
  • Glucose starvation was carried out by incubating cells in DPBS supplemented with amino acids from a 1OX mixture (24) and 10% FBS.
  • Amino acid starvation was carried out in DPBS containing 11 mM glucose, and DPBS was used for serum, glucose, and amino acid starvation.
  • MCF-7 cells stably expressing EGFP LC3 (25) were seeded in PerkinElmer View 96-well plates at 20,000 per well. Eighteen hours after seeding, chemicals from the Prestwick, Sigma LOPAC, Microsource Spectrum and Biomol natural products collections were added to plates at -10 ⁇ M using a Biorobotics Biogrid II robot equipped with a 0.4 mm diameter 96-pin tool. Chloroquine (30 ⁇ M) was added immediately after to all but negative control wells. Plates were incubated for 4 h at 37°C. Punctate EGFP-LC3 was determined quantitatively using a Cellomics Arrayscan VTI automated fluorescence imager and the Compartmental Analysis Bioapplication as described in detail in Balgi et al.
  • MCF-7 cells stably expressing EGFP-LC3 were cultured on glass coverslips in 6- well plates. Eighteen hours after plating, drugs were added for 4 h. The cells were then fixed with 3% paraformaldehyde in PBS and stained with 500 ng/ml Hoechst 33342. The coverslips were mounted in DABCO and viewed using the 6OX objective of an Olympus Fluoview FVlOOO laser scanning microscope equipped with Olympus-selected
  • Cells were seeded at 8,000 per well in 96-well plates and grown overnight. Cells were washed twice with DPBS and treated as described. The drugs were then removed, and cells were grown in complete medium for 48 h. Cell viability and proliferation were measured by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay (26).
  • MCF-7 cells were treated as described for 4 h at 37 0 C. After treatment, cells were washed once with cold PBS, and collected in subcellular fractionation buffer (250 mM sucrose, 20 mM hepes (pH 7.4), 10 mM KCl, 1.5 mM MgC12, 1 mM EDTA, ImM
  • subcellular fractionation buffer 250 mM sucrose, 20 mM hepes (pH 7.4), 10 mM KCl, 1.5 mM MgC12, 1 mM EDTA, ImM
  • the lysate was passed through a 26G1/2 needle 10 times and incubated on ice for 20 min.
  • the nuclear pellet was collected by centrifugation at 500 x g for 10 min.
  • the supernatant was ultracentrifuged at 100,000 x g to obtain a cytosolic supernatant and a pellet containing total membrane (27).
  • the pellet was resuspended in subcellular fractionation buffer containing 1% Triton X-100 and 10% glycerol. Identical amounts of the supernatant and the membrane fractions (-30-50 ⁇ g of protein), respectively, were resolved on a 16% polyacrylamide gel and immunoblotted as described above.
  • MCF-7 EGFP-LC3 cells were seeded in a 12-well plate at 75,000 cells per well and grown overnight. Cells were incubated for 24 h at 37°C with 1 ⁇ Ci/mL of L- [14C]valine (MP Biomedical, #10139E) in RPMI 1640 supplemented as described above. After radiolabelling, cells were washed three times with RPMI, and incubated in complete RPMI supplemented with 10 mM cold valine for 24 h. After this cold chase period where short-lived proteins were degraded, the cells were treated for 18 h as described in either complete RPMI or RPMI lacking serum.
  • L- [14C]valine MP Biomedical, #10139E
  • Radiolabeled proteins from the medium and the cells were precipitated in 10% (v/v) trichloroacetic acid and were separated from soluble radioactivity by centrifugation at 20,000 x g at 4°C.
  • the precipitated proteins were dissolved in 0.2 N NaOH and the supernatant containing soluble radioactivity was neutralized with 5 N NaOH. Radioactivity was determined by liquid scintillation counting, and protein degradation was calculated by dividing the total acid-soluble radioactivity by the total TCA-precipitated radioactivity (28).
  • FITC-dextran was scrape-loaded into MCF-7 cells as described (29), except cells were suspended in medium containing 0.1% DMSO, 10 mM 3-methyladenine, or 10 ⁇ M verteporfin after scrape loading. The suspended cells were plated on coverslips for 2 h or 24 h before fixation with 3% paraformaldehyde in PBS and DNA staining with Hoechst 33342. Slides were then analyzed using confocal microscopy. Benzoporphyrin derivatives
  • the ring A and B benzoporphyrin derivatives were synthesized using standard techniques (30-32 and US patent 5,990,149). All derivatives were characterized using IH NMR and elemental analysis.
  • the verteporfin regioisomers were separated by reversed phase HPLC. Using a 250 x 10 mm Phenomenex Synergi 4 ⁇ MAX-RP 80A C- 12 column and a mobile phase of 95 % MeOH/0.01 M ammonium formate, verteporfin isomer A eluted at 11.3 min and verteporfin isomer B eluted at 11.9 min.
  • MCF-7 EGFP-LC3 cells were seeded onto BD 24-well cell culture inserts with PET track-etched membrane at 30,000 cells per well. Thirty-six hours after seeding, cells were treated as described. Medium in transwells was replaced with fixative (1.5% paraformaldehyde, 1.5% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.3) at room temperature. After 30 minutes, membranes were cut from the transwells and immersed for an additional 2.5-3 h in fixative. The fixative was replaced with buffer (0.1 M sodium cacodylate, pH 7.3) and left overnight at room temperature.
  • EXAMPLE 1 Verteporfin and other BPD derivatives identified as early stage autophagy inhibitors
  • verteporfin was identified as an early stage autophagy inhibitor ( Figure 2). Verteporfin was shown to inhibit autophagosome accumulation induced by cell starvation. At concentrations that inhibit autophagy, verteporfin is selectively toxic to cells in conditions of starvation and is not toxic to cells in complete culture medium in vitro. The autophagy inhibitory activity occurs without light activation (photoactivation) and therefore does not involve photodynamic therapy.
  • a BPD diethylene glycol ester, A BPD monoacid, A BPD triol, A BPD diol, A BPD tri(ethylene glycol) ester, and A BPD tri(diethylene glycol) ester were found to inhibit autophagosome accumulation ( Figures 10-12).
  • EXAMPLE 2 Assay to discriminate between chemicals that cause autophagy- unrelated aggregation of intact GFP-LC3.
  • GFP-LC3-II electrophoretic mobility of the fusion protein
  • This assay enables the discrimination between chemicals that cause autophagy-unrelated aggregation of intact GFP-LC3, chemicals that stimulate autophagy, and chemicals that inhibit autophagy at a late stage after the recruitment of LC3 to autophagosomes.
  • Figure 3 shows that verteporf ⁇ n inhibition of autophagy is at an early stage (i.e. proteolytic processing and lipidation of LC3 during the recruitment of GFP-LC3 to autophagosomes) as evidenced by the lack of a downward shift in the electrophoretic mobility of the fusion protein (GFP-LC3-II) in -serum media. Accordingly, the early stage inhibition results from interference with the fusion of autophagosomes with lysosomes.
  • MCF-7 breast cancer cells stably expressing LC3 tagged to EGFP at its N-terminus (MCF-7 EGFP-LC3).
  • LC3 is a cytosolic protein that is recruited to autophagosomes via cleavage of its C- terminus to expose a glycine residue which is then conjugated to
  • Verteporfin is a benzoporphyrin derivative used clinically for photodynamic therapy of agerelated macular degeneration. Red light irradiation causes the generation of oxygen radicals that nonselectively kill cells exposed to verteporfin. Verteporfin shows little or no cellular toxicity in the absence of light activation (37, 38). Importantly, the ability of verteporfin to inhibit CQ-induced autophagosome accumulation occurred in the absence of light and was consequently unrelated to its photodynamic properties. All experiments described in this study were conducted without direct light.
  • Electron microscopy was used to examine this effect at the ultrastructural level. Sections of control cells treated with DMSO rarely if ever contained any autophagosomes (Figure 5a) while exposure to 75 ⁇ M CQ caused a significant perinuclear accumulation of autophagic vacuoles containing lamellar structures and undigested cytoplasmic material ( Figure 5c, arrowheads), as expected. Noticeably, incubation with 10 ⁇ M verteporfin alone caused the distinct appearance of small empty rounded single-membraned vesicles;
  • EGFP-LC3 recruited to the membranes of autophagosomes is degraded upon fusion with lysosomes but EGFP is less sensitive to lysosomal proteases than the LC3 moiety, leading to transient EGFP accumulation. Therefore, the relative levels of EGFP- LC3 and free EGFP reflect autophagic flux (25). MCF-7 cells treated with 30 nM rapamycin for 4 h showed a significant increase in free EGFP levels compared to controls, consistent with its stimulation of autophagy ( Figure 6A). As expected, co-treatment with 3 -MA prevented LC3 degradation and thus the appearance of the free EGFP band.
  • Bafilomycin Al a V-ATPase inhibitor that prevents lysosomal acidification and lysosomal protein degradation (35), also prevented the appearance of the free EGFP band in cells treated with rapamycin (Figure 6A).
  • Figure 6A Having established these conditions with well characterized compounds, we then tested the effects of different combinations of verteporfin and rapamycin on EGFP-LC3 processing. Exposure of cells to verteporfin alone led to a decrease in the intensity of the free EGFP band in a concentration-dependent manner, indicating that verteporfin inhibits basal autophagy (Figure 6B).
  • Verteporfin also caused a concentration-dependent decrease in the intensity of the free EGFP band when co-incubated with rapamycin (Figure 6B).
  • the effects of verteporfin on autophagic flux were also tested in serum starvation conditions. Cells exposed to serum-free medium for 4 h showed a significant increase in the free EGFP band compared to controls,
  • Verteporfin inhibits the sequestration of FITCdextran
  • phagophores into bowl-shaped structures that surround cytoplasmic material and capture it when the edges of the phagophores fuse to form doublemembraned autophagosomes (40, 41). This process may be monitored experimentally by examining the transfer of fluorescently labelled dextran from the cytoplasm into autophagic vesicles (29).
  • FITCdextran was introduced into the cytoplasm of MCF-7 cells by scrape-loading at a temperature of 4°C to prevent uptake by fluid-phase endocytosis. Two hours after loading, FITC-dextran was localized diffusely throughout the cytoplasm ( Figure 7a) but it redistributed to punctate structures within 24 h, reflecting autophagosomal sequestration (Figs. 7b & 7c). When cells were treated with 3 -MA, FITC-dextran remained diffuse in the cytoplasm 24 h after loading ( Figure 7d), consistent with its demonstrated ability to inhibit autophagic sequestration (42). In cells treated with verteporfin, FITC-dextran remained completely diffuse in the cytosol ( Figure Ie), showing that it too inhibits sequestration of cytoplasmic material into autophagosomes.
  • Verteporfin does not inhibit LC3 processing or LC3II membrane association
  • LC3II Lipidated LC3 associates with the isolation membrane of nascent autophagosomes and is believed to participate in phagophore expansion (43). LC3II was not detected in either vehicle- or rapamycin-treated MCF-7 cells, consistent with previous observations in MEF and HeLa cell lines (25, 44).
  • LC3II has been shown to specifically associate with autophagosome membranes while unprocessed LC3 is cytosolic (27). Lysates of treated cells were subjected to ultracentrifugation to separate membrane and cytosolic fractions. In both DMSO and verteporfin-treated cells, LC3 was predominantly in the supernatant cytosolic fraction, and no LC3II was detected in either cytosolic or pellet membrane fractions ( Figure 8B).
  • verteporfin as an inhibitor of autophagy, we investigated its effect on cell survival and proliferation.
  • MCF7 cells all treatments were 8h + 4Oh recovery and for the HeLa, HT29, and H460 cell lines all treatments were 24h + 48h recovery.
  • Verteporfin was then washed away and the cells were incubated in complete medium for 40 h or 48 h to monitor their ability to recover and proliferate.
  • Verteporfin had no effect on viability suggesting that transient inhibition of autophagy under nutrient- nch conditions does not affect MCF-7, HeLa, HT29 and H460 proliferation and viability ( Figures 9 A-D).
  • MCF-7 cells subjected to serum and amino acid starvation showed a greater than 50% survival after 12 hours.
  • Verteporfin is composed of an equal mixture of two regioisomers (Figure 10A), each of which consists of a pair of enantiomers.
  • the regioisomers were separated by HPLC and tested in the automated microscopy assay. They were equally active, indicating that the propionic acid and propionic acid methyl ester on rings C and D could be interchanged without affecting activity.
  • Verteporfin may be described as a derivative of protoporphyrin IX (apo-heme), bearing modifications to rings A, C and D (Figure 10A). Protoporphyrin IX itself showed no inhibition of autophagy (not shown), demonstrating the dependence on one or more of these modifications for activity. To examine this question, a number of analogues modified at these positions (Figure 10A) were tested at different concentrations for inhibition of autophagy ( Figures 1OB, 1 IA, 1 IB and 12).
  • protoporphyrin IX two propionic acid groups are attached to rings C and D while verteporfin has a propionic acid methyl ester at one of these positions.
  • Verteporfin analogue 1 with two propionic acid methyl esters at rings C and D was fully active, but analogue 2, with two propionic acid groups was essentially inactive, showing that the presence of one carboxylic acid is tolerated, but not two.
  • Verteporfin analogue 5 with propanol functionalities attached to rings C and D was also inactive. To determine whether additional C and D ring substitutions would affect activity, a number of different groups were also incorporated at these positions.
  • Verteporfin also differs from protoporphyrin IX by the presence, fused to ring A, of a cyclohexadiene bearing two methanoic acid methyl esters. Analogue 1 with one methanol c acid methyl ester and one methanoic acid group retained full activity suggesting that a variety of substitutions are likely tolerated at this position.
  • Figure 14 shows the normalized efficacy of verteporfin dosed qdxl i.v. in combination with HTI-286 dosed i.v. (administered 30 minutes post verteporfin at tumour CMAX) compared to saline and HTI-286 controls.
  • the assay demonstrated antitumour activity of Verteporfin in combination with HTI-286 (1.6 mg/kg once) as starvation-promoting drug in a xenograft tumour model using HT-29 colon carcinoma cells.
  • the for the studies will be as follows:
  • mice Five female NCR nude mice per group were used. The mice were treated when the tumours were ⁇ 0.2 cm3 and the groups were as follows: 1. untreated control (saline); 2. Verteporfin alone; 3. HTI-286 alone; and 4. HTI-286 plus 20mg/kg iv + Verteporfin single dose (20 mg/kg).
  • Drugs were delivered via i.v. administration. One dose of verteporfin followed by one dose of HTI-286 30 minutes later. Tumour growth was monitored using calipers 3 times per week up to 60 days after tumor cell implantation. Mice with tumours which have ulcerated or those mice with tumours >1 cm3 were euthanized.
  • EXAMPLE 11 Verteporfin and gef ⁇ tinib administration in a xenograft tumour model using JIMT-I human breast adenocarcinoma cells.
  • Figure 15 shows a decrease in tumor volume (A) and a decrease in mean fold increase of tumor volume (B) when verteporfin (10 mg/kg) is combined with gefitinib (i.e. Iressa 50 mg/kg) as compared to gefitinib alone or verteporfin alone in the herceptin- resistant JIMT-I breast cancer model.
  • A tumor volume
  • B mean fold increase of tumor volume
  • JIMT-I human breast adenocarcinoma cells were cultured in DMEM containing 10% FBS and were harvested at 80-90 % confluence using trypsin/EDTA. Cell viability was measured by trypan blue exclusion.
  • 0.5 xlO6 cells in 100 ⁇ l medium were injected subcutaneously into the lower back of 6- week old female Rag-2M immunodeficient mice. Animals were anesthetised with Isoflurane for inoculation. Drug treatment was initiated once the tumours reach a size of 100-150 mm 3 .
  • animals were randomized into six groups often animals each, within each group, 4 animals were sacrificed during treatment for biochemical measurement of autophagy and the remaining six animals were used for tumour growth measurements. Four groups as follows were tested: 1 saline control; 2 verteporfin 10 mg/kg; 3 gefitinib 50 mg/kg; and 4 verteporfin 10 mg/kg + gefitinib 50 mg/kg.
  • Drug administration 1 saline control
  • Tumour growth was monitored every Monday, Wednesday and Friday by measuring tumour dimensions with digital calipers once the tumours are palpable. Tumour volumes were calculated according to the equation L x W2 /2 with the length (mm) being the longer axis of the tumour. Animals were also weighed at the time of tumour measurement. Tumours were allowed to grow to a maximum of 1 cm3 before the animals were euthanized. Animals with ulcerated tumours were also euthanized. Upon
  • necropsy was performed to visually inspect major organs for signs of tumour growth. All animals were observed post administration, and at least once a day, more if deemed necessary, during the pre-treatment and treatment periods for mortality and morbidity. In particular, signs of ill health were based on body weight loss, change in appetite, and behavioural changes such as altered gait, lethargy and gross manifestations of stress. If severe toxicity or tumour-related illness was seen, the animals were euthanized (CO2 asphyxiation) and a necropsy was performed to assess other signs of toxicity. The following organs were examined: liver, gall bladder, spleen, lung, kidney, heart, intestine, lymph nodes and bladder.
  • tumour growth rates included tumour growth rates, tumour size on the day when controls were euthanized due to tumour progression (ulceration or tumour size in excess of 1 cm3), and Kaplan-Meier survival curves where survival was defined as the time when tumour size has increased 4-fold relative to the size when treatment was initiated

Abstract

Compositions comprising compounds of Formula IA and IB and cancer therapeutics are provided. Uses of and methods of using such compounds and compositions are provided for the modulation of autophagy. In particular, compounds and compositions may be for use in early stage autophagy inhibition and in the treatment of cancer. Furthermore, the treatment may be in combination with a cancer therapeutics agent.

Description

AUTOPHAGY INHIBITOR COMPOUNDS, COMPOSITIONS AND METHODS FOR THE USE THEREOF IN THE TREATMENT OF CANCER
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/272,049 entitled "COMPOSITIONS THAT INHIBIT OR INDUCE
AUTOPHAGY USEFUL FOR THE TREATMENT OF CANCER" filed on 11 August 2009.
TECHNICAL FIELD
This invention relates to therapeutics, their uses and methods for the modulation of autophagy. In particular the invention relates to compounds, compositions, therapies, and methods of treatment of cancer cells.
BACKGROUND
Autophagy is a catabolic process responsible for the bulk degradation of cytoplasmic components (1 ). It is characterized by formation of sequestering membranes called phagophores that expand, engulf cytoplasmic material and then fuse to form double- membraned autophago somes. The autophagosomes, undergo maturation via fusion with endosomal vesicles and ultimately fuse with lysosomes which deliver hydrolytic enzymes that breakdown sequestered molecules and release of the products into the cytoplasm for macromolecular synthesis. Constitutive autophagy functions as a housekeeping mechanism by controlling the turnover of long-lived proteins and organelles (2).
Autophagy also functions as an adaptive response to maintain cellular homeostasis; it is stimulated in response to cellular stresses such as nutrient depletion, oxidative stress, protein aggregation, and several cancer drugs (3, 4). By removing aberrant organelles and limiting the production of reactive oxygen species, autophagy is believed to protect cells from genotoxic stress and allow for cancer pathogenesis (5). This is supported by the observation that Beclin 1 and ultraviolet irradiation resistance-associated gene (UVRAG), which are involved in autophagosome assembly, also have tumour suppressor activity (6, 7). Furthermore, autophagy-deficient cells are susceptible to enhanced gene amplification and chromosomal instability, both of which may be considered tumourigenic
characteristics (8). It has been reported, that in established tumours autophagy is enhanced compared to corresponding noncancerous tissue (9), particularly in the tumour centre (10) which is generally poorly supplied with nutrients and oxygen. While the dependence on autophagy for tolerating nutrient deprivation is variable among different cancer cell types (3, 11), it has been demonstrated that suppression of autophagy can sensitize cells to apoptosis in a starved environment (9).
Multiple cancer therapy agents, such as temozolamide (12), tamoxifen (13), imatinib (14), and also ionising radiation, induce autophagy in human cancer cell lines (15, 16) and excessive autophagy has been implicated as a death mechanism, named type II programmed cell death (17). Accordingly, it has been proposed that stimulation of autophagy by cancer therapeutics can contribute to cancer cell death (16, 18). An alternative explanation for the increased autophagic activity observed in response to cancer therapy is that it constitutes a protective response against drug-induced cellular stress or vascular effects by clearing damaged organelles or replenishing nutrients (16). Studies using RNA interference (RNAi) technology to decrease autophagy while exposing cells to autophagy-stimulating cancer agents have been tried. For example, RNAi- mediated knockdown of the autophagy gene Atg5 resulted in decreased cell survival in tamoxi fen-treated MCF-7 cells (19) and in glioma cells treated with a DNA-damaging agent (20) or the tyrosine kinase inhibitor imatinib (14). Inhibition of autophagy also sensitizes tamoxifen-resistant T47D cells to treatment (19), further supporting a prosurvival role.
Despite the studies using RNAi to inhibit autophagy, chemical inhibitors with comparable specificity (1, 21, 22) are not well known. Most of the chemicals currently employed to inhibit autophagy act at a late stage of the process. For example,
lysosomotropic agents (chloroquine), V-ATPase inhibitors (bafilomycin Al) and lysosomal protease inhibitors (pepstatin A) all interfere with lysosomal function. They prevent the degradation of autophagosomes by lysosomal enzymes, leading to cytoplasmic accumulation of abnormal autophagosomes which can be toxic to cells (22).
Known early stage inhibitors of autophagy are compounds that inhibit
phosphatidylinosital 3 (PB)-kinases, such as 3 -MA, wortmannin, and LY294002. As PI3- kinase inhibitors, these compounds affect a number of cellular processes and are toxic after prolonged exposure (1, 21).
Verteporfin (CAS # 129497-78-5) is a benzoporphyrin derivative which has been used clinically for photodynamic therapy of age related macular degeneration (23).
Verteporfin is photoactivated for photodynamic therapy to eliminate the abnormal blood vessels in the eye associated with conditions such as the wet form of macular
degeneration. Verteporfin accumulates in these abnormal blood vessels and, when stimulated by nonthermal red light with a wavelength of 693 ran in the presence of oxygen, the photoactivated verteporfin produces highly reactive short-lived singlet oxygen and other reactive oxygen radicals, resulting in local damage to the endothelium and blockage of the vessels. Benzoporphoryrins, are described for example, in US patents 5,095,030, 5,214,036, and 6,008,241.
SUMMARY
This invention is based in part on the fortuitous discovery that benzoporphyrin derivatives (BPDs), as described herein, act as early stage autophagy inhibitors.
Specifically, compounds identified herein, show inhibition of early stage autophagy, which may be useful for the treatment of cancer. In particular, the treatment of cancer cells that use autophagy to resist other chemotherapeutic treatments or cells that are metabolically stressed cells. For example, nutrient deprived cancer cells. Furthermore, the activity of the compounds identified herein do not require any photo activation and the methods and uses do not require a photoactivation step. Accordingly, the compounds may be administered systemically or locally and may have the desired activity without the need to photoactivate in situ after administration.
The compounds described herein may be used for in vivo or in vitro research uses (i.e. non-clinical) to investigate the mechanisms of autophagy inhibition and the response of nutrient deprived cancer cells to various cancer therapeutic treatments. Furthermore, these compounds may be used individually or as part of a kit for in vivo or in vitro research to investigate autophagy inhibition, the response of metabolically stressed cancer cells maintained in culture, combination treatments of cancer cells maintained in culture, and/or animal models. Alternatively, the compounds described herein may be combined with commercial packaging and/or instructions for use.
This invention is also based in part on the discovery that the compounds described herein, may also be used to modulate autophagy activity either in vivo or in vitro for both research and therapeutic uses. The compounds may be used in an effective amount so that autophagy activity may be modulated. In particular, the compounds may be used to inhibit early stage autophagy activity. The compounds modulatory activity may be used in either an in vivo or an in vitro model for the study of cancer. For example, in early stage autophagy and in cells that are metabolically stressed (for example, nutrient deprived cancer cells or cells that have been or are about to be given a cancer chemotherapeutic agent. Furthermore, the compounds modulatory activity may be used for the treatment of cancer to sensitize cells to other cancer therapeutics or to cells that are already undergoing autophagy in response to a metabolic stress or cancer therapeutic. The cancer cells may be nutrient deprived cancer cells, hypoxic cancer cells or otherwise prone to enter autophagy in response to a cancer therapy. The autophagy may be early stage autophagy.
Furthermore, this invention is based in part on the appreciation that
benzoporphyrin derivatives, as described herein may be autophagy inhibitors, as described herein. Compounds identified herein, show inhibition of autophagy, which may be useful for the treatment of cancer in the absence of photoactivation. Compounds identified herein, may be useful as chemosensitizers for the treatment of cancer. Furthermore, the chemosensitization activity may occur in the absence of photoactivation of the compounds and in the absence of a photoactivation step. In particular, the treatment may be of nutrient deprived cancer cells or otherwise metabolically stressed cells. In particular the treatment may be of cancer cells undergoing early stage autophagy. In particular the treatment of nutrient deprived or otherwise metabolically stressed or treated cancer cells undergoing early stage autophagy or likely to undergo early stage autophagy.
In accordance with one embodiment, there is provided a method of modulating autophagy, the method including administering a compound of formula IA or IB to a cell to modulate autophagy, the compound of Formula IA or IB having the structure:
Figure imgf000005_0001
Formula IA Formula IB wherein R1 is selected from the group: CH2OH, CO2G1, CO2G1OG1,
CO2G1OG1OG1, and CO2G1OG1OG1OG1; R2 is selected from the group: CH2OH, CO2G2, CO2G2OG2, CO2G2OG2OG2, and CO2G2OG2OG2OG2; R3 is selected from the group: CH2OH, CO2G3, CO2G3OG3, CO2G3OG3OG3, and CO2G3OG3OG3OG3; wherein G1 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; G" is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and G3 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and wherein only one of R1, R , and R is COOH. The method may further include the administration of a cancer therapeutic agent.
The method modulation may occur in the absence of photoactivation. The modulation may be of early stage autophagy. The modulation may of a cancer cell. The cancer cell may be in an animal. The animal may be a human. The cancer therapeutic regimen may be selected from one or more of the agents set out in TABLE 3. The modulation may be for the treatment of cancer.
In accordance with a further embodiment, there is provided a method of sensitizing a cancerous cell to a cancer therapeutic agent, the method comprising: administering to a subject in need thereof, a compound of formula IA or IB. The sensitization occurs in the absence of photoactivation.
In accordance with a further embodiment, there is provided a compound of Formula IA or IB for the treatment of cancer.
In accordance with a further embodiment, there is provided a use of a compound of Formula IA or IB for treating cancer.
In accordance with a further embodiment, there is provided a use of a
pharmaceutical composition, the pharmaceutical composition including a compound of Formula IA or IB and a pharmaceutically acceptable carrier, for treating cancer.
In accordance with a further embodiment, there is provided a use of a compound of Formula IA or IB in the manufacture of a medicament for the treatment of cancer.
In accordance with a further embodiment, there is provided a commercial package comprising (a) the compound a compound of Formula IA or IB ; and (b) instructions for the use thereof for treating cancer.
In accordance with a further embodiment, there are provided pharmaceutical compositions which may include one or more compounds having Formula IA and IB and a cancer therapeutic agent in TABLE 3. Furthermore, the pharmaceutical compositions may further include a pharmaceutically acceptable excipient.
The treatment of cancer may be in the absence of photoactivation. The compounds and compositions described herein may be used in the absence of photoactivation. The treatment may be carried out in the absence of activating light to prevent photoactivation.
The sensitization may be of cancerous cells in early stage autophagy. The cancerous cell may be in an animal. The animal may be a human. The sensitization may precede the administration of a cancer therapeutic regimen. The sensitization may follow the administration of a cancer therapeutic regimen. Alternatively, the sensitization may be simultaneous with the administration of a cancer therapeutic regimen. The cancer therapeutic regimen may be selected from one or more of the agents set out in TABLE 3.
In accordance with a further embodiment, there is provided a pharmaceutical composition including one or more of the agents set out in TABLE 3 and a compound of Formula IA or IB. The compounds of Formula IA or IB may be active in the absence of photoactivation. The compounds of Formula IA or IB may be selected from any one or more of the compounds of TABLE 2.
R1 may be selected from the group: CH2OH, CO2G1, CO2G1OG1, and
CO2G1OG1OG1. R2 may be selected from the group: CH2OH, CO2G2, CO2G2OG2, and CO2G2OG2OG2. R3 may be selected from the group: CH2OH, CO2G3, CO2G3OG3, and CO2G3OG3OG3.
G1 may be hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group. G may be hydrogen or a linear, or branched, saturated or unsaturated one to ten carbon alkyl group. G1 may be hydrogen or a linear saturated or unsaturated one to ten carbon alkyl group. G1 may be hydrogen or a linear saturated one to ten carbon alkyl group. G1 may be hydrogen or a one to 9 carbon alkyl group. G1 may be hydrogen or a one to 8 carbon alkyl group. G1 may be hydrogen or a one to 7 carbon alkyl group. G1 may be hydrogen or a one to 6 carbon alkyl group. G1 may be hydrogen or a one to 5 carbon alkyl group. G1 may be hydrogen or a one to 4 carbon alkyl group. G may be hydrogen or a one to 3 carbon alkyl group. G1 may be hydrogen or a one to 2 carbon alkyl group.
G2 may be hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group. G may be hydrogen or a linear, or branched, saturated or unsaturated one to ten carbon alkyl group. G2 may be hydrogen or a linear saturated or unsaturated one to ten carbon alkyl group. G may be hydrogen or a linear saturated one to ten carbon alkyl group. G may be hydrogen or a one to 9 carbon alkyl group. G may be hydrogen or a one to 8 carbon alkyl group. G2 may be hydrogen or a one to 7 carbon alkyl group. G2 may be hydrogen or a one to 6 carbon alkyl group. G2 may be hydrogen or a one to 5 carbon alkyl group. G may be hydrogen or a one to 4 carbon alkyl group. G2 may be hydrogen or a one to 3 carbon alkyl group. G2 may be hydrogen or a one to 2 carbon alkyl group.
G3 may be hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group. G3 may be hydrogen or a linear, or branched, saturated or unsaturated one to ten carbon alkyl group. G3 may be hydrogen or a linear saturated or unsaturated one to ten carbon alkyl group. G may be hydrogen or a linear saturated one to ten carbon alkyl group. G3 may be hydrogen or a one to 9 carbon alkyl group. G3 may be hydrogen or a one to 8 carbon alkyl group. G3 may be hydrogen or a one to 7 carbon alkyl group. G3 may be hydrogen or a one to 6 carbon alkyl group. G3 may be hydrogen or a one to 5 carbon alkyl group. G3 may be hydrogen or a one to 4 carbon alkyl group. G may be hydrogen or a one to 3 carbon alkyl group. G3 may be hydrogen or a one to 2 carbon alkyl group.
1 9 ^
R , R", and R may be independently selected from one of more of the following: COOH; CH2OH; CO2CH2OH; CO2CH2OCH2OH; CO2CH2OCH2OCH2OH;
CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; CO2(CH2)2O(CH2)2O(CH2)2OH; CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH; CO2(CH2)3O(CH2)3O(CH2)3OH; CO2(CH2)4OH;
CO2(CH2)4θ(CH2)4θH; CO2(CH2)4O(CH2)4O(CH2)4OH; CO2(CH2)5OH;
CO2(CH2)5O(CH2)5OH; CO2(CH2)5O(CH2)5O(CH2)5OH; CO2(CH2)6OH;
CO2(CH2)6O(CH2)6OH; CO2(CH2)6O(CH2)6O(CH2)6OH; CO2(CH2)7OH;
CO2(CH2)7O(CH2)7OH; CO2(CH2)7O(CH2)7O(CH2)7OH; CO2(CH2)8OH;
CO2(CH2)gO(CH2)8OH; CO2(CH2)8O(CH2)8O(CH2)8OH; CO2(CH2)9OH;
CO2(CH2)9O(CH2)9OH; CO2(CH2)9O(CH2)9O(CH2)9θH; CO2(CH2)10OH;
C02(CH2),00(CH2)ioOH; CO2(CH2)10O(CH2)10O(CH2)10OH; CO2CH3; CO2CH2OCH3; CO2CH2OCH2CH3; CO2CH2OCH2OCH2CH3; CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3; CO2(CH2)2O(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; CO2(CH2)3O(CH2)3CH3;
CO2(CH2)3O(CH2)3O(CH2)3CH3; CO2(CH2)4CH3; CO2(CH2)4O(CH2)4CH3;
CO2(CH2)4O(CH2)4O(CH2)4CH3; CO2(CH2)5CH3; CO2(CH2)5O(CH2)5CH3;
CO2(CH2)5O(CH2)2O(CH2)5CH3; CO2(CH2)6CH3; CO2(CH2)6O(CH2)6CH3; CO2(CH2)6θ(CH2)6O(CH2)6CH3; CO2(CH2)7CH3; CO2(CH2)7O(CH2)7CH3;
CO2(CH2)7O(CH2)7O(CH2)7CH3; CO2(CH2)8CH3; CO2(CH2)8O(CH2)8CH3;
CO2(CH2)8θ(CH2)8θ(CH2)8CH3; CO2(CH2)9CH3; CO2(CH2)9O(CH2)9CH3;
Cθ2(CH2)9θ(CH2)9θ(CH2)9CH3; CO2(CH2)!0CH3; CO2(CH2) ,0O(CH2) 10CH3; and C02(CH2)ioO(CH2)ioO(CH2)10CH3; provided only one of R1, R2, and R3 is COOH.
R1, R2, and R3 may be independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH;
CO2CH2OCH2OH; CO2CH2OCH2OCH2OH; CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH; CO2CH2OCH3; CO2CH2OCH2CH3; CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3;
CO2(CH2)3CH3; and CO2(CH2)3O(CH2)3CH3; provided only one of R1, R2, and R3 is COOH.
R1, R~, and R3 may be independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2CH2OCH3; CO2(CH2)2OH;
CO2(CH2)2O(CH2)2OH; CO2CH2OCH2OH; CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH; CO2CH2OCH2CH3; CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; and CO2(CH2)3O(CH2)3CH3; provided only one of R1, R2, and R3 is COOH.
R1, R2, and R3 may be independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; and CO2CH2OCH2OH; provided only one of R1, R2, and R3 is COOH.
The compound may be selected from any one or more of the following compounds:
Figure imgf000009_0001
Figure imgf000010_0001
(also shown in TABLE 2). BRIEF DESCRIPTION OF THE DRAWINGS
Figure IA. shows a schematic model of Autophagy.
Figure IB. shows a schematic early and late stage (punctuate accumulation of
autophago somes) autophagy in MCF7 cells stably transfected with pEGFP- LC3.
Figure 2 A. shows a cell-based assay for the identification of inhibitors of
autophagosome accumulation and identification of verteporfin as an inhibitor of autophagy. MCF-7 EGFP-LC3 cells were exposed to 0.1 % DMSO (a, b), 10 mM 3-MA (c, d), or 10 μM verteporfin (e, f) without (a, c, e) or with 30 μM chloroquine (b, d, f) for 4 h in complete cell culture medium. The images were acquired by confocal microscopy. Scale bar, 10 μm.
Figure 2B. Cells were exposed to 30 μM chloroquine and different concentrations of verteporfin for 4 h in complete medium. The cells were fixed and stained and punctate EGFP-LC3fluorescence was quantitated using a Cellomics Arrayscan VTI automated imager. The level of punctuate EGFP-LC3 fluorescence observed in control DMSO-treated cells is indicated by the dotted line, (mean ± S.D., n=3).
Figure 3. shows that verteporfin inhibts autophagosome formation in serum starved cells.
Figure 4. shows that verteporfin inhibits autophagosome accumulation stimulated by rapamycin or serum starvation. MCF-7 EGFP-LC3 cells were exposed for 4 h to 30 nM rapamcycin without (a) or with 10 μM verteporfin (b) in complete cell culture medium or to medium lacking serum without (c) or with 10 μM verteporfin (d). Images were acquired by confocal microscopy. Scale bar, 10 μm.
Figure 5. shows an ultrastructural examination of inhibition of CQ-induced
autophagosome accumulation by verteporfin. MCF-7 EGFP-LC3 cells were exposed for 4 h to 0.1% DMSO (a, b) or 75 μM CQ (c, d) without (a, c) or with 10 μM verteporfin (b, d) and then images were acquired by transmission electron microscopy, with arrowheads pointing to autophagic vesicles (Scale bar, 0.5 μm). Figure 6. shows inhibition of EGFP-LC3 degradation and long-lived protein degradation by verteporfin. (A-C), MCF-7 EGFP-LC3 cells were treated for 4 h with (A) 30 nM rapamycin without or with 10 niM 3 -MA or 100 nM bafilomycin Al in complete medium; (B) different concentrations of verteporfin without or with 30 nM rapamycin in complete medium; (C) 10 μM verteporfin in complete medium or in serum-free medium. (A-C), cells were exposed to 0.1% DMSO as a vehicle control and EGFP-LC3 processing and degradation was monitored by western blotting with anti- GFP antibody. (D) The amount of [14C]valine-labeled long-lived protein degradation was measured in MCF-7 EGFP-LC3 cells treated with or without 10 μM verteporfin in complete medium or in serum-free medium for 18 h. (mean± S.D., n=3). *, p<0.05 versus corresponding DMSO treatment.
Figure 7. shows inhibition of the sequestration of cytosolic FITC-dextran into
vesicles by verteporfin. FITC-dextran was scrape-loaded into MCF-7 cells and its distribution was analyzed by confocal microscopy, wherein DMSO- treated MCF-7 cells were fixed 2 h (a) or 24 h (b, c) after FITC-dextran loading and MCF-7 cells loaded with FITC-dextran were incubated in 10 mM 3 -MA (d) or 10 μM verteporfin (e) for 24 h in complete medium, following which cells were fixed, DNA was stained and images were acquired by confocal microscopy (Scale bar, 10 μm).
Figure 8. shows verteporfin does not inhibit LC3 processing or membrane
association. (A) MCF-7 cells were exposed to 0.1% DMSO, 30 nM rapamycin, or 75 μM chloroquine without or with 10 μM verteporfin for 4 h in complete medium. Endogenous LC3 processing was examined by western blotting with anti-LC3 antibody and protein loading was monitored with anti-β tubulin antibody. (B) MCF-7 cells were exposed to 0.1% DMSO or 75 μM chloroquine without and with 10 μM verteporfin for 4 h in complete medium. Lysates were subjected to ultracentrifugation and the resulting supernatant (S) and pellet (P) fractions were immunoblotted with anti-LC3 antibody. Subcellular fractionation and protein loading was monitored by immunoblotting for the membrane-marker, Transferrin Receptor (TfR), and the cytosolic marker, β tubulin. Figure 9. shows verteporfin is able to sensitize cells to starvation in various cell lines (i.e. (A) MCF7, (B) HeLa, (C) HT29, and (D) H460 cells). Effect of verteporfin on cell survival in different starvation conditions. MCF-7 cells were incubated for 8 h and HeLa, HT29, and H460 cells were incubated for 24 h with different concentrations of verteporfin in complete medium or in the different starvation conditions as shown. Verteporfin and medium were washed away and the cells were incubated for 40 h in complete medium without verteporfin before measuring cell viability using the MTT assay. (mean ± S.D., n=4).
Figure 10. shows the effect of selected verteporfin analogues on chloroquine-induced autophagosome accumulation, wherein (A) shows the structures of protoporphyrin IX, verteporfin, and benzopophyrin derivatives tested in (B). In (B) MCF-7 EGFP-LC3 cells were incubated for 4 h in complete medium with different concentrations of each of the tested verteporfin analogues in the presence of 75 μM chloroquine. Punctate EGFP-LC3 was quantitated using the automated microscopy assay (mean ± S. D., n=3).
Figure 11. shows the effect of additional selected verteporfin analogues B BPD hexyl ester dimethyl ester, B BPD hexyl ester diacid, B BPD diethylene glycol ester, A BPD diethylene glycol ester, and verteporfin (A) and A BPD tri(ethylene glycol) ester, A BPD tri(diethylene glycol) ester, and verteporfin (B) on chloroquine-induced autophagosome accumulation, wherein autophagosome accumulation is plotted against varying concentrations of different porphyrins, including verteporfin.
Figure 12. shows a summary of autophagy inhibitors tested for autophagosome
accumulation in the presence of chloroquine (75 μM), whereby a reduction in autophagosome accumulation was considered evidence of an autophagy inhibitor.
Figure 13. (A) shows PK values for Visudyne™ (verteporfin) in HT-29 tumours in
Rag6 mice (n=4) dosed at 20 mg/kg as a single bolus compared to a single bolus followed with a dose of HTI-286 at 0.5 hrs (tumour CMAX of verteporfin following iv dosing) (B)shows PK values for Visudyne™ (verteporfin) in HT-29 tumours in Rag6 mice (n=4) dosed at 10 and 20 mg/kg as a single i.p. dose. Based on AUC, an increase of 1.6x on the doubling of dose (27464 at 10 mg/kg and 44283 at 20 mg/kg).
Figure 14. shows normalized efficacy of verteporfin dosed qdxl i.v. in combination with HTI-286 dosed i.v. (administered 30 minutes post verteporfin at tumour CMAX) compared to saline and HTI-286 controls. The study was performed nude mice (n=5) bearing HT-29 colon adenocarcinoma xenografts.
Figure 15. shows a decrease in tumor volume when verteporfin (10 mg/kg) is
combined with gefitinib (i.e. Iressa 50 mg/kg) as compared to gefitinib alone or verteporfin alone in the herceptin-resistant JIMT-I breast cancer model.
DETAILED DESCRIPTION
As used herein, "early stage autophagy", is the process of autophagosome formation and the sequestration of cytoplasmic material into autophagosomes.
As used herein, "nutrient deprived cancer cells" may be cancer cells that are undergoing autophagy, wherein autophagy may occur due to a metabolic stress such as nutrient deprivation or hypoxia. Nutrient deprivation may be induced by a metabolic stress promoting agent. Cancer cells may be nutrient deprived due to a lack of blood flow or access to sufficient nutrients, wherein nutrient deprived cells may be deficient in oxygen, serum, amino acids, sugar (for example, glucose) or any combination thereof.
As used herein, "MCF7" cells are human breast cancer cells, "HeLa" cells are human cervical cancer cells, "HT29" cells are human colorectal cancer cells, and "H460" cells are human large cell lung cancer cells.
"Cancer" as used herein refers to a proliferative or neoplastic disorder caused or characterized by the proliferation of cells which have lost susceptibility to normal growth control. The term cancer, as used in the present application, includes tumors and any other proliferative disorders. Cancers of the same tissue type usually originate in the same tissue, and may be divided into different subtypes based on their biological characteristics. Four general categories of cancers are carcinoma (epithelial tissue derived), sarcoma (connective tissue or mesodermal derived), leukemia (blood-forming tissue derived) and lymphoma (lymph tissue derived). Over 200 different types of cancers are known, and every organ and tissue of the body may be affected. Specific examples of cancers that do not limit the definition of cancer may include melanoma, pancreatic, cervical, colorectal. Examples of organs and tissues that may be affected by various cancers include pancreas, breast, brain, thyroid, ovary, uterus, testis, prostate, thyroid, pituitary gland, adrenal gland, kidney, stomach, esophagus or rectum, head and neck, bone, nervous system, skin, blood, nasopharyngeal tissue, lung, urinary tract, cervix, vagina, exocrine glands and endocrine glands. Alternatively, a cancer may be multicentric or of unknown primary site (CUPS).
As used herein, a "cancerous cell" refers to a cell that has undergone a
transformation event and whose growth is no longer regulated to the same extent as before said transformation event. A "tumor" refers to a collection of cancerous cells, often found as a solid or semi-solid lump in or on the tissue or a patient or test subject.
As used herein, a "chemosensitizer" or "sensitizer" is a medicament that may enhance the therapeutic effect of a cancer therapeutic agent, and therefore improve efficacy of such treatment or agent. The sensitivity or resistance of a tumor or cancerous cell to treatment may also be measured in an animal, such as a human or rodent, by, e.g., measuring the tumor size, tumor burden or incidence of metastases over a period of time. For example, about 2, about 3, about 4 or about 6 months for a human and about 2-4, about 3-5, or about 4-6 weeks for a mouse. A composition or a method of treatment may sensitize a tumor or cancerous cell's response to a therapeutic treatment if there is an increase in sensitivity to the treatment or where there is a reduction in the amount of therapeutic treatment needed to achieve the same result. An increase in sensitivity may be about 10% or more, for example, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more compared to the absence of such composition or method. Alternatively, a reduction in the amount of therapeutic treatment needed to achieve the same result may be about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, compared to the absence of such composition or method. The determination of sensitivity to a therapeutic treatment is routine in the art and within the skill of a person versed in the art.
Verteporfin (CAS # 129497-78-5) as used herein may include the two regioisomers as shown below:
Figure imgf000016_0001
and the 2 entantiomers of each of the two regioisomers as shown below:
Figure imgf000016_0002
Figure imgf000016_0003
The verteporfin as disclosed herein contains at least one chiral center and thus may exist in various stereoisomeric forms. If desired, such stereoisomers, including enantiomers, may be separated using techniques standard in the art (for example, chiral columns). However, racemic mixtures or mixtures containing more than one diastereomer may also be used and are contenplated herein. However, the compounds tested herein were in either of the trans entantiomers shown above. The compounds shown in Formulas IA, IB, Tables 1, 2 and Figure 10, are representative of the individual optical isomers, enantiomers or diastereomers as the case may be, as well as mixtures of these individual chiral isomers.
Visudyne™, as used herein, is the liposomal formulation of verteporfin used in humans for photodynamic therapy. Visudyne™ is given intravenously, usually within 15 minutes prior to laser treatment to eliminate the abnormal blood vessels in the eye in the treatment of wet macular degeneration. The verteporfin compound accumulates in these abnormal blood vessels and, when stimulated by a nonthermal red light laser with a wavelength of 693 nm in the presence of oxygen, produces highly reactive short-lived singlet oxygen and other reactive oxygen radicals, resulting in local damage to the endothelium and blockage of the vessels. Patients given Visudyne™ experience photosensitivity and are advised to avoid exposure to sunlight and unscreened lighting for at least 48 hours after the injection of verteporfin.
In contrast to the current use of verteporfin in photodynamic therapy, subjects administered the BPDs described herein, in accordance with the methods and uses described herein, do not require photoactivation of the BPD via nonthermal red light laser with a wavelength of 693 nm or otherwise. The activity of the BPDs to inhibit early stage autophagy is independent of the activity associated with photoactivation and would likely be hindered by photoactivation. Accordingly, a person of skill in the art would appreciate that the precautions associated with photosensitivity should also apply to the present methods and uses (i.e. avoid exposure to sunlight and unscreened lighting for at least 48 hours after the injection of of the BPD).
As used herein, the phrase "Cx-y alkyl" or "Cx-Cy alkyl" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that has a carbon skeleton or main carbon chain comprising a number from x to y (with all individual integers within the range included, including integers x and y) of carbon atoms. For example a "C1-1O alkyl" is a chemical entity that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atom(s) in its carbon skeleton or main chain.
As used herein, the term "branched" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises a skeleton or main chain that splits off into more than one contiguous chain. The portions of the skeleton or main chain that split off in more than one direction may be linear, cyclic or any combination thereof. Non-limiting examples of a branched alkyl are tert-butyl and isopropyl. As used herein, the term "unbranched" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises a skeleton or main chain that does not split off into more that one contiguous chain. Non-limiting examples of unbranched alkyls are methyl, ethyl, n-propyl, and n-butyl.
As used herein, the term "substituted" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that has one chemical group replaced with a different chemical group that contains one or more heteroatoms. Unless otherwise specified, a substituted alkyl is an alkyl in which one or more hydrogen atom(s) is/are replaced with one or more atom(s) that is/are not hydrogen(s). For example, chloromethyl is a non-limiting example of a substituted alkyl, more particularly an example of a substituted methyl. Aminoethyl is another non-limiting example of a substituted alkyl, more particularly it is a substituted ethyl. The functional groups described herein may be substituted with, for example, and without limitation, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents.
As used herein, the term "unsubstituted" is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that is a hydrocarbon and/or does not contain a heteroatom. Non-limiting examples of unsubstituted alkyls include methyl, ethyl, tert-butyl, and pentyl.
As used herein, the term "saturated" when referring to a chemical entity is used as it is normally understood to a person of skill in the art and often refers to a chemical entity that comprises only single bonds. Non-limiting examples of saturated chemical entities include ethane, tert-butyl, and N+H3.
Non-limiting examples of saturated C1-CiO alkyl may include methyl, ethyl, n- propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, i-pentyl, sec- pentyl, t-pentyl, n-hexyl, i-hexyl, 1,2-dimethylpropyl, 2-ethylproρyl, l-methyl-2- ethylpropyl, 1 -ethyl-2-methylpropyl, 1 , 1 ,2-trimethylpropyl, 1,1,2-triethylpropyl, 1,1- dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, 1,3-dimethylbutyl, 2-methylpentyl, 3- methylpentyl, sec-hexyl, t-hexyl, n-heptyl, i-heptyl, sec-heptyl, t-heptyl, n-octyl, i-octyl, sec-octyl, t-octyl, n-nonyl, i-nonyl, sec-nonyl, t-nonyl, n-decyl, i-decyl, sec-decyl and t- decyl. Non-limiting examples Of C2-CiO alkenyl may include vinyl, allyl, isopropenyl, 1- propene-2-yl, 1-butene-l-yl, l-butene-2-yl, l-butene-3-yl, 2-butene-l-yl, 2-butene-2-yl, octenyl and decenyl. Non-limiting examples OfC2-Ci0 alkynyl may include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl. Saturated Ci-Cio alkyl, C2-C)O alkenyl or C2-CiO alkynyl may be, for example, and without limitation, interrupted by one or more heteroatoms which are independently nitrogen, sulfur or oxygen.
The embodiments involving the formulae as described herein include all possible stereochemical alternatives, including those illustrated or described herein.
In some embodiments, the compounds as described herein or acceptable salts thereof above may be used for systemic treatment of a cancer. In some embodiments, the compounds as described herein or acceptable salts thereof above may be used in the preparation of a medicament or a composition for systemic treatment of a cancer. In some embodiments, methods of systemically treating any of the cancers described herein are also provided. Some embodiments, make use of compositions comprising a compound described herein and a pharmaceutically acceptable excipient or carrier. Methods of treating any of the indications described herein are also provided. Such methods may include administering a compound as described herein or a composition of a compound as described herein, or an effective amount of a compound as described herein or composition of a compound as described herein to a subject in need thereof.
Compounds as described herein may be in the free form or in the form of a salt thereof. In some embodiments, compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (Berge et al., J. Pharm. Sd. 1977, 66, 1 ). Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable).
In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, polymorphs, isomeric forms) as described herein may be in the solvent addition form, for example, solvates. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent in physical association with the compound or salt thereof. The solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.
In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, isomeric forms) as described herein may include crystalline and/or amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound.
Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and/or solubility. Those skilled in the art will appreciate that various factors including
recrystallization solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate.
In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, polymorphs) as described herein include isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and
combinations thereof, and are not limited by the description of the formula illustrated for the sake of convenience.
In some embodiments, pharmaceutical compositions in accordance with this invention may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt. Pharmaceutical preparations will typically comprise one or more carriers, excipients or diluents acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers, excipients or diluents include those known in the art for use in such modes of administration.
Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K. For enteral administration, the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable
formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogels, or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known to one of skill in the art are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20th ed., Lippencott Williams & Wilkins, (2000). Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. The formulations may be specifically prepared for intranasal delivery. For example, nasal inhalation.
Compounds or pharmaceutical compositions in accordance with this invention or for use in this invention may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.
An "effective amount" of a pharmaceutical composition as described herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduced cancer burden, increased life span or increased life expectancy. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as less severe infection or delayed or no onset, increased life span, increased life expectancy or prevention of the progression of infection. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
In some embodiments, compounds and all different forms thereof as described herein may be used, for example, and without limitation, in combination with other treatment methods.
In general, compounds described herein should be used without causing substantial toxicity. Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LDlOO (the dose lethal to 100% of the population). In some circumstances, however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions. Some compounds described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Toxicity may be evaluated by examining a particular compound's or composition's specificity across cell lines. Animal studies may be used to provide an indication if the compound has any effects on other tissues.
Compounds as described herein may be administered to a subject. As used herein, a "subject" may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or having cancer. Diagnostic methods for cancer, and the clinical delineation of cancer, are known to those of ordinary skill in the art. TABLE 1 shows compounds made and tested for early stage autophagy modulatory activity.
TABLE 1: BENZOPORPHYRIN DERIVATIVES TESTED FOR AUTOPHAGY MODULATION
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
TABLE 2 shows compounds having early stage autophagy inhibitory activity. TABLE 2: BENZOPORPHYRIN DERIVATIVES HAVING AUTOPHAGY INHIBITORY ACTIVITY
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
A "cancer therapeutic regimen" may be selected from a cancer therapeutic agent or a combination of cancer therapeutic agents.
A "cancer therapeutic agent" may be a metabolic stress promoting agent or an autophagy inducing agent, which may include a nutrient deprivation promoting agent or hypoxia inducing agent, which may include a vascular targeting agent or an anti- angiogenic agent, wherein the cancer therapeutic agent may be, for example, one or more of the agents set inTABLE 3. For example, wherein the the vascular targeting agent may be selected from one or more of combretastatin A-4-phosphate, combretastatin analogues, HTI-286, ZD6126, or OXI 4503, and wherein an anti-angiogenic agent may be selected from one or more of, endostatin, TNP-470, or VEGF trap (see also, Table 1 in Roy and Debnath Semin Immunopathol (2010) Published online 30 June 2010 or one or more of the treatments set out in Livesey K. M. et al. Current Opinion in Investigational Drugs (2009) 10(12):1269-1279).
For example, as described in Example 1 1 and Figure 15, Gefitinib (CAS # 184475- 35-2 sold by AstraZeneca™ under the trade name Iressa™) was tested in combination with verteporfin. Gefitinib inhibits the epidermal growth factor receptor (EGFR) tyrosine kinase by binding to the adenosine triphosphate (ATP)-bmding site of EGFR, which leads to inhibition of EGFR tyrosine kinase activation of the Ras signal transduction cascade, which m turn results in malignant cell inhibition Gefitmib is a selective inhibitor of the EGFR tyrosine kinase (i e Herl or ErbB-1) EGFR is known to be overexpressed in human carcinomas such as lung and breast cancers, which may lead to inappropriate activation of the apoptotic Ras signal transduction cascade and to uncontrolled cell proliferation
TABLE 3 shows cancer therapeutic agents
TABLE 3: EXAMPLES OF CANCER THERAPEUTIC AGENTS
ANGIOGENESIS INHIBITORS
Bevacizumab (Humanized anti-VEGF antibody) CAS # 216974-75-3 A recombinant humanized monoclonal IgGl antibody that binds to and inhibits the biologic activity of human vascular endothelial growth factor (VEGF) approved for the treatment of metastatic colorectal cancer
Cetuximab (Humanized anti-EGF receptor (EGFr) antibody) CAS # 205923-56-4 A chimeric IgGl monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR) preventing hgand binding and activation of the receptor This blocks downstream signaling of EGFR, inhibiting cell proliferation and angiogenesis, among other effects Metastatic colorectal cancer, head and neck cancer
Aflibercept / VEGF-trap (Antibody fusion protem) CAS # 845771-78-0: Aflibercept is a potent angiogenesis inhibitor, which blocks all VEGF-A isoforms plus VEGF-B and placental growth factor
(PIGF) additional angiogenic growth factors that appear to play a role in tumor angiogenesis
Sunitinib (N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-lH-indol-3-ylidene)methyl]-2,4-dimethyl-lH- pyrrole-3-carboxamide) CAS # 557795-19-4 approved by the FDA for the treatment of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumor (GIST)
Sorafenib (4-[4-[[4-chloro-3-(tπfluoromethyl)phenyl]carbamoylammo]phenoxy]-N-methylpyπdme-2- carboxamide) CAS # 284461-73-0 is approved for the treatment of advanced renal cell carcinoma (primary kidney cancer) and has received "Fast Track" designation by the FDA for the treatment of advanced hepatocellular carcinoma (primary liver cancer) ^^^_^^__
Vandetanib / ZD6474 (4-(4-Bromo-2-fluoroanilmo)-6-methoxy-7-[(l-methylpipendin-4- yl)methoxy]quinazolme) CAS # 443913-73-3 An orally bioavailable 4-anilinoquinazolme Vandetanib selectively inhibits the tyrosine kinase activity of vascular endothelial growth factor receptor 2 (VEGF2), thereby blocking VEGF-stimulated endothelial cell proliferation and migration and reducing tumor vessel permeability This agent also blocks the tyrosine kinase activity of epidermal growth factor receptor (EGFR) a receptor tyrosine kinase that mediates tumor cell proliferation and migration and angiogenesis This thyroid cancer drug candidate in clinical tnals
Vatalanib / PTK787 (N-(4-Chlorophenyl)-4-(4-pyridinylmethyl)-l-phthalazinamine dihydrochloπde) CAS # 212141-51-0 is a small molecule protein kinase inhibitor that inhibits angiogenesis It is being studied as a possible treatment for several types of cancer, particularly cancer that is at an advanced stage or has not responded to chemotherapy Vatalanib is orally active
Axitinib / AG-013736 (N-Methyl-2 ((3-((lE)-2-( yπdine-2-yl)ethenyl)-lH-indazol-6- yl)sulfanyl)benzamide) CAS # 319460-85-0. Axitmib is an oral selective inhibitor of vascular endothelial growth factor receptors (VEGFR) 1 , 2 and 3 Axitinib is being studied as both a single agent and in combination across many tumor types, including as a treatment for renal cell carcinoma and non-small cell lung cancer
BIBF-1120 ((Z)-methyl 3-(((4-(N-methyl-2-(4-methylpiperazin-l- yl)acetamido)phenyl)ammo)(phenyl)rnethylene)-2-oxoindohne-6-carboxylate) CAS # 928326-83-4:
Tyrosine kinase inhibitor that inhibits three growth factor receptors simultaneously vascular endothelial
Figure imgf000035_0001
Figure imgf000036_0001
Temozolamide (3-methyl-4-oxoimidazo[5,l-d][l,2,3,5]tetrazine-8-carboxamide) CAS# 85622-93-1
Tamoxifen (2-[4-[(Z)-1 , 2-di(phenyl)but-l-enyl]phenoxy]-N,N-dimethylethanamine) CAS# 10540-29-1
Trastuzumab (Humanized anti-HER2 antibody) CAS# 180288-69-1
2-deoxy-d-glucose ((3R,4S,5S)-3,4,5,6-tetrahydroxyhexanal Compound ID 10352022
Arginine deiminase (a hydrolase enzyme that catalyzes the chemical reaction L-argmine + H2O to L- citrulline + NH, ) CAS# 9027-98-9
8-aminoadenosine (2-(6,8-diammopuπn-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol) CID 259812
Temozolomide (3-methyl-4-oxoimidazo[5.1-d][l,2,3,5]tetrazine-8-carboxamide) CAS# 85622-93-1
Camptothecin / CPT (4-ethyl-4-hydroxy-l,12-dihydro-4h-2-oxa-6,12a-diaza- Dibenzo[b,h]fluorene-3,13- dione) CAS# 7689-03-4
Gemcitabine (4-amino-l-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2- one) CAS# 95058-81-4
Polyinosine-polycytidylic acid / PIC (poly[(2R,3S.4R,5R)-5-(4-ammo-2-oxopyπmidm-l-yl)-3,4- dihydroxyoxolan-2-yl]methyl dihydrogen phosphate, or poly[(2R,3S,4R,5R)-3,4-dihydroxy-5-(6-oxo-3H- punn-9-yl)oxolan-2-yl]methyl dihydrogen phosphate) CAS# 24939-03-5
Delta(9-ll)-tetrahydrocannabinol ((6aS)-6,6-dimemyl-9-methylidene-3-pentyl-7,8,10,10a-tetrahydro- 6aH-benzo[c]chromen-l-ol) CID 9883341
IxabepiIone ((lS,3S,7S,10R,l lS,12S,16R)-7,l l-dihydroxy-8,8,10,12,16-pentamethyl-3-[(E)-l-(2-methyl- l,3-thiazol-4-yl)prop-l-en-2-yl]-17-oxa-4-azabicyclo[14 1 0]heptadecane-5,9-dione) CID 6445540
Capecitabine (pentyl N-[l-f(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidm-
4-yl]carbamate) CAS# 154361-50-9
Oxaliplatin ((lR,2R)-cyclohexane-l,2-diamme, oxalate, platinum(+2) cation) CAS# 61825-94-3
Carboplatin (azanide, cyclobutane-l,l-dicarboxylic acid, platinum) CAS# 41575-94-4
Paclitaxel (5 beta,20-Epoxy-l,2a,4,7 beta, 10 beta, 13 alpha-hexahydroxytax-1 l-en-9-one 4,10-diacetate 2- benzoate 13-ester with (2 R,3S)-N-benzoyl-3-phenylisoseπne) CAS# 33069-62-4
Bortezomib ([(lS)-3-methyl-l-[[(2R)-3-phenyl-2-(pyrazine-2- carbonylamino)propanoyl]ammo]butyl]boromc acid) CAS# 179324-69-7
Gemcitabine (4-amino-l-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oλolan-2-yl]pyπmidm-2- one) CAS# 95058-81-4
Docetaxel CAS# 114977-28-5
Histone deacetylase inhibitors (HDAC)
Vorinostat / suberoylanilide hydroxamic acid (SAHA) - (N-hydroxy-N'-phenyl-octanediamide) CAS # 149647-78-9 Vormostat is marketed under the name Zolinza for the treatment of cutaneous T cell lymphoma when the disease persists, gets worse, or comes back during or after treatment with other medicines
Vitamin D3 analogs: Vitamin D3 analogs are currently in clinical trials for the treatment of various malignancies
Seocalcitol / EB-1089 ((lr,3s,5z)-5-((2e)-((lr,3as,7ar)-l-((lr,2e,4e)-6-ethyl-6-hydroxy-l-methyl-2,4- octadienyl)octahydro-7a-methyl-4h-mden-4-ylidene)ethylidene)-4-methylene- 1 ,3-cyclohexanediol) CAS # 134404-52-7 The vitamin D3 analog Seocalcitol is effective in the growth arrest and induction of cell death in various malignant cells Cell death following Seocalcitol administration in breast cancer cells is associated with autophagy
mTOR inhibitors
Rapamycin (23,27-Epoxy-3H-ρyπdo[2,l-c]fl,4]oxaazacyclohentπacontine) CAS # 53123-88-9 This macrolide antibiotic inhibits the mTORCl complex and is in advanced clinical tπals for the treatment of vaπous malignancies
Temsirolimus / CCI-779 (Rapamycm 42-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate) CAS # 162635-04-3 This rapamycm derivative is showing promising results m advanced clinical tπals
Everolimus / RADOOl (dihydroxy-12-[(2i?)-l-[(lS",3Λ,4Λ)-4-(2-hydroxyethoxy)-3- methoxycyclohexyl]propan-2-yl]- 19,30-dimethoxy- 15,17.21 ,23 ,29,35-hexamethyl- 11 ,36-dioxa-4- azatπcyclo[30 3 1 04 9]hexatπaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone) CAS # 159351-69-6 Approved for advanced renal cell carcinoma after failure of treatment with sumtinib or sorafenib
Perhexiline (2-(2,2-dicyclohexylethyl)piρeπdme) CAS # 6621-47-2
Niclosamide (5-chloro-N-(2-chloro-4-mtrophenyl)-2-hydroxybenzamide) CAS # 50-65-7
Amiodarone ((2-{4-[(2-butyl-l-benzofuran-3-yl)carbonyl]-2,6-diiodophenoxy}ethyl)diethylamme) CAS # 1951-25-3
Rottlerin / Kamalin (l-[6-[(3-acetyl-2,4,6-tπhydroxy-5-methylphenyl)methyl]-5,7-dihydroxy-2,2- dimethylchromen-8-yl]-3-phenylprop-2-en-l-one) CAS # 82-08-6
Curcumin ((lE,6E)-l,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1.6-diene-3,5-dione) CAS # 91884-86-5
Resveratrol (5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-l,3-diol) CAS # 9010-10-0
AICAR ([(2R,3 S ,4R,5R)-5-(5-amino-4-carbamoylimidazol- 1 -yl)-3 ,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate) CAS # 3031-94-5
Metformin (3-(diaminomethyhdene)-l ,l-dimethylguanidιne hydrochloride) CAS # 56258-19-6
Perifosine ((l.l-dimethylpipeπdin-l-ium-4-yl) octadecyl phosphate) CAS # 25360-07-0
GSK 690693 (4-[2-(4-amino- l,2,5-oxadiazol-3-yl)-l-ethyl-7-[[(3S)-pipeπdin-3-yl]methoxy]imidazo[4,5- c]pyπdm-4-yl]-2-methylbut-3-yn-2-ol) CAS # 937174-76-0
Triciribine (l ,5-Dihydro-5-methyl-l-beta-D-πbofuranosyl-1.4,5,6,8-pentaazaacenaphthylen-3-amme) CAS
# 35943-35-2
SB 202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-lH-imidazole) CAS # 152121-30-7
PES (2-phenylethynesulfonamide) CAS # 64984-31-2
STF-62247 (5-Pyπdin-4-yl-thiazol-2-yl)-m-tolyl-amme) CAS # 315702-99-9
It should be noted that any of the above agents may fit into one or more of the above subcategoπes of activity, whereby the above TABLE is not meant to be limiting as to the mechanism of activity. Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.
GENERAL METHODOLOGIES
Early Stage Autophagy Assay
Autophagy entails the recruitment of cytosolic LC3/Atg8 protein to the membrane of nascent autophagosomes, a criterion often used to monitor the formation and accumulation of autophagosomes. To develop a screening assay for chemicals that modulate autophagy, human breast cancer MCF-7 (or HeLa, HT29, H460) cells stably expressing LC3 linked at its N-terminus to GFP were established. In complete cell culture medium containing amino acids and serum, GFP-LC3 fluorescence was largely diffuse throughout the cytoplasm, with a few dots denoting autophagosomes generated at a basal level. The number of GFP-LC3 dots increased during serum and amino acid withdrawal, conditions that induce autophagy. The lysosomotropic agent chloroquine is known to cause the accumulation of autophagosomes through inhibiting their fusion to lysosomes and preventing their degradation. Chloroquine treatment produced extensive
redistribution of diffuse GFP-LC3 to larger punctate cytosolic structures within 4 h in complete medium.
Cell culture and transfection
MCF-7 cells were transfected with GFP-LC3 plasmid using Effectene™. The transfected cells were maintained in 400μg/ml G418 for a period of 2 months. The GFP labeled cells were sorted by FACS and further maintained in medium containing 400 μg/ml G418 as pooled stable transfectants. Cells were thereafter maintained in RPMI- 1640 supplemented with 100 units/ml penicillin/streptomycin, ImM Hepes, 10% fetal bovine serum in humidified 5% CO2 at 37°C.
Screening of autophagy modulators
MCF-7 cells stably expressing GFP-LC3 were added to wells of 96 well plates and allowed to adhere. Test drugs were added to a final concentration of 17 μM using a Biorobotics Biogrid II™ robot equipped with a 0.7mm dia 96-pin tool. The cells were incubated in the presence of test drug for 4 hours. The medium was removed and cells were fixed with 3% paraformaldehyde containing 500ng/ml of Hoechst 33342 for 15 min at room temperature. Wells were washed once with warm PBS and stored in PBS at 4°C. Plates were read using a Cellomics Arrayscan VTI™ high automated fluorescence imager. Cells were photographed using a 2Ox objective in the Hoechst and GFP (XF-100 GFP filter) channels. The Compartment Analysis algorithm was used to identify nuclei, apply a cytoplasmic mask and measure GFP spots with the threshold in the GFP channel fixed at 250. The total intensity in the GFP channel was gated at 10 to reject cells expressing very low levels of GFP-LC3 (<10 total GFP pixel intensity inside the cytoplasmic mask). The total pixel intensity for punctate GFP staining was acquired as "circ spot total intensity ch2". Wells showing at least 3-fold increase in punctate GFP-LC3 staining over controls were examined visually to eliminate any false positive readings caused by precipitation of fluorescent compounds. Compounds inducing more than 3 fold increase in punctate GFP staining were considered as hits. Wells containing fewer than 4,000 nuclei were rejected as containing toxic chemicals.
GFP-LC3 processing assay
GFP-LC3 processing was assayed by Western blotting. Cell extracts were prepared in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 raM EDTA, 1 mM EGTA, 1% Triton Xl 00™, 25 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and Roche™ protease inhibitor cocktail) and lOμg protein was subjected to 12% SDS-PAGE. Proteins were transferred to nitrocellulose and incubated with anti-GFP stabilized antibody preparation (1 :7000, Roche™) followed by HRP-conjugated anti- Mouse antibody (1 : 10000, Pierce™). Immunostained proteins were revealed using Immobilon Western Chemiluminescent HRP substrate (Millipore™) and X-ray film. Western blotting with anti β-tubulin rabbit polyclonal antibody (1 :20000, Santa Cruz™) and HRP-conjugated goat anti -rabbit antibody (1 : 10000, Pierce™) was used for loading controls.
Nutrient deprivation
Cells were seeded in 96 well plate and grown for 18 h in complete medium. The cells were washed three times with warm RPMI medium (serum deprivation) or HBSS medium (amino acid and serum deprivation) and immediately treated with drugs in the same medium. The cells were fixed and analysed for punctuate staining (as above) using the Arrayscan VTI.
Survival and proliferation assay
Cells were seeded in 96 well plates and grown for 18 h. They were then exposed to drugs diluted in appropriate medium for 4 h. Media included DPBS, DPBS containing 11 mM glucose, DPBS containing 1 1 mM glucose and 10% FBS, DPBS containing 11 mM glucose and 10% dialysed FBS, DPBS containing 10% FBS, and complete medium. The medium was removed carefully and replaced with complete medium without drug. After 48h, cell numbers were assessed using the MTT assay.
Cell culture and starvation procedures:
MCF-7 cells and MCF-7 cells stably expressing EGFPLC3 were maintained in RPMI 1640 supplemented with 100 units/ml penicillin/streptomycin, 1 mM Hepes, and 10% (v/v) FBS. EGFP-LC3 expressing cells were supplemented with 400 μg/ml G418 (Sigma, G8168). For starvation experiments, cells were washed twice with DPBS
(Invitrogen, #14040) before incubation in the denoted starvation media. Serum starvation was carried out by incubating cells in the previously described growth medium lacking FBS. Glucose starvation was carried out by incubating cells in DPBS supplemented with amino acids from a 1OX mixture (24) and 10% FBS. Amino acid starvation was carried out in DPBS containing 11 mM glucose, and DPBS was used for serum, glucose, and amino acid starvation.
Screening assay for inhibitors of autophagosome formation
MCF-7 cells stably expressing EGFP LC3 (25) were seeded in PerkinElmer View 96-well plates at 20,000 per well. Eighteen hours after seeding, chemicals from the Prestwick, Sigma LOPAC, Microsource Spectrum and Biomol natural products collections were added to plates at -10 μM using a Biorobotics Biogrid II robot equipped with a 0.4 mm diameter 96-pin tool. Chloroquine (30 μM) was added immediately after to all but negative control wells. Plates were incubated for 4 h at 37°C. Punctate EGFP-LC3 was determined quantitatively using a Cellomics Arrayscan VTI automated fluorescence imager and the Compartmental Analysis Bioapplication as described in detail in Balgi et al. (25). Briefly, cells were photographed using a 2Ox objective in the Hoechst and GFP channels (XF-100 filter) and the compartmental analysis algorithm was used to identify the nuclei, apply a cytoplasmic mask, and to quantitate GFP spots above a fixed threshold. Cells were gated such that only those with an average GFP fluorescence intensity of 10 and above were analyzed. Spots were distinguished from the background using a spot kernel radius parameter of 6 and a fixed threshold set at 500 pixel intensity units. The punctate fluorescence indicates the total intensity of all pixels inside the spot within the cytoplasmic mask. Analysis of 1 ,000 cells per well resulted in a Z factor of 0.54. Images were examined to exclude toxic compounds.
Confocal microscopy
MCF-7 cells stably expressing EGFP-LC3 were cultured on glass coverslips in 6- well plates. Eighteen hours after plating, drugs were added for 4 h. The cells were then fixed with 3% paraformaldehyde in PBS and stained with 500 ng/ml Hoechst 33342. The coverslips were mounted in DABCO and viewed using the 6OX objective of an Olympus Fluoview FVlOOO laser scanning microscope equipped with Olympus-selected
Hamamatsu photomultiplier tubes. Images were analyzed using Olympus FVlO-ASW 1.7 software.
Cell viability and proliferation assay
Cells were seeded at 8,000 per well in 96-well plates and grown overnight. Cells were washed twice with DPBS and treated as described. The drugs were then removed, and cells were grown in complete medium for 48 h. Cell viability and proliferation were measured by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay (26).
SDS-PAGE and western blotting
Cells were lysed and protein was quantified and EGFP-LC3 processing was assayed by western blotting (25). Five μg protein from each lysate was separated on a 12% polyacrylamide gel and electroblotted onto a polyvinylidene fluoride membrane, blocked in 5% (w/v) non-fat milk, and incubated in the appropriate antisera. LC3 lipidation was assessed by resolving identical amounts of cell lysates (70-100 μg of protein) on a 16% polyacrylamide gel. Electroblotting as above was followed by crosslinking using 0.2% (v/v) glutaraldehyde in PBS+0.02% (v/v) Tween 20 for 30 min. Subcellular Fractionation
MCF-7 cells were treated as described for 4 h at 370C. After treatment, cells were washed once with cold PBS, and collected in subcellular fractionation buffer (250 mM sucrose, 20 mM hepes (pH 7.4), 10 mM KCl, 1.5 mM MgC12, 1 mM EDTA, ImM
EGTA). The lysate was passed through a 26G1/2 needle 10 times and incubated on ice for 20 min. The nuclear pellet was collected by centrifugation at 500 x g for 10 min. The supernatant was ultracentrifuged at 100,000 x g to obtain a cytosolic supernatant and a pellet containing total membrane (27). The pellet was resuspended in subcellular fractionation buffer containing 1% Triton X-100 and 10% glycerol. Identical amounts of the supernatant and the membrane fractions (-30-50 μg of protein), respectively, were resolved on a 16% polyacrylamide gel and immunoblotted as described above.
Long-lived protein degradation assay
MCF-7 EGFP-LC3 cells were seeded in a 12-well plate at 75,000 cells per well and grown overnight. Cells were incubated for 24 h at 37°C with 1 μCi/mL of L- [14C]valine (MP Biomedical, #10139E) in RPMI 1640 supplemented as described above. After radiolabelling, cells were washed three times with RPMI, and incubated in complete RPMI supplemented with 10 mM cold valine for 24 h. After this cold chase period where short-lived proteins were degraded, the cells were treated for 18 h as described in either complete RPMI or RPMI lacking serum. Radiolabeled proteins from the medium and the cells were precipitated in 10% (v/v) trichloroacetic acid and were separated from soluble radioactivity by centrifugation at 20,000 x g at 4°C. The precipitated proteins were dissolved in 0.2 N NaOH and the supernatant containing soluble radioactivity was neutralized with 5 N NaOH. Radioactivity was determined by liquid scintillation counting, and protein degradation was calculated by dividing the total acid-soluble radioactivity by the total TCA-precipitated radioactivity (28).
FITC-dextran sequestration assay
FITC-dextran was scrape-loaded into MCF-7 cells as described (29), except cells were suspended in medium containing 0.1% DMSO, 10 mM 3-methyladenine, or 10 μM verteporfin after scrape loading. The suspended cells were plated on coverslips for 2 h or 24 h before fixation with 3% paraformaldehyde in PBS and DNA staining with Hoechst 33342. Slides were then analyzed using confocal microscopy. Benzoporphyrin derivatives
The ring A and B benzoporphyrin derivatives were synthesized using standard techniques (30-32 and US patent 5,990,149). All derivatives were characterized using IH NMR and elemental analysis. The verteporfin regioisomers were separated by reversed phase HPLC. Using a 250 x 10 mm Phenomenex Synergi 4μ MAX-RP 80A C- 12 column and a mobile phase of 95 % MeOH/0.01 M ammonium formate, verteporfin isomer A eluted at 11.3 min and verteporfin isomer B eluted at 11.9 min.
Electron microscopy
MCF-7 EGFP-LC3 cells were seeded onto BD 24-well cell culture inserts with PET track-etched membrane at 30,000 cells per well. Thirty-six hours after seeding, cells were treated as described. Medium in transwells was replaced with fixative (1.5% paraformaldehyde, 1.5% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.3) at room temperature. After 30 minutes, membranes were cut from the transwells and immersed for an additional 2.5-3 h in fixative. The fixative was replaced with buffer (0.1 M sodium cacodylate, pH 7.3) and left overnight at room temperature. The next morning, the membranes were washed twice (10 min each wash) with buffer and then post-fixed for 1 h on ice in 1% OsO4 in 0.1 M sodium cacodylate (pH 7.3). Membranes were washed three times for 10 min each at room temperature with dH2O, stained en bloc with 1% aqueous uranyl acetate for 1 h, washed three times with dH2O, and then dehydrated through an ascending concentration series of ETOH. The membranes were treated three times for 10 min in 100% ETOH, twice with 100% propylene oxide (15 minutes each), and then infiltrated overnight in 1 :1 propylene oxide:EMbed-812 (Electron Microscope Sciences, Hatfield, PA). After two changes of EMbed-812 (2 h each), the membranes were embedded and the resin polymerized for 24 h at 6O0C. Thin sections were stained with uranyl acetate and lead citrate and photographed on a Philips 300 electron microscope operated at 60 kV.
Synthesis
General methodologies for chemical preparation of compounds of Formulas IA and IB are known in the art (for example, US 5,990,149), described in Tovey, A. (1994) Thesis (32), or commercially available. EXAMPLES
Further embodiments are described with reference to the following, non-limiting, examples.
EXAMPLE 1: Verteporfin and other BPD derivatives identified as early stage autophagy inhibitors
Using the early stage autophagy inhibitor assay, which detects chemicals that prevent autophagosome accumulation (Figures IA and IB) in the presence of chloroquine, verteporfin was identified as an early stage autophagy inhibitor (Figure 2). Verteporfin was shown to inhibit autophagosome accumulation induced by cell starvation. At concentrations that inhibit autophagy, verteporfin is selectively toxic to cells in conditions of starvation and is not toxic to cells in complete culture medium in vitro. The autophagy inhibitory activity occurs without light activation (photoactivation) and therefore does not involve photodynamic therapy. Similarily, A BPD diethylene glycol ester, A BPD monoacid, A BPD triol, A BPD diol, A BPD tri(ethylene glycol) ester, and A BPD tri(diethylene glycol) ester were found to inhibit autophagosome accumulation (Figures 10-12).
EXAMPLE 2: Assay to discriminate between chemicals that cause autophagy- unrelated aggregation of intact GFP-LC3.
Recruitment of LC3 to autophagosomes is a requisite step in autophagy and accumulation of autophagosomes detected by GFP-LC3 may indicate enhanced autophagic flux. However, because some of the recruited LC3 is degraded along with autophagosomal contents upon fusion of autophagosomes with lysosomes, accumulation of autophagosomes containing LC3 can also result from inhibition of fusion of autophagosomes with lysosomes or inhibition of protein degradation in lysosomes.
Furthermore, punctate GFP-LC3 structures can accumulate in protein aggregates independently of autophagy. The autophagy-selective GFP-LC3 processing assay was used to distinguish between these possibilities. The assay is based on the observation that proteolytic processing and lipidation of LC3 during the recruitment of GFP-LC3 to autophagosomes generates a characteristic downward shift in the electrophoretic mobility of the fusion protein (GFP-LC3-II). A second mobility shift occurs upon fusion of autophagosomes with lysosomes, when LC3 is degraded more rapidly than the much more proteolytically resistant GFP moiety, which therefore accumulates in autolysosomes. This assay enables the discrimination between chemicals that cause autophagy-unrelated aggregation of intact GFP-LC3, chemicals that stimulate autophagy, and chemicals that inhibit autophagy at a late stage after the recruitment of LC3 to autophagosomes. Figure 3 shows that verteporfϊn inhibition of autophagy is at an early stage (i.e. proteolytic processing and lipidation of LC3 during the recruitment of GFP-LC3 to autophagosomes) as evidenced by the lack of a downward shift in the electrophoretic mobility of the fusion protein (GFP-LC3-II) in -serum media. Accordingly, the early stage inhibition results from interference with the fusion of autophagosomes with lysosomes.
EXAMPLE 3: Automated microscopy screen for chemical inhibitors of autophagosome formation
To monitor the inhibition of autophagosome formation, we used MCF-7 breast cancer cells stably expressing LC3 tagged to EGFP at its N-terminus (MCF-7 EGFP-LC3). LC3 is a cytosolic protein that is recruited to autophagosomes via cleavage of its C- terminus to expose a glycine residue which is then conjugated to
phosphatidylethanolamine, enabling its insertion into autophagosomal membranes (27). In cells grown in complete medium, EGFP-LC3 was distributed diffusely throughout the cytoplasm with a few EGFP-LC3 spots, demonstrating a low basal level of autophagy (Figure 2A). Stimulation of autophagy by exposure to medium lacking serum or to the mTORCl inhibitor rapamycin increased the punctate EGFP-LC3 fluorescence intensity but this increase was not sufficiently high to identify inhibitors in a screening assay. We overcame this problem by using chloroquine (CQ), a lysomotropic agent that neutralizes the acidic pH of lysosomes (33), thereby preventing autophagic protein degradation and causing autophagosome accumulation (34-36). Incubation of cells with 30 μM CQ caused a large increase in the number of autophagosomes (Figure 2A). 3-methyladenine (3-MA), a PI3 -kinase inhibitor, is known to prevent autophagosome formation efficaciously but with low potency and selectivity (21). As expected, 10 mM 3 -MA considerably reduced the ability of CQ to induce autophagosome accumulation (Figure 2A).
We wished to search for chemicals that, like 3 -MA, inhibit autophagosome formation. Cellular autophagosomal content was detected quantitatively by automated fluorescence microscopy in 96-well plates (25). Chloroquine caused a 5- 10-fold increase in punctate EGFP-LC3 fluorescence over that of untreated control cells. Cells were exposed for 4 h to CQ and chemicals from a collection of 3,584 drugs and pharmacological agents at a concentration of 10 μM. Compounds were designated as active if they decreased CQ-induced punctate fluorescence by >50% and induced less than 20% cell death during a 4 h incubation period. Verteporfin was the only active compound identified in this screen. It showed concentration-dependent inhibition of CQinduced punctate EGFP-LC3 with an IC50 of 1 μM, and reducing punctate fluorescence below that of DMSO controls at > 15 μM (Figure 2B).
Verteporfin is a benzoporphyrin derivative used clinically for photodynamic therapy of agerelated macular degeneration. Red light irradiation causes the generation of oxygen radicals that nonselectively kill cells exposed to verteporfin. Verteporfin shows little or no cellular toxicity in the absence of light activation (37, 38). Importantly, the ability of verteporfin to inhibit CQ-induced autophagosome accumulation occurred in the absence of light and was consequently unrelated to its photodynamic properties. All experiments described in this study were conducted without direct light.
EXAMPLE 4 Characterization of verteporfin as an inhibitor of autophagy
Having identified verteporfin as an inhibitor of autophagosome accumulation in the presence of CQ, we next asked whether this drug can inhibit autophagosome formation in response to well characterized autophagic stimuli such as rapamycin or serum deprivation. Cellular exposure to 30 nM rapamycin or to serum-free medium caused a substantial increase in punctate EGFPLC3 fluorescence (Figure 4). Simultaneous treatment with verteporfin considerably reduced both rapamycin-induced and starvation- induced punctate EGFP-LC3 fluorescence and increased diffuse cytoplasmic fluorescence (Figure 4).
Electron microscopy was used to examine this effect at the ultrastructural level. Sections of control cells treated with DMSO rarely if ever contained any autophagosomes (Figure 5a) while exposure to 75 μM CQ caused a significant perinuclear accumulation of autophagic vacuoles containing lamellar structures and undigested cytoplasmic material (Figure 5c, arrowheads), as expected. Noticeably, incubation with 10 μM verteporfin alone caused the distinct appearance of small empty rounded single-membraned vesicles;
however, it did not alter the integrity of most distinguishable organelles, such as mitochondria and multivesicular bodies (Figure 5b). Cells exposed to both 75 μM CQ and 30 μM verteporfin contained significantly fewer autophagosomes (Figure 5d, arrows) than those exposed to CQ alone. Interestingly, these cells also contained numerous empty single-membraned vesicles that were larger in size than in cells treated with verteporfin alone (Figure 5d). There was no clear accumulation of structures resembling elongation membranes in any of the samples; however, these are difficult to identify (39).
EGFP-LC3 recruited to the membranes of autophagosomes is degraded upon fusion with lysosomes but EGFP is less sensitive to lysosomal proteases than the LC3 moiety, leading to transient EGFP accumulation. Therefore, the relative levels of EGFP- LC3 and free EGFP reflect autophagic flux (25). MCF-7 cells treated with 30 nM rapamycin for 4 h showed a significant increase in free EGFP levels compared to controls, consistent with its stimulation of autophagy (Figure 6A). As expected, co-treatment with 3 -MA prevented LC3 degradation and thus the appearance of the free EGFP band.
Bafilomycin Al, a V-ATPase inhibitor that prevents lysosomal acidification and lysosomal protein degradation (35), also prevented the appearance of the free EGFP band in cells treated with rapamycin (Figure 6A). Having established these conditions with well characterized compounds, we then tested the effects of different combinations of verteporfin and rapamycin on EGFP-LC3 processing. Exposure of cells to verteporfin alone led to a decrease in the intensity of the free EGFP band in a concentration-dependent manner, indicating that verteporfin inhibits basal autophagy (Figure 6B). Verteporfin also caused a concentration-dependent decrease in the intensity of the free EGFP band when co-incubated with rapamycin (Figure 6B). The effects of verteporfin on autophagic flux were also tested in serum starvation conditions. Cells exposed to serum-free medium for 4 h showed a significant increase in the free EGFP band compared to controls,
demonstrating that starvation induces autophagic degradation of EGFP-LC3 (Figure 6C). When cells were exposed to 10 μM verteporfin in serum-free conditions, the intensity of the free EGFP band decreased, showing that verteporfin inhibits starvationinduced autophagic degradation.
The effect of verteporfin on autophagic flux was verified and quantified by monitoring the degradation of long-lived proteins. Cells were incubated with 14C- valine for 24 h followed by an additional 24 h cold chase to allow degradation of short-lived proteins before treatment. Cells exposed to verteporfin for 18 h both in complete medium and medium lacking serum showed less long-lived protein degradation than the corresponding DMSO control (Figure 6D). In serum starvation, the decrease in long-lived protein degradation was found to be statistically significant. These results further demonstrate that verteporfm inhibits degradation by both basal and starvation-induced autophagy.
EXAMPLE 5 Verteporfin inhibits the sequestration of FITCdextran
Having demonstrated that verteporfin inhibits autophagosome accumulation and autophagic protein degradation, we next investigated whether it affected the sequestration of cytoplasmic material. An early step in autophagy involves the expansion of
phagophores into bowl-shaped structures that surround cytoplasmic material and capture it when the edges of the phagophores fuse to form doublemembraned autophagosomes (40, 41). This process may be monitored experimentally by examining the transfer of fluorescently labelled dextran from the cytoplasm into autophagic vesicles (29).
FITCdextran was introduced into the cytoplasm of MCF-7 cells by scrape-loading at a temperature of 4°C to prevent uptake by fluid-phase endocytosis. Two hours after loading, FITC-dextran was localized diffusely throughout the cytoplasm (Figure 7a) but it redistributed to punctate structures within 24 h, reflecting autophagosomal sequestration (Figs. 7b & 7c). When cells were treated with 3 -MA, FITC-dextran remained diffuse in the cytoplasm 24 h after loading (Figure 7d), consistent with its demonstrated ability to inhibit autophagic sequestration (42). In cells treated with verteporfin, FITC-dextran remained completely diffuse in the cytosol (Figure Ie), showing that it too inhibits sequestration of cytoplasmic material into autophagosomes.
EXAMPLE 6 Verteporfin does not inhibit LC3 processing or LC3II membrane association
Having established that verteporfin inhibits autophagic vacuole formation, sequestration and degradation, we tested whether verteporfin inhibits the processing, lipidation and membrane association of LC3 in MCF-7 cells. Lipidated LC3 (LC3II) associates with the isolation membrane of nascent autophagosomes and is believed to participate in phagophore expansion (43). LC3II was not detected in either vehicle- or rapamycin-treated MCF-7 cells, consistent with previous observations in MEF and HeLa cell lines (25, 44). While rapamycin caused an increase in autophagosome accumulation (Figure 4) and induction of autophagic degradation (Figure 8), 4 h treatment did not cause detectable accumulation of LC3II, perhaps due to the transient nature of LC3II as a kinetic intermediate. However, treatment with CQ inhibited autophagic degradation, causing significant LC3II accumulation (Figure 8A). Interestingly, cells treated with both CQ and verteporfin showed a similar increase in LC3II despite inhibition of autophagosome accumulation, indicating that LC3 becomes lipidated. These results suggest that verteporfin acts downstream of LC3 processing.
Since verteporfin prevents autophagosome accumulation without inhibiting LC3 lipidation, we sought to establish if verteporfin affects the intracellular distribution of LC3II. LC3II has been shown to specifically associate with autophagosome membranes while unprocessed LC3 is cytosolic (27). Lysates of treated cells were subjected to ultracentrifugation to separate membrane and cytosolic fractions. In both DMSO and verteporfin-treated cells, LC3 was predominantly in the supernatant cytosolic fraction, and no LC3II was detected in either cytosolic or pellet membrane fractions (Figure 8B). In CQ-treated cells, there was a large increase in LC3II, all of which was found in the membrane fraction as expected (Figure 8B). Interestingly, in cells co-treated with verteporfin and CQ, LC3II was also only detected in the membrane fraction (Figure 8B), demonstrating that verteporfin does not prevent LC3II membrane association. In a majority of experiments, the amount of LC3II detected in the membrane pellet was similar between cells treated with CQ and those co-treated with verteporfin and CQ; however, in some experiments, there was a noticeable LC3II decrease in the membrane fraction of co- treated cells. This variability was most likely caused by washing the membrane pellet and may indicate that LC3II is not bound as tightly in the co-treated cells, resulting in a loss of material during the wash.
EXAMPLE 7 Verteporfin sensitizes cells to starvation
Having identified verteporfin as an inhibitor of autophagy, we investigated its effect on cell survival and proliferation. For the MCF7 cells all treatments were 8h + 4Oh recovery and for the HeLa, HT29, and H460 cell lines all treatments were 24h + 48h recovery. Verteporfin was then washed away and the cells were incubated in complete medium for 40 h or 48 h to monitor their ability to recover and proliferate. Verteporfin had no effect on viability suggesting that transient inhibition of autophagy under nutrient- nch conditions does not affect MCF-7, HeLa, HT29 and H460 proliferation and viability (Figures 9 A-D). MCF-7 cells subjected to serum and amino acid starvation showed a greater than 50% survival after 12 hours. This was reduced under the same conditions to less than 10% survival when 13 μM verteporfin was added (Figure 9 A). HeLa, HT29, and H460 cells subjected to starvation showed a substantial decrease in survival when 10 μM verteporfin was added (Figures 9 B-D).
Similar experiments were then carried out using different starvation conditions. Exposure of cells to medium lacking glucose, amino acids or serum (DPBS) for 8 h resulted in only a small decrease in cell numbers after 48 h, showing that transient exposure to starvation did not affect cell survival. However, when cells were exposed to DPBS and verteporfin, cell survival at 48 h decreased substantially and in a concentration dependent manner (Figure 9). Supplementing DPBS with amino acids did not rescue cells. However, supplementation of DPBS with glucose considerably increased cell survival and further addition of serum resulted in complete cell survival. Therefore, inhibiting autophagy with verteporfin in nutrient-rich conditions does not affect MCF-7 cell proliferation and viability, but verteporfin sensitizes cells to glucose and serum deprivation.
EXAMPLE 8 Structural requirements for inhibition of autophagy by benzoporphyrin derivatives
Verteporfin is composed of an equal mixture of two regioisomers (Figure 10A), each of which consists of a pair of enantiomers. The regioisomers were separated by HPLC and tested in the automated microscopy assay. They were equally active, indicating that the propionic acid and propionic acid methyl ester on rings C and D could be interchanged without affecting activity. Verteporfin may be described as a derivative of protoporphyrin IX (apo-heme), bearing modifications to rings A, C and D (Figure 10A). Protoporphyrin IX itself showed no inhibition of autophagy (not shown), demonstrating the dependence on one or more of these modifications for activity. To examine this question, a number of analogues modified at these positions (Figure 10A) were tested at different concentrations for inhibition of autophagy (Figures 1OB, 1 IA, 1 IB and 12).
In protoporphyrin IX, two propionic acid groups are attached to rings C and D while verteporfin has a propionic acid methyl ester at one of these positions. Verteporfin analogue 1 with two propionic acid methyl esters at rings C and D was fully active, but analogue 2, with two propionic acid groups was essentially inactive, showing that the presence of one carboxylic acid is tolerated, but not two. Verteporfin analogue 5 with propanol functionalities attached to rings C and D was also inactive. To determine whether additional C and D ring substitutions would affect activity, a number of different groups were also incorporated at these positions. Inhibition of autophagy by verteporfin derivatives was not affected by the presence of various large substituents on rings C and D, including some bearing distal OH groups (not shown). However, the presence of two carboxylic acid or OH groups close to the C and D rings prevented activity.
Verteporfin also differs from protoporphyrin IX by the presence, fused to ring A, of a cyclohexadiene bearing two methanoic acid methyl esters. Analogue 1 with one methanol c acid methyl ester and one methanoic acid group retained full activity suggesting that a variety of substitutions are likely tolerated at this position.
Fourteen analogues of verteporfin were generated with a disubstituted
cyclohexadiene fused to ring B instead of ring A. Interestingly, most of the derivatives showed no activity and only two showed even weak activity. Accordingly, fusing the substituted cyclohexadiene to ring B instead of ring A almost completely abolishes activity.
EXAMPLE 9 HTI-286, dosed at the tumour CMAX for verteporfin,
significantly extended the T!4 and increased the AUC for verteporfin in HT-29 cells.
A tolerability study was conducted with Visudyne® (Verteporfin) via i.v.
administration in the NCr nude mouse at 20 mg/kg. Dosing regimens of q7dX3 and qdX5 were both well tolerated, with no signs of drug induced toxicities. 20 mg/kg was highest dose possible with Visudyne® formulation. Figure 13 shows that HTI-286, dosed at the tumour CMAX for verteporfin, significantly extended the TVi and increased the area under the curve (AUC) for verteporfin. The activity presumably resulted from a slowing of the elimination of verteporfin from the tumour following the collapse of the tumour vasculature.
EXAMPLE 10 Verteporfin and HTI-286 administration in a xenograft tumour model using HT-29 colon carcinoma cells.
Figure 14 shows the normalized efficacy of verteporfin dosed qdxl i.v. in combination with HTI-286 dosed i.v. (administered 30 minutes post verteporfin at tumour CMAX) compared to saline and HTI-286 controls. This study was performed with nude mice (n=5) bearing HT-29 colon adenocarcinoma xenografts. The assay demonstrated antitumour activity of Verteporfin in combination with HTI-286 (1.6 mg/kg once) as starvation-promoting drug in a xenograft tumour model using HT-29 colon carcinoma cells. The for the studies will be as follows:
Five female NCR nude mice per group were used. The mice were treated when the tumours were ~0.2 cm3 and the groups were as follows: 1. untreated control (saline); 2. Verteporfin alone; 3. HTI-286 alone; and 4. HTI-286 plus 20mg/kg iv + Verteporfin single dose (20 mg/kg).
Drugs were delivered via i.v. administration. One dose of verteporfin followed by one dose of HTI-286 30 minutes later. Tumour growth was monitored using calipers 3 times per week up to 60 days after tumor cell implantation. Mice with tumours which have ulcerated or those mice with tumours >1 cm3 were euthanized.
EXAMPLE 11 Verteporfin and gefϊtinib administration in a xenograft tumour model using JIMT-I human breast adenocarcinoma cells.
Figure 15 shows a decrease in tumor volume (A) and a decrease in mean fold increase of tumor volume (B) when verteporfin (10 mg/kg) is combined with gefitinib (i.e. Iressa 50 mg/kg) as compared to gefitinib alone or verteporfin alone in the herceptin- resistant JIMT-I breast cancer model.
Cells, tumour implantation and animal grouping
JIMT-I human breast adenocarcinoma cells were cultured in DMEM containing 10% FBS and were harvested at 80-90 % confluence using trypsin/EDTA. Cell viability was measured by trypan blue exclusion. 0.5 xlO6 cells in 100 μl medium were injected subcutaneously into the lower back of 6- week old female Rag-2M immunodeficient mice. Animals were anesthetised with Isoflurane for inoculation. Drug treatment was initiated once the tumours reach a size of 100-150 mm3. For each study, animals were randomized into six groups often animals each, within each group, 4 animals were sacrificed during treatment for biochemical measurement of autophagy and the remaining six animals were used for tumour growth measurements. Four groups as follows were tested: 1 saline control; 2 verteporfin 10 mg/kg; 3 gefitinib 50 mg/kg; and 4 verteporfin 10 mg/kg + gefitinib 50 mg/kg. Drug administration
All treatments were administered 5 days per week for 4 weeks. Saline was injected i.p. Visudyne, the liposomal verteporfm formulation used in humans, was purchased from the BC Cancer Agency pharmacy and was reconstituted in low light conditions as per manufacturer's instructions by addition of 7 ml water, resulting in a volume of 7.5 ml with each ml containing 2 mg verteporfin. The vials were wrapped in foil and used within 72 h with storage at 4°C. Administration was i.p. and the procedure was carried out in subdued light. Prepared syringes were wrapped in foil prior to use, and covered from light. Mice were housed in cages covered with paper to avoid direct exposure to the animal room light. Light exposure at cage level was measured to 20-30 LUX at which verteporfin is not photoactivated. Gefitinib powder was purchased from LC Laboratories and dissolved in 0.5% Tween 80 in sterile milli-Q-fϊltered water and was stored at 4°C for no more than 1 week. Administration of gefitinib was by gavage using a 20 g feeding needle, 1 1/2 inches long and 2.25 mm diameter ball attached to a 1 ml syringe. The gavage volume was 200 μl/ 20 g mouse. For combination treatments, verteporfin and gefitinib were administered concurrently. Previous experiments have shown that gefitinib at these doses and administration schedules are well tolerated by mice.
Tumour growth and animal monitoring
Tumour growth was monitored every Monday, Wednesday and Friday by measuring tumour dimensions with digital calipers once the tumours are palpable. Tumour volumes were calculated according to the equation L x W2 /2 with the length (mm) being the longer axis of the tumour. Animals were also weighed at the time of tumour measurement. Tumours were allowed to grow to a maximum of 1 cm3 before the animals were euthanized. Animals with ulcerated tumours were also euthanized. Upon
euthanization, necropsy was performed to visually inspect major organs for signs of tumour growth. All animals were observed post administration, and at least once a day, more if deemed necessary, during the pre-treatment and treatment periods for mortality and morbidity. In particular, signs of ill health were based on body weight loss, change in appetite, and behavioural changes such as altered gait, lethargy and gross manifestations of stress. If severe toxicity or tumour-related illness was seen, the animals were euthanized (CO2 asphyxiation) and a necropsy was performed to assess other signs of toxicity. The following organs were examined: liver, gall bladder, spleen, lung, kidney, heart, intestine, lymph nodes and bladder. Any other unusual findings were noted Six animals per group were monitored for tumour growth Endpomts included tumour growth rates, tumour size on the day when controls were euthanized due to tumour progression (ulceration or tumour size in excess of 1 cm3), and Kaplan-Meier survival curves where survival was defined as the time when tumour size has increased 4-fold relative to the size when treatment was initiated
Although vaπous embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art Such modifications include the substitution of known equivalents for any aspect of the invention m order to achieve the same result in substantially the same way Numeric ranges are inclusive of the numbers defining the range The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise Thus, for example, reference to "a thing" includes more than one such thing Citation of references herein is not an admission that such references are pπor art to the present invention
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Claims

What is claimed is:
1. A method of modulating autophagy, the method comprising, administering a compound of formula IA or IB to a cell to modulate autophagy, the compound of formula IA or IB having the structure:
Figure imgf000058_0001
Formula IA Formula IB
wherein
R1 is selected from the group: CH2OH, CO2G1, CO2G1OG1, CO2G1OG1OG1, and CO2G1OG1OG1OG1 ;
R2 is selected from the group: CH2OH, CO2G2, CO2G2OG2, CO2G2OG2OG2, and CO2G2OG2OG2OG2;
R" is selected from the group: CH2OH,
Figure imgf000058_0002
and CO2G3OG3OG3OG3;
wherein
G1 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group;
G2 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
G3 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
wherein only one of R1, R2, and R3 is COOH.
2. The method of claim 1 , wherein the modulation is in the absence of
photoactivation.
3. The method of claim 1 or 2, wherein the modulation is of early stage autophagy.
4. The method of any one of claims 1-3, wherein the modulation is of a cancer cell.
5. The method of any one of claims 1-4, wherein the cancer cell is in an animal.
6. The method of any one of claims 1 -5, further comprising the administration of a cancer therapeutic agent.
7. The method of claim 6, wherein the animal is a human.
8. The method of claim 6, wherein cancer therapeutic regimen is selected from one or more of the agents set out in TABLE 3.
9. The method of any one of claims 1-8, wherein the modulation is for the treatment of cancer.
10. The method of any one of claims 1-9, wherein R1, R2, and R3 are independently selected from one of more of the following: COOH; CH2OH; CO2CH2OH;
CO2CH2OCH2OH; CO2CH2OCH2OCH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; CO2(CH2)2O(CH2)2O(CH2)2OH; CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH;
CO2(CH2)3O(CH2)3O(CH2)3θH; CO2(CH2)4OH; CO2(CH2)4O(CH2)4OH;
CO2(CH2)4O(CH2)4O(CH2)4OH; CO2(CH2)5OH; CO2(CH2)5O(CH2)5OH;
CO2(CH2)5O(CH2)5O(CH2)5θH; CO2(CH2)6OH; CO2(CH2)6O(CH2)6OH;
CO2(CH2)6O(CH2)6O(CH2)6OH; CO2(CH2)7OH; CO2(CH2)7O(CH2)7OH;
CO2(CH2)7O(CH2)7O(CH2)7OH; CO2(CH2)8OH; CO2(CH2)8O(CH2)8OH;
CO2(CH2)SO(CH2)SO(CH2)SOH; CO2(CH2)9OH; CO2(CH2)9O(CH2)9OH;
CO2(CH2)9O(CH2)9O(CH2)9OH; CO2(CH2)I0OH; CO2(CH2)10O(CH2)I0OH;
CO2(CHZ)1OO(CH2)IOO(CH2)IOOH; CO2CH3; CO2CH2OCH3; CO2CH2OCH2CH3;
CO2CH2OCH2OCH2CH3; CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3;
CO2(CH2)2O(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; CO2(CH2)3O(CH2)3CH3;
CO2(CH2)3O(CH2)3O(CH2)3CH3; CO2(CH2)4CH3; CO2(CH2)4O(CH2)4CH3;
CO2(CH2)4O(CH2)4O(CH2)4CH3; CO2(CH2)5CH3; CO2(CH2)SO(CH2)SCH3;
CO2(CH2)5O(CH2)2O(CH2)5CH3; CO2(CH2)6CH3; CO2(CH2)6O(CH2)6CH3;
CO2(CH2)6O(CH2)6O(CH2)6CH3; CO2(CH2)7CH3; CO2(CH2)7O(CH2)7CH3;
CO2(CH2)7O(CH2)7O(CH2)7CH3; CO2(CH2)8CH3; CO2(CH2)8O(CH2)8CH3;
CO2(CH2)gO(CH2)8O(CH2)8CH3; CO2(CH2)9CH3; CO2(CH2)9O(CH2)9CH3;
CO2(CH2)9O(CH2)9O(CH2)9CH3; CO2(CH2), 0CH3; CO2(CH2) 10O(CH2)10CH3; and C02(CH2),oO(CH2)ioO(CH2)ioCH3;
provided only one of R1, R2, and R3 is COOH.
1 1. The method of any one of claims 1-10, wherein R1, R2, and R3 are independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; CO2CH2OCH2OH; CO2CH2OCH2OCH2OH; CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH; CO2CH2OCH3; CO2CH2OCH2CH3; CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3; CO2(CHo)3CH3; and CO2(CH2)3O(CH2)3CH3; provided only one of R1, R2, and R3 is COOH.
12. The method of any one of claims 1-1 1, wherein R1, R2, and R are independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2CH2OCH3; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; CO2CH2OCH2OH;
CO2(CHz)3OH; CO2(CH2)3O(CH2)3OH; CO2CH2OCH2CH3; CO2(CH2)2CH3;
CO2(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; and CO2(CH2)3O(CH2)3CH3;
provided only one of R1, R2, and R3 is COOH.
13. The method of any one of claims 1-12, wherein R1 , R2, and R3 are independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; and CO2CH2OCH2OH;
provided only one of R1, R2, and R3 is COOH.
14. The method of any one of claims 1-13, wherein the compound is selected from any one or more of the following compounds:
Figure imgf000061_0001
Figure imgf000061_0002
15. A method of sensitizing a cancerous cell to a cancer therapeutic agent, the method comprising: administering to a subject in need thereof, a compound of formula IA or IB, the compound of formula IA or IB having the structure:
Figure imgf000062_0001
Formula IA Formula IB
wherein
R1 is selected from the group: CH2OH, CO2G1, CO2G1OG1, CO2G1OG1OG1, and
Figure imgf000062_0002
R2 is selected from the group: CH2OH, CO2G2, CO2G2OG2, CO2G2OG2OG2, and
CO1G2OG2OG2OG2;
R3 is selected from the group: CH2OH, CO2G3, CO2G 3O/~»/G~<33, / C~>ΓOΛ2 /GI33 /O~V/G-I3O/-V/G-I33, and
CO2G3OG3OG3OG3;
wherein
G1 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group;
G2 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
G3 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
and wherein only one of R , R~, and R is COOH.
16. The method of claim 15, wherein the sensitization is in the absence of
photoactivation.
17. The method of claim 15 or 16, wherein the sensitization is of cancerous cells in early stage autophagy.
18. The method of any one of claims 15-17, wherein the cancerous cell is in an animal.
19. The method of any one of claims 15-18, wherein the animal is a human.
20. The method of any one of claims 15-19, further comprising the administration of a cancer therapeutic regimen.
21. The method of claim 20, wherein the cancer therapeutic regimen is selected from one or more of the agents set out in TABLE 3.
22. The method of any one of claims 15-21 , wherein R1, R2, and R3 are independently selected from one of more of the following:
CH2OH; CO2CH2OH; CO2CH2OCH2OH; CO2CH2OCH2OCH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; CO2(CH2)2O(CH2)2O(CH2)2OH; CO2(CH2)3OH;
CO2(CH2)3O(CH2)3OH; CO2(CH2)3O(CH2)3O(CH2)3OH; CO2(CH2)4OH;
CO2(CH2)4O(CH2)4OH; CO2(CH2)4O(CH2)4O(CH2)4OH; CO2(CH2)5OH;
CO2(CH2)5O(CH2)5OH; CO2(CH2)5O(CH2)2O(CH2)5OH; CO2(CH2)6OH;
CO2(CH2)6O(CH2)6OH; CO2(CH2)6O(CH2)6O(CH2)6OH; CO2(CH2)7OH;
CO2(CH2)7O(CH2)7OH; CO2(CH2)7O(CH2)7O(CH2)7OH; CO2(CH2)8OH;
CO2(CH2)8O(CH2)8OH; CO2(CH2)8O(CH2)8O(CH2)8OH; CO2(CH2)9OH;
CO2(CH2)9O(CH2)9θH; CO2(CH2)9O(CH2)9O(CH2)9OH; CO2(CH2)10OH;
C02(CH2)ioO(CH2)ioOH; C02(CH2)ioO(CH2)100(CH2)i0OH; CO2CH3; CO2CH2OCH3; CO2CH2OCH2CH3; CO2CH2OCH2OCH2CH3; CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3; CO2(CH2)2O(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; CO2(CH2)3O(CH2)3CH3;
CO2(CH2)3O(CH2)3O(CH2)3CH3; CO2(CH2)4CH3; CO2(CH2)4O(CH2)4CH3;
CO2(CH2)4O(CH2)4O(CH2)4CH3; CO2(CH2)5CH3; CO2(CH2)5O(CH2)5CH3;
CO2(CH2)5O(CH2)2O(CH2)5CH3; CO2(CH2)6CH3; CO2(CH2)6O(CH2)6CH3;
CO2(CH2)6O(CH2)6O(CH2)6CH3; CO2(CH2)7CH3; CO2(CH2)7O(CH2)7CH3;
CO2(CH2)7O(CH2)7O(CH2)7CH3; CO2(CH2)8CH3; CO2(CH2)8O(CH2)8CH3;
CO2(CH2)8O(CH2)8O(CH2)8CH3; CO2(CH2)9CH3; CO2(CH2)9O(CH2)9CH3;
CO2(CH2)9O(CH2)9O(CH2)9CH3; CO2(CH2)I0CH3; C02(CH2)ioO(CH2)ioCH3;
C02(CH2),oO(CH2)1oO(CH2),oCH3;
provided only one of R!, R2, and R3 is COOH.
23. The method of any one of claims 15-22, wherein R 5 R , and R are independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; CO2CH2OCH2OH; CO2CH2OCH2OCH2OH; CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH; CO2CH2OCH3; CO2CH2OCH2CH3;
CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; and CO2(CH2)3O(CH2)3CH3; provided only one of R1, R2, and R3 is COOH.
24. The method of any one of claims 15-23, wherein R1, R2, and R3 are independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2CH2OCH3; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; CO2CH2OCH2OH;
CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH; CO2CH2OCH2CH3; CO2(CH2)2CH3;
CO2(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; and CO2(CH2)3O(CH2)3CH3;
provided only one of R1, R2, and R3 is COOH.
25. The method of any one of claims 15-24, wherein R1, R2, and R3 are independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; and CO2CH2OCH2OH;
provided only one of R1, R2, and R3 is COOH.
26. The method of any one of claims 15-25, wherein the compound is selected from any one or more of the compounds of TABLE 2.
27. A pharmaceutical composition comprising one or more of the agents set out in TABLE 3 and a compound of Formula IA or IB:
Figure imgf000064_0001
Formula IA Formula IB
wherein
R' is selected from the group: CH2OH, CO2G1, CO2G1OG', CO2GOG 1 1O /~\/G~* 11, and CO2G1OG1OG1OG1;
R2 is selected from the group: CH2OH, CO2G2, CO2G2OG2, CO2G2OG2OG2, and CO2G2OG2OG2OG2;
RJ is selected from the group: CH2OH, CO2G3, CO2G3OG3,
Figure imgf000064_0002
CO2G3OG3OG3OG3;
wherein
G1 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; G is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
G3 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
and wherein only one of R1, R2, and R3 is COOH.
28. The pharmaceutical composition of claim 27, wherein the one or more compounds of formula IA or IB are active in the absence of photoactivation.
29. The pharmaceutical composition of claim 27 or 28, wherein R , R , and R are independently selected from one of more of the following:
CH2OH; CO2CH2OH; CO2CH2OCH2OH; CO2CH2OCH2OCH2OH; CO2(CH2)2OH;
CO2(CH2)2O(CH2)2OH; CO2(CH2)2O(CH2)2θ(CH2)2θH; CO2(CH2)3OH;
CO2(CH2)3O(CH2)3θH; CO2(CH2)3O(CH2)3O(CH2)3OH; CO2(CH2)4OH;
CO2(CH2)4O(CH2)4OH; CO2(CH2)4O(CH2)4O(CH2)4θH; CO2(CH2)5OH;
CO2(CH2)SO(CH2)SOH; CO2(CH2)5O(CH2)2θ(CH2)5θH; CO2(CH2)6OH;
CO2(CH2)6O(CH2)6OH; CO2(CH2)6O(CH2)6O(CH2)6OH; CO2(CH2)7OH;
CO2(CH2)7O(CH2)7OH; CO2(CH2)7O(CH2)7O(CH2)7OH; CO2(CH2)SOH;
CO2(CH2)8O(CH2)8OH; CO2(CH2)8O(CH2)8O(CH2)8OH; CO2(CH2)9OH;
CO2(CH2)9O(CH2)9OH; CO2(CH2)9θ(CH2)9θ(CH2)9θH; CO2(CH2)10OH;
CO2(CH2), oO(CH2)10OH; C02(CH2),oO(CH2)100(CH2)10OH; CO2CH3; CO2CH2OCH3; CO2CH2OCH2CH3; CO2CH2OCH2OCH2CH3; CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3; CO2(CH2)2O(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; CO2(CH2)3O(CH2)3CH3;
CO2(CH2)3O(CH2)3O(CH2)3CH3; CO2(CH2)4CH3; CO2(CH2)4O(CH2)4CH3;
CO2(CH2)4O(CH2)4O(CH2)4CH3; CO2(CH2)5CH3; CO2(CH2)5O(CH2)5CH3;
CO2(CH2)5O(CH2)2O(CH2)5CH3; CO2(CH2)6CH3; CO2(CH2)6O(CH2)6CH3;
CO2(CH2)6O(CH2)6O(CH2)6CH3; CO2(CH2)7CH3; CO2(CH2)7O(CH2)7CH3;
CO2(CH2)7O(CH2)7O(CH2)7CH3; CO2(CH2)8CH3; CO2(CH2)8O(CH2)8CH3;
CO2(CH2)SO(CH2)SO(CH2)BCH3; CO2(CH2)9CH3; CO2(CH2)9O(CH2)9CH3;
C02(CH2)90(CH2)90(CH2)9CH3; C02(CH2),OCH3; C02(CH2),OO(CH2)IOCH3;
C02(CH2)100(CH2)100(CH2)ioCH3;
provided only one of R1, R2, and R3 is COOH.
30. The pharmaceutical composition of any one of claims 27-29, wherein R1, R2, and RJ are independently selected from one of more of the following: COOH; CH2OH;
CO2CH3; CO2CH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; CO2CH2OCH2OH; CO2CH2OCH2OCH2OH; CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH; CO2CH2OCH3;
CO2CH2OCH2CH3; CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3; CO2(CHo)3CH3; and
CO2(CH2)3O(CH2)3CH3;
provided only one of R1, R2, and R3 is COOH.
31. The pharmaceutical composition of any one of claims 27-30, wherein R1 , R2, and R3 are independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2CH2OCH3; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH;
CO2CH2OCH2OH; CO2(CH2)3OH; CO2(CH2)3O(CH2)3OH; CO2CH2OCH2CH3;
CO2(CH2)2CH3; CO2(CH2)2O(CH2)2CH3; CO2(CH2)3CH3; and CO2(CH2)3O(CH2)3CH3; provided only one of R1, R2, and R3 is COOH.
32. The pharmaceutical composition of any one of claims 27-31 , wherein R1, R2, and RJ are independently selected from one of more of the following: COOH; CH2OH; CO2CH3; CO2CH2OH; CO2(CH2)2OH; CO2(CH2)2O(CH2)2OH; and CO2CH2OCH2OH; provided only one of R1, R2, and R3 is COOH.
33. The pharmaceutical composition of any one of claims 27-32, wherein the compound is selected from any one or more of the compounds of TABLE 2.
34. A compound of formula IA or IB, having the structure:
Figure imgf000066_0001
Formula IA Formula IB wherein
R1 is selected from the group: CH2OH, CO2G1, CO2G1OG1, CO2G1OG1OG1, and
CO2G1OG1OG1OG1
R2 is selected from the group: CH2OH, CO2G2, CO2G2OG2, CO2G2OG2OG2, and
CO2G2OG2OG2OG2;
Rj is selected from the group: CH2OH,
Figure imgf000066_0002
CO2GOGOG', and
CO2G3OG3OG3OG3;
wherein G1 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group;
G2 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
G3 is hydrogen or a linear, branched, or cyclic, saturated or unsaturated one to ten carbon alkyl group; and
and wherein
only one of R1, R2, and R3 is COOH for the treatment of cancer, in the absence of photoactivation.
35. Use of the compound of claim 34 for treating cancer in the absence of
photoactivation.
36. Use of a pharmaceutical composition, the pharmaceutical composition comprising the compound of claim 34 and a pharmaceutically acceptable carrier for treating cancer in the absence of photoactivation.
•37. Use of the compound of claim 34 in the manufacture of a medicament for treating cancer in the absence of photoactivation.
38. A commercial package comprising (a) the compound of claim 34; and (b) instructions for the use thereof for treating cancer in the absence of photoactivation.
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