WO2014018801A1 - Diagnosis and treatment of cancer using differentially expressed starvation markers - Google Patents

Diagnosis and treatment of cancer using differentially expressed starvation markers Download PDF

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Publication number
WO2014018801A1
WO2014018801A1 PCT/US2013/052137 US2013052137W WO2014018801A1 WO 2014018801 A1 WO2014018801 A1 WO 2014018801A1 US 2013052137 W US2013052137 W US 2013052137W WO 2014018801 A1 WO2014018801 A1 WO 2014018801A1
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Prior art keywords
starvation
inhibitor
marker
subject
expression
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PCT/US2013/052137
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French (fr)
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Martha Karen Newell
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The Texas A&M University System
Scott & White Healthcare
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Publication of WO2014018801A1 publication Critical patent/WO2014018801A1/en

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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Renal Cell Carcinoma accounts for two to three percent of all adult malignant neoplasms and the disease specific mortality rate is significantly higher than other genitourinary malignancies.
  • advanced medical imaging the
  • RCC will be node positive or have evidence of metastasis at the time of diagnosis.
  • Ovarian cancer begins in the ovaries, or the female reproductive organs. Ovarian cancer often goes undetected in the earlier stages, and therefore, is
  • Skin cancer is the most common form of cancer in the US. In fact, one in five
  • the invention in some aspects is a method for selecting a course of treatment of a subject having cancer, by obtaining from the subject a tumor cell, determining the expression of one or more starvation markers which are differentially expressed in cancers under distinct metabolic conditions, and selecting a course of treatment appropriate to the cancer of the subject depending on the expression of the one or more starvation markers.
  • a method for identifying a property of a tumor involves selecting a tumor cell and determining whether the tumor cell expresses a starvation marker selected from the group consisting of LAMP and LC3, wherein expression of the starvation marker is indicative of an autophagic tumor cell.
  • the starvation marker in some embodiments is LAMP1, LAMP2 or LC3.
  • the methods may also involve selecting a course of treatment for a subject based on the expression of the starvation marker.
  • the course of treatment involves administering a starvation signal compound to the subject when the tumor cell is starvation marker negative.
  • the course of treatment involves administering an autophagy inhibitor to the subject when the tumor cell is starvation marker positive.
  • a method for treating a subject by administering to a subject who has been identified as having a starvation marker negative tumor, a therapeutically acceptable amount of a starvation signal compound to treat the tumor is provided.
  • a method for treating a subject by administering to a subject who has been identified as having a starvation marker positive tumor a therapeutically acceptable amount of an autophagy inhibitor and/or a fatty acid metabolism inhibitor.
  • the methods also involve determining the expression of one or more starvation markers in a tumor cell, following the autophagy inhibitor and/or a fatty acid metabolism inhibitor treatment to determine if the tumor cell is starvation marker inhibitor negative.
  • the methods also involve administering to the subject a therapeutically acceptable amount of a starvation signal compound to treat the tumor after the autophagy inhibitor and/or a fatty acid metabolism inhibitor treatment.
  • the starvation signal compound may be for instance, an anti-VEGF antagonist, optionally an anti-VEGF antibody such as bevacizumab, or rapamycin or a glycolysis inhibitor.
  • the tumor is selected from the group consisting of a melanoma, an ovarian tumor, a glioblastoma, a breast cancer, and a renal tumor.
  • the methods may also involve administering a chemotherapeutic agent to the subject.
  • the autophagy inhibitor in some embodiments is a 4- aminoquinoline.
  • the 4- aminoquinoline may have the structure:
  • each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration; wherein each of R 2 and R 3 is independently a hydroxalkyl, an alkyl, alkyloxy, alkylcarboxy, alkylene or alkenylene having from one to six carbon atoms.
  • the autophagy inhibitor is chloroquine or
  • the method may involve determining the expression of one or more starvation markers in a tumor cell of the subject before the treatment is administered.
  • the step of determining the expression of the starvation marker is performed by detecting the expression of starvation marker nucleic acid molecules.
  • the expression of the starvation marker nucleic acid molecules is determined by a method selected from the group consisting of nucleic acid hybridization and nucleic acid amplification.
  • the nucleic acid hybridization may be performed using a solid-phase nucleic acid molecule array.
  • the nucleic acid amplification method is real-time PCR.
  • the step of determining the expression of the starvation marker is performed by detecting the expression of starvation marker peptides.
  • the expression levels are determined by an immunological method, such as a solid-phase antibody array or an ELISA or ELISPOT assay.
  • the fatty acid metabolism inhibitor is an inhibitor of fatty acid oxidation, a fatty acid transporter inhibitor, a reductase inhibitor, or an isomerase inhibitor within the fatty acid metabolism pathway.
  • the inhibitor of fatty acid metabolism may be an inhibitor of fatty acid oxidation and is selected from the group consisting of an oxirane carboxylic acid compound, such as etomoxir (2-(6-(4- chlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester), 2-(4-(3-chlorophenoxy)- butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)- oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2- carboxylic acid ethyl ester, 2-(6-(
  • the inhibitor of fatty acid metabolism is an inhibitory nucleic acid, for instance an inhibitory nucleic acid specific for an enzyme selected from the group consisting of 2,4-dienoyl-CoA reductase, 2,4-dienoyl-CoA isomerase, and butyryl dehydrogenase.
  • the inhibitor of fatty acid metabolism is oxamate.
  • the fatty acid metabolism inhibitor is an oxirane carboxylic acid compound capable of inhibiting fatty acid metabolism, or a
  • the oxirane carboxylic acid compound is etomoxir.
  • the glycolytic inhibitor is a 2-deoxyglucose compound such as 2-deoxy-D-glucose.
  • the invention involves a method comprising providing a starvation marker from a tumor tissue sample, determining the expression level of the starvation marker, and comparing the expression level of the starvation marker in the tissue sample with threshold value of a starvation marker, wherein a lower level of expression in the tumor cell is indicative of the tumor cells susceptibility to starvation signal compound.
  • the threshold value is an expression level value of a starvation marker from a control cell.
  • the control cell is a non-cancerous cell of the same type of tissue as the tumor cell.
  • a method of treating melanoma, ovarian cancer, or renal cancer is provided in other aspects of the invention.
  • the method involves administering daily to a subject having melanoma, ovarian cancer, or renal cancer a therapeutically effective dose of an autophagy inhibitor and administering once every 2-4 months to the subject a therapeutically effective amount of bevacizumab.
  • the bevacizumab is administered in a dose range of 12-16 mg/Kg IV. In other embodiments the bevacizumab is administered in a dose of 15 mg/Kg IV.
  • the invention involves a method of treating cancer, by administering to a subject having cancer a therapeutically effective dose of an autophagy inhibitor and administering to the subject a therapeutically effective amount of rapimycin.
  • the autophagy inhibitor is administered daily.
  • the autophagy inhibitor is hydroxychloroquine or chloroquine. In yet other embodiments the autophagy inhibitor is administered in a dose range of 100-500 mg per day or 200-400 mg per day.
  • Figure 1 is a bar graph showing FSC vs SSC dot plots for WM35 human melanoma cells treated with Etomoxir, Chloroquine, 2-deoxyglucose, and Chloroquine +
  • Figure 2 is a bar graph showing the granularity of the B16F1 mouse melanoma cell line.
  • Figure 3 is a bar graph showing the relative numbers of live and dead cells FSC vs SSC for an HTB-77 cell line.
  • Figure 4 is a bar graph showing the relative numbers of live and dead cells FSC vs SSC dot plots for an HTB-77 cell line.
  • Figure 5 is a bar graph showing the relative numbers of live and dead cells FSC vs SSC dot plots for ACHN renal carcinoma.
  • Figure 6 is a bar graph depicting the results of treating T24 bladder tumor cells treated with etomoxir at 0.5mM and chloroquine at O. lmM.
  • Figure 7 is a bar graph depicting the results of treating WM35 Melanoma, L1210, and L1210 DDP cells were treated with Rapamycin and stained for MitoTracker.
  • Figure 8 is a bar graph depicting the results of treating WM35 Melanoma, L1210, and L1210 DDP cells were treated with Rapamycin and stained for LAMP-1; or LC3A/B
  • Personalized medicine in which a therapy is tailored to specific properties of an individual and their disease has been proposed as a solution to this problem.
  • one approach to personalized medicine involves the identification of the presence or absence of biomarkers to aid in cancer therapeutics, where the expression of the biomarker or lack thereof has been associated with a successful therapeutic protocol.
  • the expression of HER2 on breast cancer cells is used to guide a decision on whether to include HER2 specific drugs such as HERCEPTIN® and TYKERB® in therapy.
  • starvation markers including for instance, LAMP and LC3 are useful in predicting therapeutic outcome in response to certain anti-cancer therapies.
  • Low levels of expression of the starvation markers indicates that a tumor cell is not experiencing conditions of starvation.
  • Such tumor cells are susceptible to anti-cancer therapies without the requirement for further manipulation.
  • High levels of starvation markers are indicative of tumor cells that are autophagic.
  • Autophagic tumor cells should be treated with autophagy inhibitors and/or fatty acid oxidation inhibitors to render the cells susceptible to starvation signal compound.
  • the invention relates to methods for characterizing a tumor cell, such as for instance, selecting a course of treatment for a subject having cancer or for identifying a property of a tumor.
  • the methods can be used on any type of tumor cell.
  • Tumor cells from a variety of sources can be used.
  • a tumor cell may be an in vitro tumor cell line or a primary tumor cell that has been preserved for a period of time or one that has just been removed from a subject.
  • a tumor cell can be obtained from a subject, for instance, as part of a tissue biopsy or as a cancerous tissue removed from the subject. The cell may then be analyzed immediately or preserved for a period of time prior to analysis.
  • the expression of a starvation marker in the tumor cell is assessed in order to determine the metabolic state or condition of the cell.
  • the starvation marker is indicative of whether the cell is experiencing conditions of starvation, such that it is autophagic.
  • autophagy When cancer cells are deprived of nutrient, many undergo a form of survival strategy known as autophagy.
  • Autophagy literally “self-eating,” is a cellular survival strategy that cells use when confronted with conditions of starvation, oxidative stress, including the stresses of chemotherapeutic agents or irradiation.
  • Autophagy provides a mechanism for survival in the face of stress and involves auto-digestion of the cells own organelles into the lysosomal system, thereby providing the engulfed organellar contents as a source of energy.
  • a starvation marker is a detectable compound, i.e., a protein or nucleic acid, the expression of which is altered under conditions of cellular starvation.
  • Starvation markers include but are not limited to LAMP, LC3, Beclin (BCNl or BCN2), AMBRA1, ATG12, ATG16L1, ATG4A, ATG4B, ATG4C, ATG4D, ATG5, ATG9A, ATG9B, BECN1, GABARAP, GABARAPL1, GABARAPL2, IRGM, MAP1LC3A, MAP1LC3B, RGS1, ULK1, ATG4A, ATG4B, ATG4C, ATG4D, GABARAP, ATG10, ATG16L1, ATG16L2, ATG3, ATG4A, ATG4B, ATG4C, ATG4D, ATG7, ATG9A, GABARAP, GABARAPL2, RAB24, DRAM, GABARAP,
  • LAMP is Lysosomal-associated membrane protein. In some instances it is also known as CD 107 antigen-like family member.
  • LAMP includes the various forms of LAMP such as LAMP-1, LAMP-2, and LAMP-3.
  • the protein encoded by this gene is a member of a family of membrane glycoproteins that provides selectins with carbohydrate ligands.
  • LAMP 1 _HUM AN The protein sequence for human LAMP-1 is disclosed in UniProt, reference number PI 1279 (LAMP 1 _HUM AN) and is as follows: MAAPGSARRP LLLLLLLLLL GLMHCASAAM FMVKNGNGTA CIMANFSAAF SVNYDTKSGP KNMTFDLPSD ATVVLNRSSC GKENTSDPSL VIAFGRGHTL TLNFTRNATR YSVQLMSFVY NLSDTHLFPN ASSKEIKTVE SITDIRADID KKYRCVSGTQ VHMNNVTVTL HDATIQAYLS NSSFSRGETR CEQDRPSPTT APPAPPSPSP SPVPKSPSVD KYNVSGTNGT CLLASMGLQL NLTYERKDNT TVTRLLNINP NKTSASGSCG AHLVTLELHS EGTTVLLFQF GMNASSSRFF LQGIQLNTIL PDARDPAFKA ANGSLRALQA TVGNSYKCNA EEHVRVTK
  • the protein sequence for human LAMP-2 is disclosed in UniProt, reference number PI 3473 (LAMP2_HUMAN) and is as follows: MVCFRLFPVP GSGLVLVCLV LGAVRSYALE LNLTDSENAT CLYAKWQMNF TVRYETTNKT YKTVTISDHG TVTYNGSICG DDQNGPKIAV QFGPGFSWIA NFTKAASTYS IDSVSFSYNT GDNTTFPDAE DKGILTVDEL LAIRIPLNDL FRCNSLSTLE KNDVVQHYWD VLVQAFVQNG TVSTNEFLCD KDKTSTVAPT IHTTVPSPTT TPTPKEKPEA GTYSVNNGND TCLLATMGLQ LNITQDKVAS VININPNTTH STGSCRSHTA LLRLNSSTIK YLDFVFAVKN ENRFYLKEVN ISMYLVNGSV FSIANNNLSY WDAPLGSSYM CNKEQTVSVS GAFQINTFDL RVQPFNVTQG
  • the protein sequence for human LAMP-3 is disclosed in UniProt, reference number Q9UQV4 (LAMP3_HUMAN) and is as follows: MPRQLSAAAA
  • LC3 is an autophagy sensitive marker. It includes at least LC3B, LC3A, and LC3C.
  • the full name for LC3B, for instance, is microtubule-associated proteins 1A/1B light chain 3B.
  • the protein sequence for human LC3B is disclosed in UniProt, reference number Q9GZQ8 (MLP3B_HUMAN) and is as follows: MPSEKTFKQR RTFEQRVEDV RLIREQHPTK IPVIIERYKG EKQLPVLDKT KFLVPDHVNM SELIKIIRRR LQLNANQAFF LLVNGHSMVS VSTPISEVYE SEKDEDGFLY MVYASQETFG MKLSV (SEQ ID NO. 4).
  • the protein sequence for human LC3A is disclosed in UniProt, reference number Q9H492 (MLP3 A_HUM AN) and is as follows: MPSDRPFKQR RSFADRCKEV QQIRDQHPSK IPVIIERYKG EKQLPVLDKT KFLVPDHVNM SELVKIIRRR LQLNPTQAFF LLVNQHSMVS VSTPIADIYE QEKDEDGFLY MVYASQETFG F
  • the protein sequence for human LC3C is disclosed in UniProt, reference number Q9BXW4 (MLP3C_HUMAN) and is as follows: MPPPQKIPSV RPFKQRKSLA IRQEEVAGIR AKFPNKIPVV VERYPRETFL PPLDKTKFLV PQELTMTQFL SIIRSRMVLR ATEAFYLLVN NKSLVSMSAT MAEIYRDYKD EDGFVYMTYA SQETFGCLES AAPRDGSSLE DRPCNPL (SEQ ID NO. 6).
  • nucleic acid sequence for human LAMP-1 is disclosed in GENBANK, accession number NM_005561 and is as follows: tgcggggagc cgagccgccg gcgctcgacg cgcgctct cgcgagaccc gcgggatcac gtgacgcccg ggcgcggcgc agctcacgtg acaagcgctg ccggccgcgg tgtctctc gtgtgcgcagtggcc gggctcgt cgcagtggcc gggctctg cgtcgtaa cgccgctgtc tctaacgcca gcccttggcg ccgccccc gcaccgcag cgcccggcag tcgc
  • the nucleic acid sequence for human LAMP-3 is disclosed in GENBANK, accession number NM_014398 and is as follows: ccgggccccg gcggctgcgc cgagtccccg cccctccctg ctccgtaggg gtaggagggg gccggcggag tttcccccccccccgcccagcgg ccctgggcgg gctttcggc tgcttctcat aagcaggtgg ttcgtttctctccggcacagg taggtttctc tggcaccgat tcggggcctg cccggacttc gccgcacgcgct gcagaacctc gcccagcgcccccccggacttc gccgcacgct gc
  • nucleic acid sequence for human LC3B is disclosed in GENBANK, accession number NM_022818 and is as follows: acgctgcgtg ccgctgctgg gttccgccac gcccgtcatg gcggcggccc cggccggctc tggccccgcc cctcggtgac gcgtcgcgag tcacctgacc aggctgcggg ctgaggagat acaagggaag tggctatcgc cagagtcgga ttcgcgccg cagcagccgcccccggg agccgcggg accctcgcgt cgcgcgcgcgcgcggg agccgcgcgcggg agccgccgcggg
  • the nucleic acid sequence for human LC3A variant 1 is disclosed in GENBANK, accession number NM_032514 and is as follows: accgggcgag ttacctcccg cagccgcagc cgccgtgctc agcgcgagcc ccggagccct tgagcgcgag gcgcggagcccc ccaaaccgca gacacatcccc cgccccag agccccggcc tgcgcgccca gccgggcccg cgcgatgccc tcagaccggc ctttcaagca gcggcggagc ttcgccgacc gctgtaagga ggtacagcag atccgcgaccccag caaaatcc
  • the nucleic acid sequence for human LC3C is disclosed in GENBANK, accession number NM_001004343 and is as follows: ggaggaatga gttaggttcc cggttgcggg acagttttttttttttt aaaacagaca cagctactga gtgcaatgcc gcctccacag aaaatcccaa gcgtcagacc cttcaagcag aggaaagct tggcaatcag acaagaggaa gttgctggaa tccgggcaaa gttccccaac aaaatcccgg tggtagtgga gcgctaccccc agggagacgt tcctgcccccc gctggacaaa accaaagtttcccc
  • the tumor cell is starvation marker negative.
  • a "starvation marker negative" tumor cell as used herein refers to any cancer that is not starvation marker positive.
  • a starvation marker positive cancer is one which involves production of significant levels of a starvation marker.
  • Methods for classifying a cancer cell as a starvation marker positive or negative cell are known in the art. For instance, starvation marker positivity may be analyzed by means of immunohistochemical analysis with a monoclonal antibody and categorized according to whether there were enough positive cells. For instance, when the cells are analyzed by flow cytometry a value of at least 90% starvation marker positive cells in comparison to isotype control would indicate a population of cells is starvation marker positive.
  • the value in comparison to isotype controls is at least 85%, 80%, 75%, 70%, 60%, or 50%. In contrast, when the cells are analyzed by flow cytometry a value of 10% or fewer starvation marker positive cells in comparison to isotype control would indicate a population of cells is starvation marker negative. In some embodiments the value in comparison to isotype controls is 40%, 30%, 25%, 20%, 5%, 3%, 2%, 1% or fewer.
  • the isolated cells can be treated with rapimycin or avastin or other starvation signal compound and the changes in expression levels of the starvation markers can be assessed. If the increase in starvation marker level in response to the starvation signal is 25% or less then the patient should be treated with the starvation signal compound. In some embodiments the signal should be 20%, 15%, 10%, 5%, 3%, 2%, 1% or less. If the increase in starvation marker level in response to the starvation signal is greater than 25% then the patient should be treated with the starvation signal compound. In some embodiments the signal should be greater than 50%, 60%, 70%, 80%, 90%, 95%, or 98%.
  • the expression of one or more starvation markers in the tumor cell is determined using methods known to the skilled artisan.
  • the detection methods generally involve contacting a starvation marker binding molecule with a sample in or from a subject or in an in vitro cell.
  • the sample is first harvested from the subject, although in vivo detection methods are also envisioned.
  • the sample may include any body tissue or fluid that is suspected of harboring the cancer cells.
  • the cancer cells are commonly found in or around a tumor mass for solid tumors.
  • the binding molecules are referred to herein as isolated molecules that selectively bind to a starvation marker.
  • a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In all embodiments human subjects are preferred.
  • the subject is a human either suspected of having the cancer, or having been diagnosed with cancer.
  • Methods for identifying subjects suspected of having cancer may include physical examination, subject's family medical history, subject's medical history, biopsy, or a number of imaging technologies such as ultrasonography, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy, or positron emission tomography. Diagnostic methods for cancer and the clinical delineation of cancer diagnoses are well known to those of skill in the medical arts.
  • tissue sample is tissue obtained from a tissue biopsy, a surgically resected tumor, or any other tumor cell mass removed from the body using methods well known to those of ordinary skill in the related medical arts.
  • the phrase "suspected of being cancerous" as used herein means a cancer tissue sample believed by one of ordinary skill in the medical arts to contain cancerous cells. Methods for obtaining the sample from a biopsy include gross apportioning of a mass,
  • microdissection laser-based microdissection, or other art-known cell-separation methods.
  • the sample size required for analysis may range from 1, 10, 50, 100, 200, 300, 500, 1000, 5000, 10,000, to 50,000 or more cells.
  • the appropriate sample size may be determined based on the cellular composition and condition of the biopsy and the standard preparative steps for this determination and subsequent isolation of the nucleic acid for use in the invention are well known to one of ordinary skill in the art.
  • RNA conversion and/or amplification methods or other methods to enhance resolution of the nucleic acid molecules or proteins.
  • methods which allow use of limited biopsy materials, are well known to those of ordinary skill in the art and include, but are not limited to: direct RNA amplification, reverse transcription of RNA to cDNA, amplification of cDNA, or the generation of radio-labeled nucleic acids or proteins.
  • the phrase determining the expression of a starvation marker nucleic acid molecule in the tissue means identifying RNA transcripts in the tissue sample by analysis of nucleic acid or protein expression in the tissue sample.
  • the phrase determining the expression of a starvation marker protein or peptide molecule in the tissue means identifying proteins or peptides in the tissue sample by analysis of protein expression in the tissue sample.
  • the expression of the starvation marker nucleic acid molecules in the sample can be compared to the expression of starvation marker nucleic acid molecules in a sample of tissue that is non-cancerous, i.e. a reference value.
  • tissue that is non-cancerous
  • non-cancerous tissue means tissue determined by one of ordinary skill in the medical art to have no evidence of the particular tumor being analyzed based on standard diagnostic methods including, but not limited to, histologic staining and microscopic analysis.
  • the reference value is the expression level of the gene or protein in a reference sample.
  • a reference value may be a predetermined value and may also be determined from reference samples (e.g., control biological samples) tested in parallel with the test samples.
  • a reference value may be a positive or negative control level.
  • a reference value can be a single cut-off value, such as a median or mean or a range of values, such as a confidence interval.
  • Reference values can be established for various subgroups of individuals, such as individuals free of cancer, individuals having early or late stage cancer, male and/or female individuals, or individuals undergoing cancer therapy. The level of the reference value will depend upon the particular population or subgroup selected. For example, an apparently healthy population may have a different "normal" value than will a population which has cancer, or a population that has cancer but has received cancer treatment. Appropriate ranges and categories for reference values can be selected with no more than routine
  • threshold values may vary for different tumors or under different circumstances, such as the conditions of the assay to determine expression. However, the skilled artisan would be able to identify the correct threshold values based on the circumstances. For example threshold values could easily be generated using normal non-cancerous tissue under similar circumstances. In each instance, the comparison of the expression levels of starvation markers to a reference value is useful in determining the relative levels of starvation or autophagic conditions in the test tumor cells.
  • the reference sample can be any of a variety of biological samples against which a diagnostic assessment may be made.
  • Examples of reference samples include biological samples from control populations or control samples. Reference samples may be generated through manufacture to be supplied for testing in parallel with the test samples, e.g. , reference sample may be supplied in diagnostic kits. Appropriate reference samples will be apparent to the skilled artisan.
  • the biomarker based methods are based in part on a comparison of expression levels of starvation marker genes or proteins between test samples and reference sample. In some embodiments, if the expression level of the starvation marker gene or protein in the test sample is about equal to the expression level of the starvation marker gene or protein in the reference sample, then the test sample and reference sample likely have similar metabolic conditions.
  • the magnitude of the difference between the test sample and reference sample that is sufficient to indicate a determination of therapeutic strategy will depend on a variety of factors such as the particular starvation marker gene or protein being evaluated, the type of cancer, the type of therapeutic to be applied, heterogeneity in healthy or disease populations from which samples are drawn, the type of reference sample, the assay being used, etc. It is well within the purview of the skilled artisan to determine the appropriate magnitude of difference between the test sample and reference sample that is sufficient to indicate a determination of the autophagic state of the cell.
  • the expression level of the starvation marker gene or protein in the test sample may be determined based on a direct comparison to a reference level in absolute values. For instance, at least 10%, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% or more higher than the expression level of the gene or protein in the reference sample. In other embodiments, the expression level of the starvation marker in the test sample is at least 10%, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% or more lower than the expression level of the gene or protein in the reference sample.
  • the starvation marker levels may be measured using any of a number of techniques available to the person of ordinary skill in the art for protein or nucleic acid, e.g., direct physical measurements (e.g., mass spectrometry), binding assays (e.g., immunoassays, agglutination assays, and immunochromatographic assays), Polymerase Chain Reaction (PCR) technology, branched oligonucleotide technology, Northern blot technology, oligonucleotide hybridization technology, and in situ hybridization technology.
  • direct physical measurements e.g., mass spectrometry
  • binding assays e.g., immunoassays, agglutination assays, and immunochromatographic assays
  • PCR Polymerase Chain Reaction
  • the method may also comprise measuring a signal that results from a chemical reaction, e.g., a change in optical absorbance, a change in fluorescence, the generation of chemiluminescence or electrochemiluminescence, a change in reflectivity, refractive index or light scattering, the accumulation or release of detectable labels from the surface, the oxidation or reduction or redox species, an electrical current or potential, changes in magnetic fields, etc.
  • a chemical reaction e.g., a change in optical absorbance, a change in fluorescence, the generation of chemiluminescence or electrochemiluminescence, a change in reflectivity, refractive index or light scattering, the accumulation or release of detectable labels from the surface, the oxidation or reduction or redox species, an electrical current or potential, changes in magnetic fields, etc.
  • Suitable detection techniques may detect binding events by measuring the participation of labeled binding reagents through the measurement of the labels via their photoluminescence (e.g., via measurement of fluorescence, time- resolved fluorescence, evanescent wave fluorescence, up-converting phosphors, multi- photon fluorescence, etc.), chemiluminescence, electrochemiluminescence, light scattering, optical absorbance, radioactivity, magnetic fields, enzymatic activity (e.g., by measuring enzyme activity through enzymatic reactions that cause changes in optical absorbance or fluorescence or cause the emission of chemiluminescence).
  • photoluminescence e.g., via measurement of fluorescence, time- resolved fluorescence, evanescent wave fluorescence, up-converting phosphors, multi- photon fluorescence, etc.
  • chemiluminescence e.g., via measurement of fluorescence, time- resolved fluorescence, evanescent wave fluorescence, up-converting phosphors, multi- photo
  • detection techniques may be used that do not require the use of labels, e.g., techniques based on measuring mass (e.g., surface acoustic wave measurements), refractive index (e.g., surface plasmon resonance measurements), or the inherent luminescence of an analyte.
  • the methods may involve the steps of isolating nucleic acids from the sample and/or an amplification step.
  • a nucleic acid comprising a sequence of interest can be obtained from a biological sample, more particularly from a sample comprising DNA (e.g. gDNA or cDNA) or RNA (e.g. mRNA). Release, concentration and isolation of the nucleic acids from the sample can be done by any method known in the art.
  • the nucleic acid of interest may be amplified.
  • amplification procedures can be accomplished by those methods known in the art, including, for example, the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, T7-polymerase amplification, and reverse transcription polymerase reaction (RT-PCR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • strand displacement amplification strand displacement amplification
  • rolling circle amplification rolling circle amplification
  • T7-polymerase amplification T7-polymerase amplification
  • RT-PCR reverse transcription polymerase reaction
  • PCR Polymerase chain reaction
  • PCR technology allows for the rapid generation of multiple copies of DNA sequences by providing 5' and 3' primers that hybridize to sequences present in an RNA or DNA molecule, and further providing free nucleotides and an enzyme which fills in the complementary bases to the nucleotide sequence between the primers with the free nucleotides to produce a complementary strand of DNA.
  • PCR primers can be designed routinely by those having ordinary skill in the art using sequence information.
  • the mRNA or cDNA is combined with the primers, free nucleotides and enzyme following standard PCR protocols. The mixture undergoes a series of temperature changes. If the test gene transcript or cDNA generated therefrom is present, that is, if both primers hybridize to sequences on the same molecule, the molecule comprising the primers and the intervening complementary sequences will be exponentially amplified.
  • the amplified DNA can be easily detected by a variety of well known means. If no gene transcript or cDNA generated therefrom is present, no PCR product will be exponentially amplified.
  • PCR product may be detected by several well known means.
  • One method for detecting the presence of amplified DNA is to separate the PCR reaction material by gel electrophoresis and stain the gel with ethidium bromide in order to visual the amplified DNA if present.
  • a size standard of the expected size of the amplified DNA is preferably run on the gel as a control.
  • RNA In some instances, such as when unusually small amounts of RNA are recovered and only small amounts of cDNA are generated therefrom, it is desirable to perform a PCR reaction on the first PCR reaction product.
  • the second PCR can be performed to make multiple copies of DNA sequences of the first amplified DNA.
  • a nested set of primers are used in the second PCR reaction. The nested set of primers hybridize to sequences downstream of the 5' primer and upstream of the 3' primer used in the first reaction.
  • Branched chain oligonucleotide hybridization may be performed as described in
  • nucleic acid molecules hybridize under stringent conditions to nucleic acid markers expressed in cancer cells.
  • the nucleic acid markers disclosed herein are known genes and fragments thereof.
  • Targets are nucleic acids selected from the group, including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and may be natural or synthetic. In all embodiments, nucleic acid molecules from human tissue are preferred. The tissue may be obtained from a subject or may be grown in culture. Binding assays for measuring starvation marker levels may use solid phase or homogenous formats. Suitable assay methods include sandwich or competitive binding assays. Examples of sandwich immunoassays are described in U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al., both of which are incorporated herein by reference. Examples of competitive immunoassays include those disclosed in U.S. Pat.
  • Multiple starvation markers may be measured using a multiplexed assay format, e.g., multiplexing through the use of binding reagent arrays, multiplexing using spectral discrimination of labels, multiplexing by flow cytometric analysis of binding assays carried out on particles (e.g., using the Luminex system
  • Detection of a protein in a test sample involves routine methods. The skilled artisan can detect the presence or absence of a protein using well known methods.
  • One such method is an immunoassay.
  • immunoassays involve the binding of proteins in a sample to a solid phase support such as a plastic surface. Detectable antibodies are then added which selectively binding to the protein of interest. Detection of the antibody indicates the presence of the protein.
  • the detectable antibody may be a labeled or an unlabeled antibody. Unlabeled antibody may be detected using a second, labeled antibody that specifically binds to the first antibody or a second, unlabeled antibody which can be detected using labeled protein A, a protein that complexes with antibodies.
  • Various immunoassay procedures are described in Immunoassays for the 80's, A. Voller et al., Eds., University Park, 1981, which is incorporated herein by reference.
  • Simple immunoassays such as a dot blot and a Western blot involve the use of a solid phase support which is contacted with a test sample. Any proteins present in the test sample bind the solid phase support and can be detected by a specific, detectable antibody preparation. The intensity of the signal can be measured to obtain a quantitative readout.
  • Other more complex immunoassays include forward assays for the detection of a protein in which a first anti-protein antibody bound to a solid phase support is contacted with the test sample. After a suitable incubation period, the solid phase support is washed to remove unbound protein. A second, distinct anti-protein antibody is then added which is specific for a portion of the specific protein not recognized by the first antibody. The second antibody is preferably detectable.
  • the solid phase support After a second incubation period to permit the detectable antibody to complex with the specific protein bound to the solid phase support through the first antibody, the solid phase support is washed a second time to remove the unbound detectable antibody.
  • a third detectable antibody which binds the second antibody is added to the system.
  • Other types of immunometric assays include simultaneous and reverse assays.
  • a simultaneous assay involves a single incubation step wherein the first antibody bound to the solid phase support, the second, detectable antibody and the test sample are added at the same time. After the incubation is completed, the solid phase support is washed to remove unbound proteins. The presence of detectable antibody associated with the solid support is then determined as it would be in a conventional assays.
  • a reverse assay involves the stepwise addition of a solution of detectable antibody to the test sample followed by an incubation period and the addition of antibody bound to a solid phase support after an additional incubation period.
  • the solid phase support is washed in conventional fashion to remove unbound protein/antibody complexes and unreacted detectable antibody.
  • antibodies against LAMP are well known in the art and are commercially available from several companies including but not limited to the following LAMP-1 antibodies in the Santa Cruz Catalog 1D4B (sc- 19992, mouse), 4E151 ( sc-71489, rat), 5K53 (sc-71488, mouse), C-20 (sc-8098, m, r, h), E-5 (sc- 17768, h > m, r), H-228 (sc- 5570, h > m, r), H4A3 (sc-20011, human), H5G11 (sc-18821, human), LY1C6 (sc- 65236, rat), B-T47 (sc-65331, human), and N-19 (sc-8099, human); the following LAMP-2 antibodies in the Santa Cruz Catalog 4E152 (sc-71491, mouse), 5K54 (s
  • antibodies can be detectably labeled by linking the antibodies to an enzyme and subsequently using the antibodies in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), such as a capture ELISA.
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Enzymes which can be used to detectably label antibodies include, but are not limited to malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • a detectable label is a moiety, the presence of which can be ascertained directly or indirectly.
  • detection of the label involves an emission of energy by the label.
  • the label can be detected directly by its ability to emit and/or absorb photons or other atomic particles of a particular wavelength (e.g., radioactivity, luminescence, optical or electron density, etc.).
  • a label can be detected indirectly by its ability to bind, recruit and, in some cases, cleave another moiety which itself may emit or absorb light of a particular wavelength (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, etc.).
  • An example of indirect detection is the use of a first enzyme label which cleaves a substrate into visible products.
  • the label may be of a chemical, peptide or nucleic acid molecule nature although it is not so limited.
  • Other detectable labels include radioactive isotopes such as P 32 or H 3 , luminescent markers such as fluorochromes, optical or electron density markers, etc., or epitope tags such as the FLAG epitope or the HA epitope, biotin, avidin, and enzyme tags such as horseradish peroxidase, ⁇ -galactosidase, etc.
  • the label may be bound to a peptide during or following its synthesis. There are many different labels and methods of labeling known to those of ordinary skill in the art.
  • Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds.
  • Those of ordinary skill in the art will know of other suitable labels for the peptides described herein, or will be able to ascertain such, using routine experimentation.
  • the coupling or conjugation of these labels to the peptides of the invention can be performed using standard techniques common to those of ordinary skill in the art.
  • haptens can then be specifically altered by means of a second reaction.
  • haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.
  • detectable labels include diagnostic and imaging labels (generally referred to as in vivo detectable labels) such as for example magnetic resonance imaging (MRI): Gd(DOTA); for nuclear medicine: 201 Tl, gamma-emitting radionuclide 99mTc; for positron-emission tomography (PET): positron-emitting isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F- fluoride, copper-64, gadodiamide, and radioisotopes of Pb(II) such as 203Pb; 11 lln.
  • MRI magnetic resonance imaging
  • DOTA positron-emission tomography
  • PET positron-emitting isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F- fluoride, copper-64, gadodiamide, and radioisotopes of Pb(II) such as 203Pb;
  • conjugation means two entities stably bound to one another by any physiochemical means. It is important that the nature of the attachment is such that it does not impair substantially the effectiveness of either entity. Keeping these parameters in mind, any covalent or non-covalent linkage known to those of ordinary skill in the art may be employed. In some embodiments, covalent linkage is preferred.
  • Noncovalent conjugation includes hydrophobic interactions, ionic interactions, high affinity interactions such as biotin-avidin and biotin- strep tavidin complexation and other affinity interactions. Such means and methods of attachment are well known to those of ordinary skill in the art.
  • the methods may also be accomplished using in situ hybridization or in vivo imaging methods.
  • In situ hybridization technology involves the addition of detectable probes which contain a specific nucleotide sequence to fixed cells. If the cells contain complementary nucleotide sequences, the probes, which can be detected, will hybridize to them.
  • In vivo Imaging involves the labeling of nucleic acids or proteins in vivo. The probes used in such methods are typically labeled for detection.
  • the methods of the invention are useful in determining the course of treatment of a variety of cancers, including but not limited to ovarian cancer, renal cancer, skin cancer, and breast cancer.
  • Other cancers treatable according to the invention include but are not limited to glioblastoma multiforme, leukemias, lymphomas, Hodgkins and Non- Hodgkins, thymomas.
  • Other cancers are well known to the skilled artisan.
  • ovarian cancer is predominately surgical resection. This includes removal of the uterus, ovaries, fallopian tubes, cancer in the abdomen or pelvis, removal of lymph nodes Chemotherapeutic treatment is
  • chemotherapeutic agents include paclitaxel, carboplatin or cisplatin. These treatment options cause many undesirable side effects; and unfortunately, between 50% to 70% of women who are treated for ovarian cancer with surgery and chemotherapy have cancer return within 5 years.
  • Ovarian cancer is highly vascularized, meaning that there is a critical dependence on blood supply for growth. Because ovarian cancer is highly vascularized, the rationale for using drugs that block blood vessel formation that, in turn, prevents required nutrients to feed the tumor cells, seems a rational proposition.
  • Blood vessel formation results from stimulation of endothelial cells by vascular endothelial growth factor (VEG-F) and currently available treatment consists of an antibody, a protein complex that neutralizes the growth factor VEG-F.
  • the antibody is known as AVASTIN® or bevacizumab.
  • RCC metastatic Renal cell Carinoma
  • RCC is also known as a very vascular tumor and newer treatment paradigms focus on the use of tyrosine kinase inhibitors.
  • VEGF vascular endothelial growth factor
  • angiogenesis in RCC has become increasingly available.
  • This transition in the treatment of RCC has led to less treatment associated toxicity as well as increased response rates.
  • bevacuzimab has been shown to have increased inhibition of VEGF and upregulation in the immunosuppressive cells, the response rates of patient' s have failed to be as high as initially hoped.
  • Ipilumubab VERVOX®
  • stage III unresectable melanoma Stage IV melanoma
  • This agent is the first approved since IL-2 in 1996.
  • Ipilumumab is expensive and has some serious immune-mediated side effects, such as endocrinopathy, hepatitis, and colitis.
  • melanoma is also highly vascularized, recent work has been aimed at blocking blood vessel formation that, in turn, prevents required nutrients to feed the tumor cells.
  • a treatment strategy to inhibit the growth factor effects of VEGF may provide an effective solution.
  • the assays described herein for identifying expression levels of starvation biomarkers assist in selecting a course of treatment for these difficult to treat cancers as well as other cancers.
  • the expression of the starvation marker is indicative of an autophagic tumor cell. If the tumor cell is an autophagic tumor cell, then autophagy inhibitors and/or fatty acid metabolism inhibitors should be administered prior to the starvation signal compound. According to one set of embodiments, the cells are exposed to an autophagy inhibitor.
  • An "autophagy modulator,” as used herein, is a lysosomotropic agent, meaning that it accumulates preferentially in the lysosomes of cells in the body and blocks pathways involved in break down of cellular components.
  • An autophagy inhibitor is any compound which blocks the collection or metabolism of lipids in the lysosome.
  • the inhibitor is effective for killing cells by inhibiting autophagy in cells that depend on autophagy to survive. While no one knows exactly the mechanism by which autophagy inhibitors function, it may well be through the inhibition of the acidic hydrolases (enzymes in the lysosomes) that are necessary to break down proteins, lipids, etc. for processing and removal by increasing the pH to decrease the necessary acidity for the enzymes to work.
  • the autophagy inhibitor is selected from the group consisting of: chloroquine compounds, 3-methyladenine, bafilomycin Al, 5-amino-4- imidazole carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1, analogues of cAMP, and drugs which elevate cAMP levels, adenosine, N6-mercaptopurine riboside, wortmannin, and vinblastine.
  • AICAR 5-amino-4- imidazole carboxamide riboside
  • the autophagy inhibitor is preferably a chloroquine compound.
  • Chloroquine is a synthetically manufactured drug containing a quinoline nucleus (The Merck Index, p. 2220, 1996).
  • the chloroquine compounds useful according to the invention include chloroquine analogs and derivatives. A number of chloroquine analogs and derivatives are well known.
  • suitable compounds include but are not limited to chloroquine, chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate, hydroxychloroquine sulphate, quinacrine, primaquine, mefloquine, halofantrine, lumefantrine and tafenoquine or enantiomers, derivatives, analogs, metabolites, pharmaceutically acceptable salts, and mixtures thereof.
  • Chloroquine and hydroxychloroquine are generally racemic mixtures of (-)- and (-i-)-enantiomers.
  • the (-)-enantiomers are also known as (R)-enantiomers (physical rotation) and 1 -enantiomers (optical rotation).
  • the (-i-)-enantiomers are also known as (S)-enantiomers (physical rotation) and r-enantiomers (optical rotation).
  • the (-)-enantiomer of chloroquine is used.
  • hydroxychloroquine can be prepared by procedures known to the art.
  • the compounds of the invention, such as, chloroquine may exhibit the
  • the invention covers any tautomeric, conformational isomeric, optical isomeric and/or geometric isomeric forms of the compounds described herein, as well as mixtures of these various different forms.
  • the autophagy inhibitor useful in the invention is a 4- aminoquinoline.
  • 4-aminoquinolines include compounds having the following structure:
  • each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration
  • Ri is 1 or 2 hydrogens, alkyl, cycloalkyl, aryl, substituted alkyl, substituted cycloalkyl or substituted aryl.
  • the 4- aminoquinoline has the following structure:
  • each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration
  • R 2 and R 3 is independently a hydroxalkyl, an alkyl, alkyloxy, alkylcarboxy, alkylene or alkenylene having from one to six carbon atoms.
  • 4-aminoquinolines useful according to the invention include but are not limited to chloroquine, 2-hydroxychloroquine, amodiaquine,
  • the cells are exposed to a fatty acid metabolism inhibitor.
  • Metabolic disruption of fatty acids can be achieved using inhibitors of fatty acid metabolism.
  • a "fatty acid metabolism inhibitor,” as used herein, is a compound able to inhibit (e.g., prevent, or at least decrease or inhibit the activity by an order of magnitude or more) a reaction within the fatty acid metabolism pathway, such as an enzyme-catalyzed reaction within the pathway.
  • the inhibitor may inhibit the enzyme, e.g., by binding to the enzyme or otherwise interfering with operation of the enzyme (for example, by blocking an active site or a docking site, altering the configuration of the enzyme, competing with an enzyme substrate for the active site of an enzyme, etc.), and/or by reacting with a coenzyme, cofactor, etc. necessary for the enzyme to react with a substrate.
  • the fatty acid metabolism pathway is the pathway by which fatty acids are metabolized within a cell for energy (e.g., through the synthesis of ATP and the breakdown of fatty acids into simpler structures, such as C0 2 , acyl groups, etc.) or to produce a carbohydrate source.
  • inhibitors of fatty acid metabolism include inhibitors of fatty acid oxidation, fatty acid transporter inhibitors, reductase inhibitors, and isomerase inhibitors within the fatty acid metabolism pathway.
  • the fatty acid metabolism inhibitor in some embodiments is an inhibitor of fatty acid oxidation, a fatty acid transporter inhibitor, a reductase inhibitor, or an isomerase inhibitor within the fatty acid metabolism pathway.
  • the reductase is 2,4-dienoyl-CoA reductase.
  • the isomerase is 2,4-dienoyl-CoA isomerase.
  • the inhibitor of fatty acid metabolism is an inhibitor of fatty acid oxidation and is any one or more of the following: oxirane carboxylic acid compound, such as etomoxir (2-(6-(4-chlorophenoxy)-hexyl)-oxirane-2- carboxylic acid ethyl ester), 2-(4-(3-chlorophenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(3,4- dichlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(4-fluorophenoxy)-
  • the fatty acid metabolism pathway includes several enzymatic reactions, which use various enzymes such as reductases or isomerases. Specific examples of enzymes within the fatty acid metabolism pathway include 2,4-dienoyl-CoA reductase, 2,4- dienoyl-CoA isomerase, butyryl dehydrogenase, etc, as further discussed below.
  • the fatty acid metabolism inhibitor is an inhibitor able to inhibit a beta- oxidation reaction in the fatty acid metabolism pathway.
  • the inhibitor is an inhibitor for a fatty acid transporter (e.g., a transporter that transports fatty acids into the cell, or from the cytoplasm into the mitochondria for metabolism).
  • the inhibitor may react or otherwise inhibit key steps within the fatty acid metabolism pathway.
  • the inhibitor may be an inhibitor of fatty acids as a source of energy in the mitochondria.
  • the inhibitor may inhibit the breakdown of intermediates such as butyryl CoA, glutaryl CoA, or isovaleryl CoA.
  • 2,4-dienoyl-CoA reductase is an enzyme within the fatty acid metabolism pathway that catalyzes reduction reactions involved in the metabolism of polyunsaturated fatty acids.
  • Certain fatty acids are substrates for 2,4-dienoyl-CoA reductases located within the mitochondria.
  • fatty acids may be transported into the mitochondria through uncoupling proteins.
  • the uncoupling protein may, in certain instances, increase the mitochondrial metabolism to increase the availability of fatty acids within the mitochondria and/or increase the throughput of beta- oxidation within the mitochondria.
  • the enzyme 2,4-dienoyl-CoA isomerase is an enzyme within the fatty acid metabolism pathway that catalyzes isomerization of certain fatty acids.
  • One step in the metabolism of certain polyunsaturated fatty acids may be protective against reactive oxygen intermediates ("ROI").
  • ROI reactive oxygen intermediates
  • fatty acid metabolism inhibitors compounds useful for inhibiting fatty acid metabolism are also useful for altering cellular production of reactive oxygen; compounds described in reference to fatty acid metabolism inhibition should also be understood herein to be able to alter reactive oxygen production within a cell.
  • fatty acid metabolism inhibitors By altering the ability of a cell to metabolize a fatty acid, the ability of the cell to produce reactive oxygen may also be affected, since one pathway for a cell to produce reactive oxygen intermediates is through the metabolism of fatty acids.
  • the production of reactive oxygen can be affected by exposing a cell to, or removing a cell from, a fatty acid metabolism inhibitor.
  • the inhibitor of fatty acid metabolism may be an inhibitory nucleic acid.
  • the inhibitory nucleic acid may be, for instance, specific for an enzyme selected from the group consisting of 2,4-dienoyl-CoA reductase, 2,4-dienoyl-CoA isomerase, and butyryl dehydrogenase.
  • the inhibitor of fatty acid metabolism is oxamate.
  • the oxamate may be, for instance an alkyl oxamate such as, ethyl oxamate or sodium oxamate.
  • the inhibitor of fatty acid metabolism is a compound having the following structure:
  • the fatty acid inhibitor is an oxamate including, for example, each of the following:
  • the method involves the use of a fatty acid metabolism inhibitor that is an oxirane carboxylic acid compound capable of inhibiting fatty acid metabolism, or a pharmacologically acceptable salt thereof in some embodiments.
  • a fatty acid metabolism inhibitor that is an oxirane carboxylic acid compound capable of inhibiting fatty acid metabolism, or a pharmacologically acceptable salt thereof in some embodiments.
  • the subject may not have an indication otherwise indicated for treatment with the compound.
  • the oxirane carboxylic acid compound has the formula:
  • R 5> R 6 and R 7 are herein; wherein R5 represents a hydrogen atom, a halogen atom, a 1-4C alkyl group, a 1-4C alkoxy group, a nitro group or a trifluoromethyl group, R 6 has one of the meanings of R 5 , R 7 represents a hydrogen atom or a 1-4C alkyl group, Y represents the grouping— O— (CH 2 ) m — , m is 0 or a whole number from 1 to 4, and n is a whole number from 2 to 8 wherein the sum of m and n is a whole number from 2 to 8.
  • R 5 in some embodiments is a halogen atom
  • R 6 is a hydrogen atom
  • m is 0, and n is 6.
  • R 7 is an ethyl group.
  • the oxirane carboxylic acid compound is etomoxir in some embodiments.
  • etomoxir i.e., 2-(6-(4-chlorophenoxy)- hexyl)-oxirane-2-carboxylic acid ethyl ester.
  • examples of other oxirane carboxylic acid compounds useful in the invention are 2-(4-(3-chlorophenoxy)-butyl)-oxirane-2- carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2-carboxylic acid ethyl ester, 2- (6-(3,4-dichlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(4- fluorophenoxy)-hexyl)-oxirane-2-carboxylic acid eth
  • oxirane carboxylic acid compounds including etomoxir
  • Horst Wolf and Klaus Eistetter in United States Patent 4,946,866 for the prevention and treatment of illnesses associated with increased cholesterol and/or triglyceride concentration
  • Horst Wolf in United States Patent 5,739,159 for treating heart insufficiency.
  • the preparation of oxirane carboxylic acid compounds, and their use for blood glucose lowering effects as an ant diabetic agent, is described in Jew et al United States Patent 6,013,666.
  • Etomoxir has been described as an inhibitor of mitochondrial carnitine palmitoyl transferase-I by Mannaerts, G. P., L. J. Debeer, J.
  • fatty acid metabolism inhibitors include fatty acid transporter inhibitors, beta- oxidation process inhibitors, reductase inhibitors, and/or isomerase inhibitors within the fatty acid metabolism pathway.
  • fatty acid metabolism inhibitors include, but are not limited to, cerulenin, 5-
  • the inhibitor may be a non- hydrolyzable analog of carnitine.
  • the fatty acid metabolism inhibitor is a carboxylic acid.
  • the carboxylic acid may have the structure:
  • R 14 comprises an organic moiety, as further described below.
  • R 14 may include at least two nitrogen atoms, or R 14 may include an aromatic moiety (as further described below), such as a benzene ring, a furan, etc.
  • the fatty acid metabolism inhibitor has the structure:
  • each of R 5 and R 16 independently comprises organic moiety.
  • either or both of R15 and R 16 may independently be an alkyl, such as a straight- chain alkyl, for instance, methyl, ethyl, propyl, etc.
  • R 16 may have at least 5 carbon atoms, at least 10 carbon atoms, or at least 15 or more carbon atoms.
  • R 16 may be a tetradecyl moiety.
  • R 16 may include an aromatic moiety, for example, a benzene ring.
  • R 16 may have the structure:
  • R 3 comprises an organic moiety and Ar 1 comprises an aromatic moiety.
  • R 17 may be a an alkyl, such as a straight-chain alkyl.
  • Ar 1 may be a benzene ring or a derivative thereof, i.e., having the structure:
  • each of R 18 , R 19 , R 2 o, R 2 i, and R 22 is hydrogen, a halogen, an alkyl, an alkoxy, etc.
  • the fatty acid metabolism inhibitor has the structure:
  • each of R 2 , R 24 , R 25 , R % , R 27 R 2 8 and R 29 independently comprises hydrogen, a halogen, or an organic moiety, such as an alkyl, an alkoxy, etc.
  • R 23 and R 24 together may define an organic moiety, such as a cyclic group.
  • the fatty acid metabolism inhibitor may have the structure:
  • R 3 o comprises an organic moiety, such as an alkyl, an alkoxy, an aromatic moiety, an amide, etc.
  • An exam le, of R 0 is:
  • Ar comprises an aromatic moiety, such as a benzene ring or a benzene derivative, as previously described.
  • the cells may be exposed to an agent that inhibits the synthesis or production of one or more enzymes within the fatty acid metabolism pathway. Exposure of the cells to the agent thus inhibits fatty acid metabolism within the cell.
  • an inhibitory oligonucleotide such as a RNAi or antisense oligonucleotide may be used that selectively binds to regions encoding enzymes present within the fatty acid metabolism pathway, such as 2,4-dienoyl-CoA reductase or 2,4-dienoyl-CoA isomerase.
  • agents that inhibit enzymes of the fatty acid metabolism pathway include enzymes of the fatty acid metabolism pathway expression inhibitors.
  • a enzymes of the fatty acid metabolism pathway expression inhibitor as used herein is molecule that knocks down expression of an enzyme of the fatty acid metabolism pathway.
  • the invention also features the use of small nucleic acid molecules, referred to as short interfering nucleic acid (siNA) that include, for example: microRNA (miRNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), and short hairpin RNA (shRNA) molecules to knockdown expression of proteins such as enzymes of the fatty acid metabolism pathway.
  • siNA of the invention can be unmodified or chemically- modified.
  • siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized.
  • the instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating gene expression or activity in cells by RNA interference (RNAi).
  • siNA synthetic short interfering nucleic acid
  • RNAi RNA interference
  • the use of chemically-modified siNA improves various properties of native siNA molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake.
  • siNA having multiple chemical modifications may retain its RNAi activity.
  • the siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic applications.
  • nucleic acid molecules with modifications that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065;
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996,
  • one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target RNA or a portion thereof
  • the second strand of the double- stranded siNA molecule comprises a nucleotide sequence identical to the nucleotide sequence or a portion thereof of the targeted RNA.
  • one of the strands of the double- stranded siNA molecule comprises a nucleotide sequence that is substantially complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double- stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the target RNA.
  • each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
  • an siNA is an shRNA, shRNA-mir, or microRNA molecule encoded by and expressed from a genomically integrated transgene or a plasmid-based expression vector.
  • a molecule capable of inhibiting mRNA expression, or microRNA activity is a transgene or plasmid-based expression vector that encodes a small-interfering nucleic acid.
  • Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siRNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue-specific expression systems.
  • transgenes and expression vectors are controlled by tissue specific promoters.
  • transgenes and expression vectors are controlled by inducible promoters, such as tetracycline inducible expression systems.
  • a small interfering nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • the recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid- specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters.
  • Developmentally-regulated promoters are also encompassed, for example the murine hox promoters and the a-fetoprotein promoter.
  • inhibitor molecules that can be used include sense and antisense nucleic acids (single or double stranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, antibodies, and aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins.
  • Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al.,
  • Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9,1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al., Nucleic Acids Symp Ser. (29): 121-2, 1993).
  • Anti-sense oligonucleotides may be designed to hybridize to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of an enzymes of the fatty acid metabolism pathway encoded by a given DNA sequence (e.g. either native polypeptide or a mutant form thereof), so that its expression is reduce or prevented altogether.
  • Anti-sense techniques may be used to target a coding sequence; a control sequence of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with control sequences.
  • Anti-sense oligonucleotides may be DNA or RNA and may be of around 14-23 nucleotides, particularly around 15-18 nucleotides, in length. The construction of antisense sequences and their use is described in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).
  • sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence.
  • the sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective sense RNA molecules to hybridize. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene.
  • Triple helix approaches have also been investigated for sequence- specific gene suppression. Triple helix forming oligonucleotides have been found in some cases to bind in a sequence- specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A.
  • suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz Nature 329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6, 1989; Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989).
  • suppression strategies have led to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein.
  • the diverse array of suppression strategies that can be employed includes the use of DNA and/or RNA aptamers that can be selected to target, for example, a protein of interest such as enzymes of the fatty acid metabolism pathway.
  • 2,4-dienoyl-CoA reductase has been described in for instance Koivuranta et al Biochemical Journal 1994, 304, p. 787. It is also disclosed in NCBI gene ID 1666 (DECR1) as well as NCBI genbank Accession number U78302. The sequence of 2,4- dienoyl-CoA isomerase is disclosed in NCBI gene ID 1891 (ECH1).
  • Nucleic acid sequence for 2,4-dienoyl-CoA reductase is taagctttaa aaacatgtaa aaaggacatt aaattgacat ctttttgtg ttaggtcacc aaggagcagt gggacaccat agaagaactc atcaggaaga caaaaggttc ctaagaccac tttggccttc atcttggtta cagaaaaggg aatagaaatg aaacaaatta tctctcatct tttgactatt tcaagtctaa taaattctta attaacaaaac attcattgaaa tatgtattat gtgccaggcc agtgatagcc attgtatattt caaagataaaaatgaat
  • the cells may be treated with both an autophagic inhibitor and an inhibitor of fatty acid metabolism. In some embodiments when both are administered it is preferred to administer the autophagic inhibitor first.
  • a starvation signal compound refers to a cancer therapy involving the blocking of a metabolic pathway or blood vessel formation.
  • a starvation signal compound as used herein includes VEGF antagonists, rapimycin, and glycolytic inhibitors.
  • a VEGF antagonist is a compound that inhibits the activity of VEGF.
  • a VEGF antagonists is an anti-VEGF antibody.
  • Anti-VEGF antibodies refer to peptides that bind to VEGF with sufficient affinity and specificity to prevent VEGF from interacting with VEGF receptor and reduce VEGF signaling.
  • the term "VEGF” refers to the vascular endothelial cell growth factor, as described by Leung et al. Science, 246: 1306 (1989), and Houck et al. Mol. Endocrin., 5: 1806 (1991), together with the naturally occurring allelic and processed forms thereof.
  • VEGF is also used to refer to known truncated forms of the polypeptide.
  • the anti-VEGF antibody of the invention can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein the VEGF activity is involved.
  • An anti-VEGF antibody will usually not bind to other VEGF homologues such as VEGF-B or VEGF-C, nor other growth factors such as PIGF, PDGF or bFGF.
  • a preferred anti- VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599, including but not limited to the antibody known as bevacizumab (AVASTIN®).
  • Bevacizumab is a major drug developed for treating cancer, including metastatic cancer, and has the trade name AVASTIN ®, by Genentech/Roche.
  • Bevacizumab is a humanized monoclonal antibody, and was the first commercially available angiogenesis inhibitor. It stops tumor growth by preventing the formation of new blood vessels (angiogenesis) by targeting and inhibiting the function of a natural protein called vascular endothelial growth factor that stimulates new blood vessel formation.
  • the drug was first developed as a genetically engineered version of a mouse antibody that contains both human and mouse components, a monoclonal antibody against VEGF-A.
  • the VEGF antibody is not VEGFR-3 mAb disclosed by Imclone Systems Inc., New York, N.Y.
  • variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three or four CDRs or "hypervariable regions" in both in the light-chain and the heavy-chain variable domains, as discussed above.
  • the starvation signal compound of the invention may also be an mTOR
  • mTOR inhibitors include but are not limited to rapimycin (sirolimus). Rapimycin is an immunosuppressant that has been shown to be useful in the treatment of cancer.
  • a glycolytic inhibitor may also be used in the methods of the invention.
  • Glycolytic inhibitors are inhibitors of
  • glycolytic inhibitors are 2-deoxyglucose compounds, defined herein as homologs, analogs, and/or derivatives of 2-deoxy-D-glucose.
  • Glycolytic inhibitors particularly useful herein can have the formula:
  • Rg , Rjo, Rn, R 12 , and R 13 are herein; wherein X represents an O or S atom; R 9 represents a hydrogen atom or a halogen atom; R 10 represents a hydroxyl group, a halogen atom, a thiol group, or CO-R 6 ; Rn, Ri 2 , and R 13 each represent a hydroxyl group, a halogen atom, or CO- R 14 , R 14 represents an alkyl group of from 1 to 20 carbon atoms, and at least two of R 11; R 12 , and R 1 are hydroxyl groups.
  • the 2-deoxyglucose compound is 2-deoxy-D-glucose.
  • the invention also encompasses a method of treating cancer by administering a mTOR inhibitor such as rapamycin and an autophagy inhibitor and/or a fatty acid metabolism inhibitor.
  • a mTOR inhibitor such as rapamycin and an autophagy inhibitor and/or a fatty acid metabolism inhibitor.
  • the compounds may be administered simultaneously, in one or separate administrations. Alternatively they may be administered at different times. For instance the autophagy inhibitor and/or a fatty acid metabolism inhibitor may be administered before or after the mTOR inhibitor.
  • a combined therapy of VEGF antagonist i.e. AVASTIN® to block VEGF-mediated blood vessel formation
  • autogphagy inhibitor i.e.
  • hydroxy-chloroquine in a specific dose and timing of administration protocol is another aspect of the invention.
  • a preferred treatment protocol involves AVASTIN, administered at 15 mg/Kg IV every 3 months; and Hydroxychloroquine administered at 200-400 mg per day.
  • AVASTIN neutralizes VEGF and lead to decreased nutrition to the tumor, while Hydroxychloroquine blocks autophagy.
  • the molecules useful herein are isolated molecules. As used herein, the term
  • isolated means that the referenced material is removed from its native environment, e.g., a cell.
  • an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material is occurs naturally (e.g., cytoplasmic or membrane component).
  • the isolated molecules may be
  • the molecules are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing.
  • an isolated peptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the peptide may comprise only a small percentage by weight of the preparation.
  • the peptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.
  • the peptide is a synthetic peptide.
  • purified in reference to a protein or a nucleic acid, refers to the separation of the desired substance from contaminants to a degree sufficient to allow the practitioner to use the purified substance for the desired purpose. Preferably this means at least one order of magnitude of purification is achieved, more preferably two or three orders of magnitude, most preferably four or five orders of magnitude of purification of the starting material or of the natural material.
  • a purified thymus derived peptide is at least 60%, at least 80%, or at least 90% of total protein or nucleic acid, as the case may be, by weight.
  • a purified thymus derived peptide is purified to homogeneity as assayed by, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis, or agarose gel electrophoresis.
  • the therapeutic compounds described herein can be administered in combination with other therapeutic agents and such administration may be simultaneous or sequential.
  • the other therapeutic agents When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time.
  • the administration of the other therapeutic agent including chemotherapeutics and the chloroquine, hydroxychloroquine, or AVASTIN® can also be temporally separated, meaning that the therapeutic agents are administered at a different time, either before or after, the administration of the therapeutics described herein.
  • the separation in time between the administration of these compounds may be a matter of minutes or it may be longer.
  • the invention also involves administering another cancer treatment (e.g. , radiation therapy, chemotherapy or surgery) to a subject.
  • cancer therapies include treatment of the cancer with agents such as All- trans retinoic acid, Actinomycin D, Adriamycin, anastrozole, Azacitidine, Azathioprine, Alkeran, Ara-C, Arsenic Trioxide (Trisenox), BiCNU Bleomycin, , Busulfan, CCNU, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Cytoxan, DTIC, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, 5-flurouracil, Epirubicin, Epothilone, Etoposide, exemestane, Erlotinib, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Herceptin, Hydrea, Ifos
  • the conjugates include an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (i.e., a radioconjugate).
  • chemotherapeutic agent e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin
  • radioactive isotope i.e., a radioconjugate
  • Enzymatically active toxins and fragments thereof which can be used in the conjugates include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • the antibody may comprise a highly radioactive atom.
  • a variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 ,
  • Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu may comprise a radioactive atom for scintigraphic studies, for example tc 99 m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-I l l, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • the radio- or other labels may be incorporated in the conjugate in known ways.
  • the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen.
  • Labels such as tc 99m or I 123 , .Re 186 , Re 188 and In 111 can be attached via a cysteine residue in the peptide.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in
  • Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP) , succinimidyl-4- (N-maleimidomethyl)cyclohexane- 1 - carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
  • SPDP N-succinimidyl-3-(2- pyridyldithio)propionate
  • IT imin
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
  • Carbon- 14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
  • the linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell.
  • an acid-labile linker for example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
  • the dosages of known therapies may be reduced in some instances, to avoid side effects.
  • the therapeutic compounds of the invention are formulated into a pharmaceutical composition that further comprises one or more additional anticancer agents.
  • an effective amount for instance, is an amount necessary or sufficient to realize a desired biologic effect.
  • an effective amount is that amount sufficient to prevent or inhibit autophagy.
  • An effective amount for treating precancerous tissue may be an amount sufficient to prevent, delay or inhibit the development of a tumor in the subject compared to the levels in the absence of treatment.
  • an effective amount is that amount of a compound of the invention alone or in combination with another
  • prevention or inhibition refers to any reduction or delay in tumor formation as a result of the treatment when compared to an untreated subject.
  • the effective amount of a compound of the invention in the treatment of a subject may vary depending upon the specific compound used, the mode of delivery of the compound, and whether it is used alone or in combination.
  • the effective amount for any particular application can also vary depending on such factors as the type and/or amount of catastrophic trigger to which the subject is exposed, the particular compound being administered for treatment, the size of the subject, or the severity of the disorder.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular molecule of the invention without necessitating undue experimentation.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity in and of itself and yet is entirely effective to treat the particular subject.
  • Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays, animal studies and human studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans.
  • the dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • DCA Dichloroacetate
  • Chloroquine typically is administered in a dosage of 300mg-600mg to adults for the treatment of malarial infection.
  • DCA can be used, for example, in dosages of 1- 25 mg/kg of body weight per day, 1- 15 mg/kg of body weight per day, or 5- 10 mg/kg of body weight per day.
  • treat, treated, or treating when used with respect to a disorder refers to a prophylactic treatment which increases the resistance of a subject to development of the disease or, in other words, decreases the likelihood that the subject will develop the disease as well as a treatment after the subject has developed the disease in order to fight the disease, prevent the disease from becoming worse, or slow the progression of the disease compared to in the absence of the therapy.
  • Certain compounds as described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
  • the compounds provided herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • the compounds as described herein are enantiopure compounds.
  • mixtures of stereoisomers are provided.
  • certain compounds, as described herein may have one or more double bonds that can exist as either the cis or trans, or the E or Z isomer, unless otherwise indicated.
  • the invention additionally encompasses the compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers, e.g., racemic mixtures of E/Z isomers or mixtures enriched in one E/Z isomer.
  • an enantiomerically enriched preparation of the (S)-enantiomer means a preparation of the compound having greater than 50% by weight of the (S)-enantiomer relative to the (R)- enantiomer, more preferably at least 75% by weight, and even more preferably at least 80% by weight.
  • the enrichment can be much greater than 80% by weight, providing a "substantially enantiomerically enriched," "substantially
  • enantiomerically pure or a "substantially non-racemic" preparation, which refers to preparations of compositions which have at least 85% by weight of one enantiomer relative to other enantiomer, more preferably at least 90% by weight, and even more preferably at least 95% by weight.
  • the enantiomerically enriched composition has a higher potency with respect to therapeutic utility per unit mass than does the racemic mixture of that composition.
  • Enantiomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred enantiomers can be prepared by asymmetric syntheses.
  • alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 6 carbon atoms ("Cl-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("Cl-5 alkyl").
  • an alkyl group has 1 to 4 carbon atoms ("CI— 4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("Cl-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("Cl-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“CI alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”).
  • Cl-6 alkyl groups include methyl (CI), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6).
  • each instance of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") are substituted with one or more substituents.
  • the alkyl group is an unsubstituted Cl-6 alkyl (e.g., -CH3).
  • the alkyl group is a substituted Cl-6 alkyl.
  • alkyloxy refers to an alkyl group, as defined herein, substituted with an oxygen atom, wherein the point of attachment is the oxygen atom.
  • the alkyl group has 1 to 6 carbon atoms ("Cl-6 alkyloxy”).
  • the alkyl group has 1 to 4 carbon atoms (“Cl-4 alkyloxy”). Examples of Cl-4 alkyloxy groups include methoxy (CI), ethoxy (C2), propoxy (C3), isopropoxy (C3), butoxy (C4), tert-butoxy (C5) and the like.
  • Cl-6 alkyloxy groups include the aforementioned Cl-4 alkyloxy groups as well as pentyloxy (C5), isopentyloxy (C5), neopentyloxy (C5), hexyloxy (C6) and the like. Unless otherwise specified, each instance of the alkyl moiety of the alkyloxy group is independently unsubstituted (an "unsubstituted alkyloxy") or substituted (a "substituted alkyloxy”) with one or more substituents. In certain embodiments, the alkyloxy group is an unsubstituted Cl-6 alkyloxy. In certain embodiments, the alkyloxy group is a substituted Cl-6 alkyloxy.
  • the alkyl of the alkylcarboxy group has 1 to 6 carbon atoms ("Cl-6 alkylcarboxy”).
  • the alkyl of the alkylcarboxy group has 1 to 5 carbon atoms ("Cl-5 alkylcarboxy”).
  • the alkyl of the alkylcarboxy group has 1 to 4 carbon atoms ("CI— 4 alkylcarboxy").
  • the alkyl of the alkyl of the alkyl of the alkyl of the alkylcarboxy has 1 to 6 carbon atoms ("Cl-6 alkylcarboxy").
  • the alkyl of the alkylcarboxy group has 1 to 5 carbon atoms ("Cl-5 alkylcarboxy”).
  • the alkyl of the alkylcarboxy group
  • alkylcarboxy group has 1 to 3 carbon atoms (“Cl-3 alkylcarboxy").
  • the alkyl of the alkylcarboxy group has 1 to 2 carbon atoms ("Cl-2 alkylcarboxy"). Unless otherwise specified, each instance of the alkyl of the
  • alkylcarboxy group is independently unsubstituted (an "unsubstituted alkylcarboxy") or substituted (a "substituted alkylcarboxy") with one or more substituents.
  • the alkylcarboxy group is an unsubstituted Cl-6 alkylcarboxy.
  • the alkylcarboxy group is a substituted Cl-6 alkylcarboxy.
  • alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 6 carbon atoms and one or more carbon-carbon double bonds (“C2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl”).
  • the one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
  • C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4) and the like.
  • C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6) and the like.
  • each instance of an alkenyl group is independently unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents.
  • the alkenyl group is an unsubstituted C2-6 alkenyl.
  • the alkenyl group is a substituted C2-6 alkenyl.
  • alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 6 carbon atoms and one or more carbon-carbon triple bonds (“C2-6 alkynyl”).
  • an alkynyl group has 2 to 5 carbon atoms ("C2-5 alkynyl”).
  • an alkynyl group has 2 to 4 carbon atoms ("C2-4 alkynyl”).
  • an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”).
  • an alkynyl group has 2 carbon atom (“C2 alkynyl”).
  • the one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
  • C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4) and the like.
  • C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6) and the like.
  • each instance of an alkynyl group is independently unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents.
  • the alkynyl group is an unsubstituted C2-6 alkynyl.
  • the alkynyl group is a substituted C2-6 alkynyl.
  • a "saturated or unsaturated acyclic hydrocarbon” refers to radical of a saturated or unsaturated, straight-chain or branched, hydrocarbon group having from 1 to 20 carbon atoms and optionally one or more carbon-carbon double or triple bonds.
  • the hydrocarbon group is saturated.
  • the hydrocarbon group is unsaturated, and contains one or more carbon-carbon double or triple bonds.
  • the hydrocarbon group contains 1-10 carbon atoms.
  • the hydrocarbon group contains 1-5 carbon atoms.
  • the hydrocarbon group contains 1-4 carbon atoms.
  • the hydrocarbon group contains 1-3 carbon atoms.
  • the hydrocarbon group contains 1-2 carbon atoms.
  • hydroxyl or “hydroxy” refers to the group -OH.
  • Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl referred to without the suffix "-ene,” describe a monoradical of alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, respectively, and as defined herein, wherein the monoradical is attached to another group by only one single bond.
  • Groups referred to with the suffix "-ene”, such as alkylene, alkenylene, alkynylene, carbocyclylene, heterocyclylene, arylene and heteroarylene groups, describe a diradical of alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, respectively, and as defined herein, wherein the diradical is attached to one or two groups by two single bonds.
  • the term "pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(Cl-4alkyl)4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • prodrug means a biologically active derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide the pharmacologically active compound.
  • the "prodrug” is a compound administered to a subject, and the pharmacologically active compound is the "active metabolite thereof.”
  • a prodrug has improved physical and/or delivery properties over the parent compound.
  • Prodrugs are typically designed to enhance pharmaceutically and/or pharmacokinetically based properties associated with the parent compound. The advantage of a prodrug can lie in its physical properties, such as enhanced water solubility for parenteral administration at
  • a sub-therapeutic dosage of either or both of the molecules may be used.
  • a "sub-therapeutic dose” as used herein refers to a dosage which is less than that dosage which would produce a therapeutic result in the subject if administered in the absence of the other agent.
  • compositions of the present invention comprise an effective amount of one or more agents, dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • animal e.g. , human
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • the compounds are generally suitable for administration to humans. This term requires that a compound or
  • composition be nontoxic and sufficiently pure so that no further manipulation of the compound or composition is needed prior to administration to humans.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. , antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and
  • the compounds may be sterile or non-sterile.
  • the agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally,
  • intraperitoneal injection is contemplated.
  • the composition may comprise various antioxidants to retard oxidation of one or more components.
  • the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g. , methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g. , methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the agent may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g. , those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g. , glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g. , triglycerides, vegetable oils, liposomes) and combinations thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods.
  • isotonic agents such as, for example, sugars, sodium chloride or combinations thereof.
  • the compounds of the invention may be administered directly to a tissue.
  • Direct tissue administration may be achieved by direct injection.
  • the compounds may be administered once, or alternatively they may be administered in a plurality of administrations. If administered multiple times, the compounds may be administered via different routes. For example, the first (or the first few) administrations may be made directly into the affected tissue while later administrations may be systemic.
  • compositions of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • a pharmaceutical composition comprises the compound of the invention and a pharmaceutically-acceptable carrier.
  • Pharmaceutically- acceptable carriers for peptides, monoclonal antibodies, and antibody fragments are well- known to those of ordinary skill in the art.
  • a pharmaceutically- acceptable carrier means a non-toxic material that does not interfere with the
  • Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Exemplary pharmaceutically acceptable carriers for peptides in particular are described in U.S. Patent No. 5,211,657. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically- acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • the compounds of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections, and usual ways for oral, parenteral or surgical administration.
  • the invention also embraces pharmaceutical compositions which are formulated for local administration, such as by implants.
  • Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as a syrup, an elixir or an emulsion.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
  • polyvinylpyrrolidone PVP
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethan
  • the compounds when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g. , by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g. , in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer' s, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
  • the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient.
  • exemplary bioerodible implants that are useful in accordance with this method are described in PCT International Application No. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, 1994).
  • PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing a biological macromolecule. The polymeric matrix may be used to achieve sustained release of the agent in a subject.
  • the agent described herein may be encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agent is stored in the core of a polymeric shell).
  • Other forms of the polymeric matrix for containing the agent include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted.
  • the size of the polymeric matrix device further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular, pulmonary, or other surface.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the agents of the invention to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
  • the agents of the invention may be delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly- vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose prop
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters,
  • polyurethanes poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, ze
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compound, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non- polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the platelet reducing agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • Therapeutic formulations of the peptides or antibodies or other therapeutic may be prepared for storage by mixing a peptide or antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • chelating agents such as EDTA
  • sugars such as sucrose, mannitol, trehalose or sorbitol
  • salt-forming counter-ions such as sodium
  • metal complexes e.g. Zn-protein complexes
  • non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • the peptide or other therapeutic may be administered directly to a cell or a subject, such as a human subject alone or with a suitable carrier.
  • a peptide may be delivered to a cell in vitro or in vivo by delivering a nucleic acid that expresses the peptide to a cell.
  • Various techniques may be employed for introducing nucleic acid molecules of the invention into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid molecule-calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the foregoing viruses including the nucleic acid molecule of interest, liposome-mediated transfection, and the like.
  • a vehicle used for delivering a nucleic acid molecule of the invention into a cell can have a targeting molecule attached thereto.
  • a targeting molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid molecule delivery vehicle.
  • proteins that bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.
  • proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.
  • Polymeric delivery systems also have been used successfully to deliver nucleic acid molecules into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acid molecules.
  • a peptide of the invention may also be expressed directly in mammalian cells using a mammalian expression vector.
  • a mammalian expression vector can be delivered to the cell or subject and the peptide expressed within the cell or subject.
  • the recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art.
  • tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid- specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters.
  • Developmentally-regulated promoters are also encompassed, for example the murine hox promoters and the a-fetoprotein promoter.
  • a "vector" may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
  • the invention also includes articles, which refers to any one or collection of components.
  • the articles are kits.
  • the articles include
  • the article may include instructions or labels promoting or describing the use of the compounds of the invention.
  • a kit comprises antibodies against the starvation markers being measured in a method of the invention.
  • the kit may further comprise assay diluents, standards, controls and/or detectable labels.
  • the assay diluents, standards and/or controls may be optimized for a particular sample matrix.
  • promoted includes all methods of doing business including methods of education, hospital and other clinical instruction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with compositions of the invention in connection with treatment of infections, cancer, and autoimmune disease.
  • Instructions can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.
  • kits may include one or more containers housing the components of the invention and instructions for use.
  • kits may include one or more agents described herein, along with instructions describing the intended therapeutic application and the proper administration of these agents.
  • agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.
  • the kit may be designed to facilitate use of the methods described herein by physicians and can take many forms.
  • Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder).
  • compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • a suitable solvent or other species for example, water or a cell culture medium
  • "instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the invention. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc.
  • the written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflects approval by the agency of manufacture, use or sale for human administration.
  • the kit may contain any one or more of the components described herein in one or more containers.
  • the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject.
  • the kit may include a container housing agents described herein.
  • the agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely.
  • the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.
  • Example 1 shows bar graphs of FSC vs SSC dot plots for WM35 human melanoma cells treated with Etomoxir, Chloroquine, 2-deoxyglucose, and Chloroquine + Etomoxir.
  • the dot plots demonstrate sensitivity to chloroquine and etomoxir treatment.
  • the cells were treated with chloroquine at O.lmM, and Etomoxir at 0.5mM.
  • the expression of LC3 is higher in the dead population of cells when treated with chloroquine and 2-deoxyglucose, but is decreased with etomoxir and the
  • LC3 expression is relatively lower in the treated cells when compared to the no treatment in the live population of cells.
  • LAMP expression is increased with Chloroquine and Etomoxir in the live population.
  • the bar graph shows the relative numbers of live and dead cells in response to the treatments.
  • Example 2 B16F1 mouse melanoma cells were used to conduct similar experiments to those shown in Example 1.
  • Figure 2 is FSC vs SSC dot plots showing the relative size and granularity of the B16F1 mouse melanoma cell line.
  • the dot plots include: no treatment control, chloroquine, chloroquine + 2-deoxyglucose, 2- deoxyglucose.
  • the addition of chloroquine demonstrates death in the B16F1 cells as measured by FSC vs SSC.
  • trypan blue counts show the same result as the FSC vs. SSC dot plots.
  • Chloroquine was treated at 0.05mM and 2-deoxyglucose was treated at 0.4mM.
  • B16F1 cells express a relatively large amount of LAMP and LC3 before treatment.
  • the graphs show that the addition of the compounds chloroquine and 2- deoxyglucose decreased the expression of both LAMP and LC3.
  • Example 3 Similar experiments were performed on an HTB-77 cell line as were performed in Examples 1 and 2. The data is shown in Figure 3.
  • the HTB-77 cells were treated with chloroquine at O.lmM and etomoxir at 0.5mM.
  • Figure 3 shows the results of the FSC vs SSC dot plots, along with the percent death graph demonstrating that etomoxir and chloroquine (and the combination) cause more death in the HTB-77s when compared to the no treatment control.
  • the MFI of LC3 in the live cell population is decreased when treated with etomoxir, chloroquine, and the combination of both compounds.
  • the dead population of HTB-77 shows an increase in LC3 expression with the cells treated with etomoxir and chloroquine.
  • Example 4 Similar experiments were performed on an HTB-77 cell line as were performed in Examples 1 and 2. The data is shown in Figure 4. The HTB-77 cells were treated with chloroquine at O.lmM and etomoxir at 0.5mM. This data shows an increase in death when treated with chloroquine first, followed by etomoxir treatment. The LC3 expression on the live cells is higher when treated with etomoxir first and the LC3 expression on the dead cells is decreased when treated with chloroquine first. The
  • LAMP expression is increased in the live population of cells when they are treated with chloroquine first.
  • the dead population demonstrates a relative higher expression of LAMP when treated with etomoxir first.
  • Example 5 Similar experiments were performed on ACHN renal carcinoma cells as were performed in the Examples above. The data is shown in Figure 5.
  • the FSC vs SSC dot plots show ACHN renal carcinoma cells treated with Etomoxir at 0.5mM for 24 hours followed by Chloroquine at O. lmM for an additional 24 hours, and vice versa (shown in bar graph format in Figure 5).
  • the renal cells treated with chloroquine first followed by etomoxir show more death.
  • the LAMP expression in the dead population is higher in the cells treated with etomoxir first, then followed by chloroquine.
  • the LC3 expression in both the dead and live cell populations is higher in the etomoxir treated first followed by chloroquine treatment cells.
  • Example 6 T24 bladder tumor cells treated with etomoxir at 0.5mM and chloroquine at 0. ImM. The data is shown in Figure 6. These trypan blue counts indicated that the T24 cells are sensitive to chloroquine and etomoxir. These cells were treated with etomoxir, chloroquine and etomoxir + chloroquine for 24 hours, and counted at 24 hours. The counts are shown in the first 4 bars of the graph starting from the left. The last two bars show the cells that were treated with etomoxir first for 24 hours, followed by chloroquine for an additional 24 hours and vice versa. At this point, they were counted for percent cell death. The cells that were first treated with chloroquine followed by etomoxir showed an increased rate in cell death.
  • Example 7 WM35 Melanoma, L1210, and L1210 DDP cells were treated according to the methods described below and levels of starvation markers were analyzed. L1210 and L1210DDP cells were grown in T25 flasks at 1.0X106 cells/mL in lOmL of 5% FBS complete RPMI. Cells were treated with Rapamycin (LC
  • WM35 cells were grown in a 6 well plate at 1.0X106 cells/mL in 5mL of 10% FBS complete RPMI. Cells were treated with Rapamycin (LC Laboratories) at a final concentration of 10 ⁇ . After 24 hours, cells were harvested and counted using trypan blue on a hemocytometer. Cells were stained for the following: MitoTracker Red; LAMP-1; and LC3A/B.
  • the cells were resuspended in warm (37o C PBS containing a final concentration of 0.5 ⁇ dye. The cells were incubated for 20 minutes, pelleted, and resuspended in PBS for analysis.
  • Cells were fist fixed using the Cytoperm/Cytofix Kit (BDBioscience) According to the manufacturer's directions. Next non-specific binding was blocked using FC:Block (BDBioscience) for 10 minutes on ice. Cells were fist stained using monoclonal antibodies to either LAMP-1 or LC3A/B (Abeam) for 20minutes on ice. Cells are then pelleted and responded in cold staining buffer (BDBioscience). Next cells were stained with their respective fluorochrome conjugated second step antibodies and incubated for 20 minutes on ice. Cells were finally pelleted and resuspended in cold staining buffer (BDBioscience) for analysis. Cells were analyzed on a BD FACS Canto II flow cytometer. Data was analyzed using FlowJo software and the raw data is shown in Figure 14 as dot plots.

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Abstract

The invention relates to methods and related products for diagnosis and treatment of cancer. The methods involve the detection of markers such as LAMP and LC3, and administration of an appropriate therapeutic regimen based on the presence or absence of the markers.

Description

DIAGNOSIS AND TREATMENT OF CANCER USING DIFFERENTIALLY EXPRESSED STARVATION MARKERS
RELATED APPLICATIONS
This application claims priority under 35 U.S.C. § 119(e) from U.S. provisional application serial number 61/675,375, filed July 25, 2012, the entire contents of which is incorporated by reference herein.
BACKGROUND OF INVENTION
While significant progress has been made in the treatment of some types of
cancers, effective therapeutic strategies have not been developed for a number of forms of cancer. Renal Cell Carcinoma, for instance, accounts for two to three percent of all adult malignant neoplasms and the disease specific mortality rate is significantly higher than other genitourinary malignancies. In the era of advanced medical imaging, the
incidence of RCC has continued to rise over the last thirty years. Over thirty thousand new diagnoses are made in the United States each year and over ten thousand patients die secondary to RCC.
Although there are several potential etiologic risk factors for RCC, there is little evidence in human models about definitive etiologic agents. The standard of care for
treatment of RCC is currently surgical resection. While this is a potentially curative
therapy, disease progression and late diagnosis of some RCC's continues to drive the
need for improved systemic therapy. Unfortunately, one quarter of the patient' s with
RCC will be node positive or have evidence of metastasis at the time of diagnosis.
Ovarian cancer causes more deaths than any other cancer of the female
reproductive system. Ovarian cancer begins in the ovaries, or the female reproductive organs. Ovarian cancer often goes undetected in the earlier stages, and therefore, is
typically diagnosed once it has metastasized to the abdomen and pelvis. There were
approximately 25,000 new cases and around 14,000 associated deaths in 2010.
Skin cancer is the most common form of cancer in the US. In fact, one in five
Americans will develop skin cancer in the course of a lifetime. While melanoma
accounts for only about 3 percent of skin cancer cases, it is the most aggressive form of cancer and causes more than 75% of skin cancer deaths. The incidence of melanoma is increasing in the United States as a result of higher exposure to UV light, with
projections made by the American Cancer Society/SEER indicating there will be 68,130 new cases this year, with a slight predominance in males, and some 8,700 deaths, 5,600 of those in men. Surgery has proven curative in melanoma for superficial lesions but not for deeper tumors, and after twenty years, the use of Interferon remains controversial and of little benefit. SUMMARY OF INVENTION
The invention, in some aspects is a method for selecting a course of treatment of a subject having cancer, by obtaining from the subject a tumor cell, determining the expression of one or more starvation markers which are differentially expressed in cancers under distinct metabolic conditions, and selecting a course of treatment appropriate to the cancer of the subject depending on the expression of the one or more starvation markers.
A method for identifying a property of a tumor is also provided. The method involves selecting a tumor cell and determining whether the tumor cell expresses a starvation marker selected from the group consisting of LAMP and LC3, wherein expression of the starvation marker is indicative of an autophagic tumor cell.
The starvation marker in some embodiments is LAMP1, LAMP2 or LC3.
The methods may also involve selecting a course of treatment for a subject based on the expression of the starvation marker. In some embodiments the course of treatment involves administering a starvation signal compound to the subject when the tumor cell is starvation marker negative. In other embodiments the course of treatment involves administering an autophagy inhibitor to the subject when the tumor cell is starvation marker positive.
According to other aspects of the invention a method for treating a subject, by administering to a subject who has been identified as having a starvation marker negative tumor, a therapeutically acceptable amount of a starvation signal compound to treat the tumor is provided.
In another aspect a method is provided for treating a subject by administering to a subject who has been identified as having a starvation marker positive tumor a therapeutically acceptable amount of an autophagy inhibitor and/or a fatty acid metabolism inhibitor.
In some embodiments the methods also involve determining the expression of one or more starvation markers in a tumor cell, following the autophagy inhibitor and/or a fatty acid metabolism inhibitor treatment to determine if the tumor cell is starvation marker inhibitor negative.
In other embodiments the methods also involve administering to the subject a therapeutically acceptable amount of a starvation signal compound to treat the tumor after the autophagy inhibitor and/or a fatty acid metabolism inhibitor treatment.
The starvation signal compound, may be for instance, an anti-VEGF antagonist, optionally an anti-VEGF antibody such as bevacizumab, or rapamycin or a glycolysis inhibitor.
In some embodiments the tumor is selected from the group consisting of a melanoma, an ovarian tumor, a glioblastoma, a breast cancer, and a renal tumor.
The methods may also involve administering a chemotherapeutic agent to the subject.
The autophagy inhibitor in some embodiments is a 4- aminoquinoline. The 4- aminoquinoline may have the structure:
Figure imgf000004_0001
or a pharmaceutically acceptable salt or prodrug thereof; each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration; wherein each of R2 and R3 is independently a hydroxalkyl, an alkyl, alkyloxy, alkylcarboxy, alkylene or alkenylene having from one to six carbon atoms. In other embodiments the autophagy inhibitor is chloroquine or
hydroxychloroquine .
In some embodiments the method may involve determining the expression of one or more starvation markers in a tumor cell of the subject before the treatment is administered. Optionally, the step of determining the expression of the starvation marker is performed by detecting the expression of starvation marker nucleic acid molecules. In some embodiments the expression of the starvation marker nucleic acid molecules is determined by a method selected from the group consisting of nucleic acid hybridization and nucleic acid amplification. The nucleic acid hybridization may be performed using a solid-phase nucleic acid molecule array. In some embodiments the nucleic acid amplification method is real-time PCR. Alternatively, the step of determining the expression of the starvation marker is performed by detecting the expression of starvation marker peptides. In some embodiments the expression levels are determined by an immunological method, such as a solid-phase antibody array or an ELISA or ELISPOT assay.
In some embodiments the fatty acid metabolism inhibitor is an inhibitor of fatty acid oxidation, a fatty acid transporter inhibitor, a reductase inhibitor, or an isomerase inhibitor within the fatty acid metabolism pathway. The inhibitor of fatty acid metabolism may be an inhibitor of fatty acid oxidation and is selected from the group consisting of an oxirane carboxylic acid compound, such as etomoxir (2-(6-(4- chlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester), 2-(4-(3-chlorophenoxy)- butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)- oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2- carboxylic acid ethyl ester, 2-(6-(3,4-dichlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(4-fluorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6- phenoxyhexyl)-oxirane-2-carboxylic acid ethyl ester, cerulenin, 5-(tetradecyloxy)-2- furoic acid, oxfenicine, methyl palmoxirate, metoprolol, amiodarone, perhexiline, aminocamitine, hydrazonopropionic acid, 4-bromocrotonic acid, trimetazidine, ranolazine, hypoglycin, dichloroacetate, methylene cyclopropyl acetic acid, beta-hydroxy butyrate, and a non-hydrolyzable analog of carnitine or pharmacologically acceptable salts thereof. In some embodiments the inhibitor of fatty acid metabolism is an inhibitory nucleic acid, for instance an inhibitory nucleic acid specific for an enzyme selected from the group consisting of 2,4-dienoyl-CoA reductase, 2,4-dienoyl-CoA isomerase, and butyryl dehydrogenase. In other embodiments the inhibitor of fatty acid metabolism is oxamate. In yet other embodiments the fatty acid metabolism inhibitor is an oxirane carboxylic acid compound capable of inhibiting fatty acid metabolism, or a
pharmacologically acceptable salt thereof. In some embodiments the oxirane carboxylic acid compound is etomoxir. In some embodiments the glycolytic inhibitor is a 2-deoxyglucose compound such as 2-deoxy-D-glucose.
According to other aspects the invention involves a method comprising providing a starvation marker from a tumor tissue sample, determining the expression level of the starvation marker, and comparing the expression level of the starvation marker in the tissue sample with threshold value of a starvation marker, wherein a lower level of expression in the tumor cell is indicative of the tumor cells susceptibility to starvation signal compound. In some embodiments the threshold value is an expression level value of a starvation marker from a control cell. In other embodiments the control cell is a non-cancerous cell of the same type of tissue as the tumor cell. In yet other
embodiments there is at least a 2-fold difference in mean expression levels between the at starvation marker and the threshold level.
A method of treating melanoma, ovarian cancer, or renal cancer, is provided in other aspects of the invention. The method involves administering daily to a subject having melanoma, ovarian cancer, or renal cancer a therapeutically effective dose of an autophagy inhibitor and administering once every 2-4 months to the subject a therapeutically effective amount of bevacizumab.
In some embodiments the bevacizumab is administered in a dose range of 12-16 mg/Kg IV. In other embodiments the bevacizumab is administered in a dose of 15 mg/Kg IV.
In other aspects the invention involves a method of treating cancer, by administering to a subject having cancer a therapeutically effective dose of an autophagy inhibitor and administering to the subject a therapeutically effective amount of rapimycin.
In some embodiments the autophagy inhibitor is administered daily.
In other embodiments the autophagy inhibitor is hydroxychloroquine or chloroquine. In yet other embodiments the autophagy inhibitor is administered in a dose range of 100-500 mg per day or 200-400 mg per day.
This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
BRIEF DESCRIPTION OF DRAWINGS
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
Figure 1 is a bar graph showing FSC vs SSC dot plots for WM35 human melanoma cells treated with Etomoxir, Chloroquine, 2-deoxyglucose, and Chloroquine +
Etomoxir.
Figure 2 is a bar graph showing the granularity of the B16F1 mouse melanoma cell line.
Figure 3 is a bar graph showing the relative numbers of live and dead cells FSC vs SSC for an HTB-77 cell line.
Figure 4 is a bar graph showing the relative numbers of live and dead cells FSC vs SSC dot plots for an HTB-77 cell line.
Figure 5 is a bar graph showing the relative numbers of live and dead cells FSC vs SSC dot plots for ACHN renal carcinoma.
Figure 6 is a bar graph depicting the results of treating T24 bladder tumor cells treated with etomoxir at 0.5mM and chloroquine at O. lmM.
Figure 7 is a bar graph depicting the results of treating WM35 Melanoma, L1210, and L1210 DDP cells were treated with Rapamycin and stained for MitoTracker.
Figure 8 is a bar graph depicting the results of treating WM35 Melanoma, L1210, and L1210 DDP cells were treated with Rapamycin and stained for LAMP-1; or LC3A/B
DETAILED DESCRIPTION
A problem associated with current medical treatments arises from different responses to medicine by individual patients. Personalized medicine, in which a therapy is tailored to specific properties of an individual and their disease has been proposed as a solution to this problem. For instance, one approach to personalized medicine involves the identification of the presence or absence of biomarkers to aid in cancer therapeutics, where the expression of the biomarker or lack thereof has been associated with a successful therapeutic protocol. The expression of HER2 on breast cancer cells is used to guide a decision on whether to include HER2 specific drugs such as HERCEPTIN® and TYKERB® in therapy.
A new set of biomarkers for guiding therapeutic decisions has been discovered herein. Specifically it has been discovered that starvation markers, including for instance, LAMP and LC3 are useful in predicting therapeutic outcome in response to certain anti-cancer therapies. Low levels of expression of the starvation markers indicates that a tumor cell is not experiencing conditions of starvation. Such tumor cells are susceptible to anti-cancer therapies without the requirement for further manipulation. High levels of starvation markers, however, are indicative of tumor cells that are autophagic. Autophagic tumor cells should be treated with autophagy inhibitors and/or fatty acid oxidation inhibitors to render the cells susceptible to starvation signal compound.
Patient outcome using a specific set of therapeutics depends on the phenotype of individual tumors at the molecular level, and this is reflected in the status of the starvation marker expression. Therefore, in some aspects the invention relates to methods for characterizing a tumor cell, such as for instance, selecting a course of treatment for a subject having cancer or for identifying a property of a tumor. The methods can be used on any type of tumor cell. Tumor cells from a variety of sources can be used. For example a tumor cell may be an in vitro tumor cell line or a primary tumor cell that has been preserved for a period of time or one that has just been removed from a subject. A tumor cell can be obtained from a subject, for instance, as part of a tissue biopsy or as a cancerous tissue removed from the subject. The cell may then be analyzed immediately or preserved for a period of time prior to analysis.
The expression of a starvation marker in the tumor cell is assessed in order to determine the metabolic state or condition of the cell. The starvation marker is indicative of whether the cell is experiencing conditions of starvation, such that it is autophagic. When cancer cells are deprived of nutrient, many undergo a form of survival strategy known as autophagy. Autophagy, literally "self-eating," is a cellular survival strategy that cells use when confronted with conditions of starvation, oxidative stress, including the stresses of chemotherapeutic agents or irradiation. Autophagy provides a mechanism for survival in the face of stress and involves auto-digestion of the cells own organelles into the lysosomal system, thereby providing the engulfed organellar contents as a source of energy.
A starvation marker, as used herein, is a detectable compound, i.e., a protein or nucleic acid, the expression of which is altered under conditions of cellular starvation. Starvation markers include but are not limited to LAMP, LC3, Beclin (BCNl or BCN2), AMBRA1, ATG12, ATG16L1, ATG4A, ATG4B, ATG4C, ATG4D, ATG5, ATG9A, ATG9B, BECN1, GABARAP, GABARAPL1, GABARAPL2, IRGM, MAP1LC3A, MAP1LC3B, RGS1, ULK1, ATG4A, ATG4B, ATG4C, ATG4D, GABARAP, ATG10, ATG16L1, ATG16L2, ATG3, ATG4A, ATG4B, ATG4C, ATG4D, ATG7, ATG9A, GABARAP, GABARAPL2, RAB24, DRAM, GABARAP, TMEM166, ATG3, ATG7, ATG4A, ATG4B, ATG4C, ATG4D, AKT1, APP, ATG12, ATG5, BAD, BAK1, BAX, BCL2, BCL2L1, BECN1, BID, BNIP3, CASP3, CASP8, CDKN1B, CDKN2A, CLN3, CTSB, CXCR4, DAPK1, DRAM, EIF2AK3, FADD, FAS, HDAC1, HTT, IFNA2, IFNG, IGF1, INS, MAPK8, NFKB1, PIK3CG, PRKAA1, PTEN, SNCA, SQSTM1, TGFB1, TGM2, TNF, TNFSF10, TP53, TP73, BAX, CDKN1B, CDKN2A, IFNG, PTEN, RBI, TGFB1, TP53, TP73, EIF2AK3, IFNA2, IFNA4, IFNG, ARSA, CTSS, EIF4G1, ESR1, GAA, HGS, MAPK14, PIK3C3, PIK3R4, PRKAA2, RPS6KB1, TMEM74, TMEM77, ULK2, UVRAG, HSP90AA1, and HSPA8.
LAMP is Lysosomal-associated membrane protein. In some instances it is also known as CD 107 antigen-like family member. The term LAMP includes the various forms of LAMP such as LAMP-1, LAMP-2, and LAMP-3. The protein encoded by this gene is a member of a family of membrane glycoproteins that provides selectins with carbohydrate ligands.
The protein sequence for human LAMP-1 is disclosed in UniProt, reference number PI 1279 (LAMP 1 _HUM AN) and is as follows: MAAPGSARRP LLLLLLLLLL GLMHCASAAM FMVKNGNGTA CIMANFSAAF SVNYDTKSGP KNMTFDLPSD ATVVLNRSSC GKENTSDPSL VIAFGRGHTL TLNFTRNATR YSVQLMSFVY NLSDTHLFPN ASSKEIKTVE SITDIRADID KKYRCVSGTQ VHMNNVTVTL HDATIQAYLS NSSFSRGETR CEQDRPSPTT APPAPPSPSP SPVPKSPSVD KYNVSGTNGT CLLASMGLQL NLTYERKDNT TVTRLLNINP NKTSASGSCG AHLVTLELHS EGTTVLLFQF GMNASSSRFF LQGIQLNTIL PDARDPAFKA ANGSLRALQA TVGNSYKCNA EEHVRVTKAF SVNIFKVWVQ AFKVEGGQFG SVEECLLDEN SMLIPIAVGG ALAGLVLIVL lAYLVGRKRS HAGYQTI (SEQ ID NO. 1).
The protein sequence for human LAMP-2 is disclosed in UniProt, reference number PI 3473 (LAMP2_HUMAN) and is as follows: MVCFRLFPVP GSGLVLVCLV LGAVRSYALE LNLTDSENAT CLYAKWQMNF TVRYETTNKT YKTVTISDHG TVTYNGSICG DDQNGPKIAV QFGPGFSWIA NFTKAASTYS IDSVSFSYNT GDNTTFPDAE DKGILTVDEL LAIRIPLNDL FRCNSLSTLE KNDVVQHYWD VLVQAFVQNG TVSTNEFLCD KDKTSTVAPT IHTTVPSPTT TPTPKEKPEA GTYSVNNGND TCLLATMGLQ LNITQDKVAS VININPNTTH STGSCRSHTA LLRLNSSTIK YLDFVFAVKN ENRFYLKEVN ISMYLVNGSV FSIANNNLSY WDAPLGSSYM CNKEQTVSVS GAFQINTFDL RVQPFNVTQG KYSTAQDCSA DDDNFLVPIA VGAALAGVLI LVLLAYFIGL KHHHAGYEQF (SEQ ID NO. 2).
The protein sequence for human LAMP-3 is disclosed in UniProt, reference number Q9UQV4 (LAMP3_HUMAN) and is as follows: MPRQLSAAAA
LFASLAVILH DGSQMRAKAF PETRDYSQPT AAATVQDIKK PVQQPAKQAP HQTLAARFMD GHITFQTAAT VKIPTTTPAT TKNTATTSPI TYTLVTTQAT PNNSHTAPPV TEVTVGPSLA PYSLPPTITP PAHTTGTSSS TVSHTTGNTT QPSNQTTLPA TLSIALHKST TGQKPVQPTH APGTTAAAHN TTRTAAPAST VPGPTLAPQP SSVKTGIYQV LNGSRLCIKA EMGIQLIVQD KESVFSPRRY FNIDPNATQA SGNCGTRKSN LLLNFQGGFV NLTFTKDEES YYISEVGAYL TVSDPETIYQ GIKHAVVMFQ TAVGHSFKCV SEQSLQLSAH LQVKTTDVQL QAFDFEDDHF GNVDECSSDY TP LPVIGAI VVGLCLMGMG VYKIRLRCQS SGYQRI (SEQ ID NO. 3).
LC3 is an autophagy sensitive marker. It includes at least LC3B, LC3A, and LC3C. The full name for LC3B, for instance, is microtubule-associated proteins 1A/1B light chain 3B.
The protein sequence for human LC3B is disclosed in UniProt, reference number Q9GZQ8 (MLP3B_HUMAN) and is as follows: MPSEKTFKQR RTFEQRVEDV RLIREQHPTK IPVIIERYKG EKQLPVLDKT KFLVPDHVNM SELIKIIRRR LQLNANQAFF LLVNGHSMVS VSTPISEVYE SEKDEDGFLY MVYASQETFG MKLSV (SEQ ID NO. 4).
The protein sequence for human LC3A is disclosed in UniProt, reference number Q9H492 (MLP3 A_HUM AN) and is as follows: MPSDRPFKQR RSFADRCKEV QQIRDQHPSK IPVIIERYKG EKQLPVLDKT KFLVPDHVNM SELVKIIRRR LQLNPTQAFF LLVNQHSMVS VSTPIADIYE QEKDEDGFLY MVYASQETFG F
(SEQ ID NO. 5).
The protein sequence for human LC3C is disclosed in UniProt, reference number Q9BXW4 (MLP3C_HUMAN) and is as follows: MPPPQKIPSV RPFKQRKSLA IRQEEVAGIR AKFPNKIPVV VERYPRETFL PPLDKTKFLV PQELTMTQFL SIIRSRMVLR ATEAFYLLVN NKSLVSMSAT MAEIYRDYKD EDGFVYMTYA SQETFGCLES AAPRDGSSLE DRPCNPL (SEQ ID NO. 6).
The nucleic acid sequence for human LAMP-1 is disclosed in GENBANK, accession number NM_005561 and is as follows: tgcggggagc cgagccgccg gcgctcgacg cgcgcgctct cgcgagaccc gcgggatcac gtgacgcccg ggcgcggcgc agctcacgtg acaagcgctg ccggccgcgg tgtcttcttc gtgccggcgt cgcagtggcc gggcctcttg cgtctggtaa cgccgctgtc tctaacgcca gcccttggcg cccgcgcccc gccaccgcag cgcccggcag tccgcggccc aaccgccgcc cgcgcccccg ctccccgcac cgtacccggc cgcctcgcgc catggcggcc cccggcagcg cccggcgacc cctgctgctg ctactgctgt tgctgctgct cggcctcatg cattgtgcgt cagcagcaat gtttatggtg aaaaatggca acgggaccgc gtgcataatg gccaacttct ctgctgcctt ctcagtgaac tacgacacca agagtggccc taagaacatg acctttgacc tgccatcaga tgccacagtg gtgctcaacc gcagctcctg tggaaaagag aacacttctg accccagtct cgtgattgct tttggaagag gacatacact cactctcaat ttcacgagaa atgcaacacg ttacagcgtc cagctcatga gttttgttta taacttgtca gacacacacc ttttccccaa tgcgagctcc aaagaaatca agactgtgga atctataact gacatcaggg cagatataga taaaaaatac agatgtgtta gtggcaccca ggtccacatg aacaacgtga ccgtaacgct ccatgatgcc accatccagg cgtacctttc caacagcagc ttcagcaggg gagagacacg ctgtgaacaa gacaggcctt ccccaaccac agcgccccct gcgccaccca gcccctcgcc ctcacccgtg cccaagagcc cctctgtgga caagtacaac gtgagcggca ccaacgggac ctgcctgctg gccagcatgg ggctgcagct gaacctcacc tatgagagga aggacaacac gacggtgaca aggcttctca acatcaaccc caacaagacc tcggccagcg ggagctgcgg cgcccacctg gtgactctgg agctgcacag cgagggcacc accgtcctgc tcttccagtt cgggatgaat gcaagttcta gccggttttt cctacaagga atccagttga atacaattct tcctgacgcc agagaccctg cctttaaagc tgccaacggc tccctgcgag cgctgcaggc cacagtcggc aattcctaca agtgcaacgc ggaggagcac gtccgtgtca cgaaggcgtt ttcagtcaat atattcaaag tgtgggtcca ggctttcaag gtggaaggtg gccagtttgg ctctgtggag gagtgtctgc tggacgagaa cagcatgctg atccccatcg ctgtgggtgg tgccctggcg gggctggtcc tcatcgtcct catcgcctac ctcgtcggca ggaagaggag tcacgcaggc taccagacta tctagcctgg tgcacgcagg cacagcagct gcaggggcct ctgttccttt ctctgggctt agggtcctgt cgaaggggag gcacactttc tggcaaacgt ttctcaaatc tgcttcatcc aatgtgaagt tcatcttgca gcatttacta tgcacaacag agtaactatc gaaatgacgg tgttaatttt gctaactggg ttaaatattt tgctaactgg ttaaacatta atatttacca aagtaggatt ttgagggtgg gggtgctctc tctgaggggg tgggggtgcc gctgtctctg aggggtgggg gtgccgctgt ctctgagggg tgggggtgcc gctctctctg agggggtggg ggtgccgctt tctctgaggg ggtgggggtg ccgctctctc tgagggggtg ggggtgctgc tctctccgag gggtggaatg ccgctgtctc tgaggggtgg gggtgccgct ctaaattggc tccatatcat ttgagtttag ggttctggtg tttggtttct tcattcttta ctgcactcag atttaagcct tacaaaggga aagcctctgg ccgtcacacg taggacgcat gaaggtcact cgtggtgagg ctgacatgct cacacattac aacagtagag agggaaaatc ctaagacaga ggaactccag agatgagtgt ctggagcgct tcagttcagc tttaaaggcc aggacgggcc acacgtggct ggcggcctcg ttccagtggc ggcacgtcct tgggcgtctc taatgtctgc agctcaaggg ctggcacttt tttaaatata aaaatgggtg ttatttttat ttttttttgt aaagtgattt ttggtcttct gttgacattc ggggtgatcc tgttctgcgc tgtgtacaat gtgagatcgg tgcgttctcc tgatgttttg ccgtggcttg gggattgtac acgggaccag ctcacgtaat gcattgcctg taacaatgta ataaaaagcc tctttctttt aaaaaaaaaa aaaaaaaaaa (SEQ ID NO. 7). The nucleic acid sequence for human LAMP-2, variant A, is disclosed in
GENBANK, accession number NM_002294 and is as follows: aagaaagagc cccgccccta gtcttatgac tcgcactgaa gcgccgattc ctggcttttg caaggctgtg gtcggtggtc atcagtgctc ttgacccagg tccagcgagc cttttccctg gtgttgcagc tgttgttgta ccgccgccgt cgccgccgtc gccgcctgct ctgcggggtc atggtgtgct tccgcctctt cccggttccg ggctcagggc tcgttctggt ctgcctagtc ctgggagctg tgcggtctta tgcattggaa cttaatttga cagattcaga aaatgccact tgcctttatg caaaatggca gatgaatttc acagtacgct atgaaactac aaataaaact tataaaactg taaccatttc agaccatggc actgtgacat ataatggaag catttgtggg gatgatcaga atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg aattttacca aggcagcatc tacttattca attgacagcg tctcattttc ctacaacact ggtgataaca caacatttcc tgatgctgaa gataaaggaa ttcttactgt tgatgaactt ttggccatca gaattccatt gaatgacctt tttagatgca atagtttatc aactttggaa aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagcttttgt ccaaaatggc acagtgagca caaatgagtt cctgtgtgat aaagacaaaa cttcaacagt ggcacccacc atacacacca ctgtgccatc tcctactaca acacctactc caaaggaaaa accagaagct ggaacctatt cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag ctgaacatca ctcaggataa ggttgcttca gttattaaca tcaaccccaa tacaactcac tccacaggca gctgccgttc tcacactgct ctacttagac tcaatagcag caccattaag tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac atcagcatgt atttggttaa tggctccgtt ttcagcattg caaataacaa tctcagctac tgggatgccc ccctgggaag ttcttatatg tgcaacaaag agcagactgt ttcagtgtct ggagcatttc agataaatac ctttgatcta agggttcagc ctttcaatgt gacacaagga aagtattcta cagctcaaga ctgcagtgca gatgacgaca acttccttgt gcccatagcg gtgggagctg ccttggcagg agtacttatt ctagtgttgc tggcttattt tattggtctc aagcaccatc atgctggata tgagcaattt tagaatctgc aacctgattg attatataaa aatacatgca aataacaaga ttttcttacc tctcagttgt tgaaacactt tgcttcttaa aattgatatg ttgaaacttt aattctttta tcaatcccag cattttgaga tcagtcttta ttaataaaac ctgttctctt taatcagctt aaaatccaaa gtgtcatatt tactggtcct ggagacaaac ttgttcaaaa gaacatcaac gtgcaatgtt ttaaggtcta tcttaagaag ccctggccaa attttgatcc taaccttgaa gtatgccttg aacttattaa catggccatt ataagaataa aatatgtagt tgtgtcttaa tggaattaat aaatgtcatt tcactactgg tgttctgttt caatgtataa ggactatagt gatttaaact catcaatgtg cctttgcata aagttcatta aataaatatt gatgtggtat aaatgcccat cagatatgct taaacttggt tttcagttga atgaagtaga gaatgtcctc aggaccatca gcattttaaa ggttatgtga cttttgctga tttctctgag ttcaagttaa gcatgaagtt agtacctcaa gcctgtgatt tttccctagg gatgatacag acccaagagg ctacaacaga acttaaactg gcttcgtaat tagagttttt aagataattg tttgtttttc agcaatatag actgaaaaga tccaagcata tttagccact tgcttttttg tttcttgttt tgttcttctt tggatgcctg attagtattg aaagatagaa atattctatg aactaattag gacagattgt gttgtgtttc tctacctcat cttgttgatc tctggagcat taaaatctat ttagtgttgt catcagtgtg gtacttatga aatgtaagct aacagcaatc tcagaaggga ggcagtgaag catagcaact aggctcttgt ttcttcaaga tggcccctgt ggggcagtgc atagatgggg gtgtaaagag aagctgttgg cattaaaatg agctagataa tcagcccttg ttgaagcata ttccatggta taagagtagc acagacatga aacatagata aagaaggaag gcttaataga ctagaagact tccacattga agtattatta acccattgta tgtatatagg ggcatgatca gagtctctat aacttcctga ttaacaatac agtgtatctt gttacccagc tgtcagtctt tgagagcttt cagtaaaata tagtaaattc tttcagcata ggctaatgtg tggttactga gatgagtgtt gtgtactcag aaccgtagca acatttttat gaatggtaaa agtacaagag gaggaaaagt taaaattaga agaaaagtac aagttattgc ttaatcataa atcacaccag ataacacatt ttgttaattt cattagctat tactggaaag gaccttaacg attatttaca gaaaggggag tgaaattcat tgaggttcca tatcaagtgg gcaacaaaac tattactagc attttgataa aaattgcccc taatgaaatc tagtcactca acagtaaaac aacagctggt ttacacttga aattatgaga tcagaattgg gcactttggg cttccgtact atgttttgct taagtttttt ttttaatact aatatgggct ttttcagtag taatatacca aaacacttct attttaatct ctgtttgcta cttcaaaacc taatcctcct cagatgggat catgagcata agaggaaaag agaagagaat gaatacttgt tgacctcttg atgtgtatca gatgctttag aaatgtaatt gtatttaatc ctcaaatacc ttataggtat tattatcccc ttcttacaga tgaggaaagt gaggcccagt ttaaataact tgcccaaggt cctttggcta gtactggaag gagtcaagat ttaaacacag ttctgtctga atccagaact caaaatctac attgcacatg ttgctttcct ggtggttcgg tggaatggac tgcaacgcat tagatactgc tgttattctt ccaggccacc gctcagctaa aaataattgt gtgtgtgtgt atatatatat atatgtacac acacacacat atatatacac acacacatat atatacacat atacatatat atacacatat atatacacac acatatatat gtatatatat actacattct tgatcctaag tcttttttaa cttaaatttt attacttata cagaattctt atttatactt taattatagg tgtgacgaag agaaagagag tagggaaata cacaggcagt ggttttaagt gtagatgatg gctccttaac ccagtgtcat tagataatca aacctaaagt cttcccatat taggcaagcg caattctcta ttttggaccc ttcccattct tcccttacct tctgcttttc gtactgagga atttcgtgtg attttagata aagtgataat gagatattga gcaaataaga aaatagaggt aatgctataa aaaactaagc tatgtacact ttcaaaatgc atgtttcttg catgcttttt actacttaat tgcattcttt gctaatttcc tttccttgct gtctgttctt ttctaacagc tgaagaatgt tctgctgact ctgacctcaa ctttcttatt cctgttgcag tgggtgtggc cttgggcttc cttataattg ttgtctttat ctcttatatg attggaagaa ggaaaagtcg tactggttat cagtctgtgt aatcagttaa atctagtgtt tgtttgtttt tttcaattag aagttacgtt tccattggct aaaagccagg acatgctgtg caatagattg tttaagatat gcagactaac ttcagtgagt tcctagctaa cttgggcatg agtacactta tttaagacaa aatatattag gaccaatttt tttctgtttt ttttcttcct ttgttaaagt ataattaaaa gaaaaattgt ggcttagaat tttttaagta aataatgatt ttaagcccct ggatccaatt atgaaagcat ttttgctgat gtgtaatttt atatgttaca gttacttata ttttactact ttgatgttat ttgcaaaatc aaaggtgtta aagaatttaa cttgcttcag gaaataaatt caagaacata gtggattcat tttcattggt ggcagacacg aaatttggtt catgataaga cttcctttcc ccacctcctg atcagcatta tttaaatctg tatttttctg ttagttaaga aagaaatggc ttcatgatat tgtatttaat agcaaaagtt tggctgtctt cttcattact gttaatagct actatatttt aacaaggaga tttctttttt tgttgttgtt gttctagagt ttggaatata ctgattatct cagacttgac atttatactg aaggatgaag taagacctcc agcttttttt aaaaaaggtg ttgatttgga acacctgtat gggttatggt ttattaaggt tatggtttag aaagtttttt tccctcagag ccttaacttg ttaagaaggt tcatttatcc tgcactgaaa acaaaaactc tatatacttt gtttgtgtgc ctcctgcact ctcccattcc ctatgtgaat atgctctagt tgatattttt aatatattga tttctttttt ctcacagcaa caagtgctta ctctagaggt tagtgggccc tgatatgtca tcagtcagat gcctgcctag ccaaagctgg actaagatta ttctgtacat ttgttgatct tgatatagac ttatatccct gtagggactg ctaatggctc cggcttctgg agtaaggtac tggagaccac tcatccctgt gtctgcttga ttggttcagc tgttgaattg cccttttatt tggaagcagt gttgaagttg tctagggttc aaatggctgc tttgtacacc tgtcattagt ataaggcaga tgtttatttt atcaagctat tttatctcta catttaacta aaaacaaaag ttcccaaaga tctgccttca cttcagaaat tttttttgga ttaaaaaaat taagcctgaa ccttaaataa agtgagttgg ttattcattc caaggattaa gtcccaatct acctctcagc acaatgcaga agctcaccac tgtattgctg ccattaactc atgccagaac cctttgccaa taactggaat tacaaatttt tgttaaagaa aatttatcaa gatctttctt tactgccttc tctatatgta catctcaaaa acatgtacat ctcaaaaact ggagtagaaa gttagattgc tcaactacaa ctcctctaga actctatagc tctgacatac agattcacac tctcctctat ttgctaagta tgtaaagaat gttttctttt aaaatgttct cttttgagaa caactgctta tttgttataa aagcatttgg ttaaaatgat gtcatcataa aaaacagtgg ctttgtttca atacatattt ttgagatgat tatctagaag ccagattaat aaaatcagct tgtgaccttg ctaagcatat aaactggaaa ttcagataca ttcaaaatta tgggttcatt taaaagtgtt ctaccttttg ggtatgagac taatatcact aattcctcaa tagttatcat ggctctatct taattaatta gaaaatatgt gtgtttaatt ctttgagaat taaaatagag aatattaaca gagggttaaa aactgcttca actccaataa gataaaggaa gctcaaaatc tatgagctga gtgttcaatt agctttgcct actgagttca attttatgtc aatacaacag tggatcagac agtacgactt tgaactggtg aatgtaaaca attgtttttc acctaagctg ctttggaaga actgatgctt gctgctaact aaagttttgg atgtatcgat ttagagaacc aattaatacc tgcaaaataa agcatactgt ggtacttctg tttgatctag tatgtgtgat tttagattga tggattaaaa attaataaag atcatacatt ccataccaaa aaaaaaaaaa aaa (SEQ ID NO. 8).
The nucleic acid sequence for human LAMP-3 is disclosed in GENBANK, accession number NM_014398 and is as follows: ccgggccccg gcggctgcgc cgagtccccg cccctccctg ctccgtaggg gtaggagggg gccggcggag tttccctccc cgcccagcgg ccctgggcgg gcttttcggc tgcttctcat aagcaggtgg tttcgtttct ccggcacagg taggtttctc tggcaccgat tcggggcctg cccggacttc gccgcacgct gcagaacctc gcccagcgcc caccatgccc cggcagctca gcgcggcggc cgcgctcttc gcgtccctgg ccgtaatttt gcacgatggc agtcaaatga gagcaaaagc atttccagaa accagagatt attctcaacc tactgcagca gcaacagtac aggacataaa aaaacctgtc cagcaaccag ctaagcaagc acctcaccaa actttagcag caagattcat ggatggtcat atcacctttc aaacagcggc cacagtaaaa attccaacaa ctaccccagc gactacaaaa aacactgcaa ccaccagccc aattacctac accctggtca caacccaggc cacacccaac aactcacaca cagctcctcc agttactgaa gttacagtcg gccctagctt agccccttat tcactgccac ccaccatcac cccaccagct catacaactg gaaccagttc atcaaccgtc agccacacaa ctgggaacac cactcaaccc agtaaccaga ccacccttcc agcaacttta tcgatagcac tgcacaaaag cacaaccggt cagaagcctg ttcaacccac ccatgcccca ggaacaacgg cagctgccca caataccacc cgcacagctg cacctgcctc cacggttcct gggcccaccc ttgcacctca gccatcgtca gtcaagactg gaatttatca ggttctaaac ggaagcagac tctgtataaa agcagagatg gggatacagc tgattgttca agacaaggag tcggtttttt cacctcggag atacttcaac atcgacccca acgcaacgca agcctctggg aactgtggca cccgaaaatc caaccttctg ttgaattttc agggcggatt tgtgaatctc acatttacca aggatgaaga atcatattat atcagtgaag tgggagccta tttgaccgtc tcagatccag agacaattta ccaaggaatc aaacatgcgg tggtgatgtt ccagacagca gtcgggcatt ccttcaagtg cgtgagtgaa cagagcctcc agttgtcagc ccacctgcag gtgaaaacaa ccgatgtcca acttcaagcc tttgattttg aagatgacca ctttggaaat gtggatgagt gctcgtctga ctacacaatt gtgcttcctg tgattggggc catcgtggtt ggtctctgcc ttatgggtat gggtgtctat aaaatccgcc taaggtgtca atcatctgga taccagagaa tctaattgtt gcccgggggg aatgaaaata atggaattta gagaactctt tcatcccttc caggatggat gttgggaaat tccctcagag tgtgggtcct tcaaacaatg taaaccacca tcttctattc aaatgaagtg agtcatgtgt gatttaagtt caggcagcac atcaatttct aaatactttt tgtttatttt atgaaagata tagtgagctg tttattttct agtttccttt agaatatttt agccactcaa agtcaacatt tgagatatgt tgaattaaca taatatatgt aaagtagaat aagccttcaa attataaacc aagggtcaat tgtaactaat actactgtgt gtgcattgaa gattttattt tacccttgat cttaacaaag cctttgcttt gttatcaaat ggactttcag tgcttttact atctgtgttt tatggtttca tgtaacatac atattcctgg tgtagcactt aactcctttt ccactttaaa tttgtttttg ttttttgaga cggagtttca ctcttgtcac ccaggctgga gtacagtggc acgatctcgg cttatggcaa cctccgcctc ccgggttcaa gtgattctcc tgcttcagct tcccgagtag ctgggattac aggcacacac taccacgcct ggctaatttt tgtattttta ttatagacgg ggtttcacca tgttggccag actggtcttg aactcttgac ctcaggtgat ccacccacct cagcctccca aagtgctggg attacaggca tgagccattg cgcccggcct taaatgtttt ttttaatcat caaaaagaac aacatatctc aggttgtcta agtgttttta tgtaaaacca acaaaaagaa caaatcagct tatatttttt atcttgatga ctcctgctcc agaatcgcta gactaagaat taggtggcta cagatggtag aactaaacaa taagcaagag acaataataa tggcccttaa ttattaacaa agtgccagag tctaggctaa gcactttatc tatatctcat ttcattctca caacttatag gtgaatgagt aaactgagac ttaagggaac tgaatcactt aaatgtcacc tggctaactg atggcagagc cagagcttga attcatgttg gtctgacatc aaggtctttg gtcttctccc tacaccaagt tacctacaag aacaatgaca ccacactctg cctgaaggct cacacctcat accagcatac gctcacctta cagggaaatg ggtttatcca ggatcatgag acattagggt agatgaaagg agagctttgc agataacaaa atagcctatc cttaataaat cctccactct ctggaaggag actgaggggc tttgtaaaac attagtcagt tgctcatttt tatgggattg cttagctggg ctgtaaagat gaaggcatca aataaactca aagtattttt aaattttttt gataatagag aaacttcgct aaccaactgt tctttcttga gtgtatagcc ccatcttgtg gtaacttgct gcttctgcac ttcatatcca tatttcctat tgttcacttt attctgtaga gcagcctgcc aagaatttta tttctgctgt tttttttgct gctaaagaaa ggaactaagt caggatgtta acagaaaagt ccacataacc ctagaattct tagtcaagga ataattcaag tcagcctaga gaccatgttg actttcctca tgtgtttcct tatgactcag taagttggca aggtcctgac tttagtctta ataaaacatt gaattgtagt aaaggttttt gtaataaaaa cttactttgg a (SEQ ID NO. 9).
The nucleic acid sequence for human LC3B is disclosed in GENBANK, accession number NM_022818 and is as follows: acgctgcgtg ccgctgctgg gttccgccac gcccgtcatg gcggcggccc cggccggctc tggccccgcc cctcggtgac gcgtcgcgag tcacctgacc aggctgcggg ctgaggagat acaagggaag tggctatcgc cagagtcgga ttcgccgccg cagcagccgc cgcccccggg agccgccggg accctcgcgt cgtcgccgcc gccgccgccc agatccctgc accatgccgt cggagaagac cttcaagcag cgccgcacct tcgaacaaag agtagaagat gtccgactta ttcgagagca gcatccaacc aaaatcccgg tgataataga acgatacaag ggtgagaagc agcttcctgt tctggataaa acaaagttcc ttgtacctga ccatgtcaac atgagtgagc tcatcaagat aattagaagg cgcttacagc tcaatgctaa tcaggccttc ttcctgttgg tgaacggaca cagcatggtc agcgtctcca caccaatctc agaggtgtat gagagtgaga aagatgaaga tggattcctg tacatggtct atgcctccca ggagacgttc gggatgaaat tgtcagtgta aaaccagaaa aaatgcagct cttctagaat tgtttaaacc cttaccaagg aaaaaaaagg gatgttacca actgagatcg atcagttcat ccaatcacag atcatgaaac agtagtgttc ccacctagga gtgttaggaa gttgtgtttg tgtttcaagc agaaaaactg agctccaagt gagcacattc agctttggaa actatattat ttaatgtagg ctagcttgtt ttcaaatttt aaaagtttaa aaataaaata ctttgcattc taagttgcca ataaaataga ccttcaagtt attttaatgc tcttttctca ctaataggaa cttgtaattc cagcagtaat ttaaaggctt tcagagagac cctgagtctt ctcttcaggt tcacagaacc cgccgccttt ttgggtagaa gttttctact cagctagaga gatctcccta agaggatctt taggcctgag ttgtgaagcg caacccccgc aaaacgcatt tgccatcaca gttggcacaa acgcagggta aacgggctgt gtgagaaaac ggccctgact gtaaactgct gaaggtccct gactcctaag agaaccacac ccaaagtcct cactcttgca ggggtagaca tttctggttt ggtttgttct ctagatagtt acacacataa agacaccact caaaaggaaa cttgaataat ttataatttt gatcgagttt cttaaaagac cctggagaaa gagtggcatt tcttctgttt caggttttgt ctgagttcaa actagtgcct gtgttgttac ggaaagcagc agtgtaccag tgtcactctg gagtacagcg ggagaaacac aaaatagtat aactgaaaac attaacattc agacacactc ccttctgcct tccggcttaa agctgtggat gatccacgtt tttgtttttt taatgttaaa tgtgtaactc agtattactg aaaaggtacc cacattttga atagtagtta tcactcttag gtcagacagc catcagaatt ctcccacacc aagtgcatgt cagttgtgga gaaaacatag caaaaagagc cgtacgctct ttacagatac taatgtcaag agttaaacct cctcaggttc aacctgtgat aaaagactag tgcttcccag tacttgcatg gggttcacta tttatagttt tcttgggagt atcacaggaa aatcacaatt acaccacttt agaccctatg tgtagcaggt cacaacttac ccttgtgtgt ttagatgtgt atgaaatacc tgtatacgtt agtgaaagct gtttactgta acggggaaaa ccagattctt tgcatctggg ccctctactg attgttaaag gagttcctgt cacctgctcc ccccaccccc gcatgcgtct gtccacttgg ctaactttta atatgtgtat ttttacatta tgtatattct taactggact gtctcgttta gactgtatac atcatatctg acattattgt aactaccgtg tgatcagtaa gattcctgta agaaatactg ctttttaaga aaaaaaataa catgctgagg ggtgacctat atcccatgtg agtggtcact ttatttatag gatctttaaa acatttttaa tgaactaagt tgaataaagg cacaattaaa aactgtcaaa aaaaaaaaaa aaaa (SEQ ID NO. 10).
The nucleic acid sequence for human LC3A variant 1 is disclosed in GENBANK, accession number NM_032514 and is as follows: accgggcgag ttacctcccg cagccgcagc cgccgtgctc agcgcgagcc ccggagccct tgagcgcgag gcgcggagcc cccggagccc ccaaaccgca gacacatccc cgcgccccag agccccggcc tgcgcgccca gccgggcccg cgcgatgccc tcagaccggc ctttcaagca gcggcggagc ttcgccgacc gctgtaagga ggtacagcag atccgcgacc agcaccccag caaaatcccg gtgatcatcg agcgctacaa gggtgagaag cagctgcccg tcctggacaa gaccaagttt ttggtcccgg accatgtcaa catgagcgag ttggtcaaga tcatccggcg ccgcctgcag ctgaacccca cgcaggcctt cttcctgctg gtgaaccagc acagcatggt gagtgtgtcc acgcccatcg cggacatcta cgagcaggag aaagacgagg acggcttcct ctatatggtc tacgcctccc aggaaacctt cggcttctga gccagcagta ggggggctcg gcctgggagt cggggggccc cggtcaggcc ctgcccagag agctcctggt tcctgaactg agctgcctct accgtggtgg gctgggcagg catgtgcccc cctagtcaga gggcaccaac ccacctactc tgcccctggg tggatcctgg gccggtcgtg ttagggttgt ccctctgggt gctggctggt gggatggggg agggtgggga gcagctccca gcacccctgc tgtgtggttc atcttttttt taggcccctg cctgtctgcc catctgcccc tcacccaccc gaggctctgc ccaccgcctg gacctgccca cccctgaaag actggcccct ggctccccgc ccctcggtct ccacgtggtg tatggatctg tggtcattgt ccctctgcag aataaagatt gctcaggcct gcctggcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa (SEQ ID NO. 11).
The nucleic acid sequence for human LC3C is disclosed in GENBANK, accession number NM_001004343 and is as follows: ggaggaatga gttaggttcc cggttgcggg acagtttttt tttctttttt aaaacagaca cagctactga gtgcaatgcc gcctccacag aaaatcccaa gcgtcagacc cttcaagcag aggaaaagct tggcaatcag acaagaggaa gttgctggaa tccgggcaaa gttccccaac aaaatcccgg tggtagtgga gcgctacccc agggagacgt tcctgccccc gctggacaaa accaagttcc tggtcccgca ggagctgacc atgacccagt tcctcagcat catccggagc cgcatggtcc tgagagccac ggaagccttt tacttgctgg tgaacaacaa gagcctggtc agcatgagcg caaccatggc agagatctac agagactaca aggatgagga tggcttcgtg tacatgacct acgcctccca ggagacattt ggctgcctgg agtcagcagc ccccagggat gggagcagcc ttgaggacag accctgcaat cctctctagc ccatgtcggg aaggatgtgt gctctgacag acgtgtcaga tgctggcaga agggattggt tttctcctgt gtatacagtg gaggagtctg agaagcaggg atgcctgggg tgatcagctc caaccagtgg cagcagagtg gtggctcttc ctagtttagt ttttgtgctc cgtgttcttg tgtccttcta agaaaaatgt gggctgctct tgaaagttat ataattatca tttttttttt tttgaaacag ggtcttgctc tattgcctag gctggagcac agtggcatga tcttggctca ctgcaacctc cgcctcctgg gttcaagtga ttcttctgcc tccacccccc gagtggctgg tattacaagc acatgccacc acaccaggct aatttttgta tttttagtag agacggggtt tcaccatgtt ggccaggctg gtctcgaact cctgacctca ggtgatccac ccacctcggc ctcccaaagt gctgggatta caggcgtgag ccactgcatc cagccatata accgtattct aaataagaaa tggttggctt gtgtgatggt tttgtgtaat gagctagaga taatatttta agtgtcttct gtggtatatg tgggagggcc attaaggagt gggtttcact ccctgcatgt gggcaggtgt cc (SEQ ID NO. 12).
The protein and nucleic acid sequences of other starvation markers are well known in the art.
In some embodiments the tumor cell is starvation marker negative. A "starvation marker negative" tumor cell as used herein refers to any cancer that is not starvation marker positive. A starvation marker positive cancer is one which involves production of significant levels of a starvation marker. Methods for classifying a cancer cell as a starvation marker positive or negative cell are known in the art. For instance, starvation marker positivity may be analyzed by means of immunohistochemical analysis with a monoclonal antibody and categorized according to whether there were enough positive cells. For instance, when the cells are analyzed by flow cytometry a value of at least 90% starvation marker positive cells in comparison to isotype control would indicate a population of cells is starvation marker positive. In some embodiments the value in comparison to isotype controls is at least 85%, 80%, 75%, 70%, 60%, or 50%. In contrast, when the cells are analyzed by flow cytometry a value of 10% or fewer starvation marker positive cells in comparison to isotype control would indicate a population of cells is starvation marker negative. In some embodiments the value in comparison to isotype controls is 40%, 30%, 25%, 20%, 5%, 3%, 2%, 1% or fewer.
It is also possible to determine a percent increase of starvation marker positive cells by examining the changes in levels of expression of the starvation markers upon stimulation with the starvation signal compounds. For example the isolated cells can be treated with rapimycin or avastin or other starvation signal compound and the changes in expression levels of the starvation markers can be assessed. If the increase in starvation marker level in response to the starvation signal is 25% or less then the patient should be treated with the starvation signal compound. In some embodiments the signal should be 20%, 15%, 10%, 5%, 3%, 2%, 1% or less. If the increase in starvation marker level in response to the starvation signal is greater than 25% then the patient should be treated with the starvation signal compound. In some embodiments the signal should be greater than 50%, 60%, 70%, 80%, 90%, 95%, or 98%.
The expression of one or more starvation markers in the tumor cell is determined using methods known to the skilled artisan. The detection methods generally involve contacting a starvation marker binding molecule with a sample in or from a subject or in an in vitro cell. Preferably, the sample is first harvested from the subject, although in vivo detection methods are also envisioned. The sample may include any body tissue or fluid that is suspected of harboring the cancer cells. For example, the cancer cells are commonly found in or around a tumor mass for solid tumors. The binding molecules are referred to herein as isolated molecules that selectively bind to a starvation marker.
As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In all embodiments human subjects are preferred. In aspects of the invention pertaining to predictive therapy in cancers, the subject is a human either suspected of having the cancer, or having been diagnosed with cancer. Methods for identifying subjects suspected of having cancer may include physical examination, subject's family medical history, subject's medical history, biopsy, or a number of imaging technologies such as ultrasonography, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy, or positron emission tomography. Diagnostic methods for cancer and the clinical delineation of cancer diagnoses are well known to those of skill in the medical arts.
As used herein, a tissue sample is tissue obtained from a tissue biopsy, a surgically resected tumor, or any other tumor cell mass removed from the body using methods well known to those of ordinary skill in the related medical arts. The phrase "suspected of being cancerous" as used herein means a cancer tissue sample believed by one of ordinary skill in the medical arts to contain cancerous cells. Methods for obtaining the sample from a biopsy include gross apportioning of a mass,
microdissection, laser-based microdissection, or other art-known cell-separation methods.
Because of the variability of the cell types in diseased-tissue biopsy material, and the variability in sensitivity of the predictive methods used, the sample size required for analysis may range from 1, 10, 50, 100, 200, 300, 500, 1000, 5000, 10,000, to 50,000 or more cells. The appropriate sample size may be determined based on the cellular composition and condition of the biopsy and the standard preparative steps for this determination and subsequent isolation of the nucleic acid for use in the invention are well known to one of ordinary skill in the art. An example of this, although not intended to be limiting, is that in some instances a sample from the biopsy may be sufficient for assessment of RNA expression without amplification, but in other instances the lack of suitable cells in a small biopsy region may require use of RNA conversion and/or amplification methods or other methods to enhance resolution of the nucleic acid molecules or proteins. Such methods, which allow use of limited biopsy materials, are well known to those of ordinary skill in the art and include, but are not limited to: direct RNA amplification, reverse transcription of RNA to cDNA, amplification of cDNA, or the generation of radio-labeled nucleic acids or proteins.
As used herein, the phrase determining the expression of a starvation marker nucleic acid molecule in the tissue means identifying RNA transcripts in the tissue sample by analysis of nucleic acid or protein expression in the tissue sample. The phrase determining the expression of a starvation marker protein or peptide molecule in the tissue means identifying proteins or peptides in the tissue sample by analysis of protein expression in the tissue sample.
The expression of the starvation marker nucleic acid molecules in the sample can be compared to the expression of starvation marker nucleic acid molecules in a sample of tissue that is non-cancerous, i.e. a reference value. As used herein with respect to diagnosis of a tumor, non-cancerous tissue means tissue determined by one of ordinary skill in the medical art to have no evidence of the particular tumor being analyzed based on standard diagnostic methods including, but not limited to, histologic staining and microscopic analysis. In some embodiments, the reference value is the expression level of the gene or protein in a reference sample. A reference value may be a predetermined value and may also be determined from reference samples (e.g., control biological samples) tested in parallel with the test samples. A reference value may be a positive or negative control level. A reference value can be a single cut-off value, such as a median or mean or a range of values, such as a confidence interval. Reference values can be established for various subgroups of individuals, such as individuals free of cancer, individuals having early or late stage cancer, male and/or female individuals, or individuals undergoing cancer therapy. The level of the reference value will depend upon the particular population or subgroup selected. For example, an apparently healthy population may have a different "normal" value than will a population which has cancer, or a population that has cancer but has received cancer treatment. Appropriate ranges and categories for reference values can be selected with no more than routine
experimentation by those of ordinary skill in the art, as these markers and their detection assays are well known to the skilled artisan. The actual numbers in the particular determination of threshold values may vary for different tumors or under different circumstances, such as the conditions of the assay to determine expression. However, the skilled artisan would be able to identify the correct threshold values based on the circumstances. For example threshold values could easily be generated using normal non-cancerous tissue under similar circumstances. In each instance, the comparison of the expression levels of starvation markers to a reference value is useful in determining the relative levels of starvation or autophagic conditions in the test tumor cells.
The reference sample can be any of a variety of biological samples against which a diagnostic assessment may be made. Examples of reference samples include biological samples from control populations or control samples. Reference samples may be generated through manufacture to be supplied for testing in parallel with the test samples, e.g. , reference sample may be supplied in diagnostic kits. Appropriate reference samples will be apparent to the skilled artisan. The biomarker based methods are based in part on a comparison of expression levels of starvation marker genes or proteins between test samples and reference sample. In some embodiments, if the expression level of the starvation marker gene or protein in the test sample is about equal to the expression level of the starvation marker gene or protein in the reference sample, then the test sample and reference sample likely have similar metabolic conditions.
The magnitude of the difference between the test sample and reference sample that is sufficient to indicate a determination of therapeutic strategy will depend on a variety of factors such as the particular starvation marker gene or protein being evaluated, the type of cancer, the type of therapeutic to be applied, heterogeneity in healthy or disease populations from which samples are drawn, the type of reference sample, the assay being used, etc. It is well within the purview of the skilled artisan to determine the appropriate magnitude of difference between the test sample and reference sample that is sufficient to indicate a determination of the autophagic state of the cell.
In other embodiments, the expression level of the starvation marker gene or protein in the test sample may be determined based on a direct comparison to a reference level in absolute values. For instance, at least 10%, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% or more higher than the expression level of the gene or protein in the reference sample. In other embodiments, the expression level of the starvation marker in the test sample is at least 10%, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% or more lower than the expression level of the gene or protein in the reference sample.
The starvation marker levels may be measured using any of a number of techniques available to the person of ordinary skill in the art for protein or nucleic acid, e.g., direct physical measurements (e.g., mass spectrometry), binding assays (e.g., immunoassays, agglutination assays, and immunochromatographic assays), Polymerase Chain Reaction (PCR) technology, branched oligonucleotide technology, Northern blot technology, oligonucleotide hybridization technology, and in situ hybridization technology. The method may also comprise measuring a signal that results from a chemical reaction, e.g., a change in optical absorbance, a change in fluorescence, the generation of chemiluminescence or electrochemiluminescence, a change in reflectivity, refractive index or light scattering, the accumulation or release of detectable labels from the surface, the oxidation or reduction or redox species, an electrical current or potential, changes in magnetic fields, etc. Suitable detection techniques may detect binding events by measuring the participation of labeled binding reagents through the measurement of the labels via their photoluminescence (e.g., via measurement of fluorescence, time- resolved fluorescence, evanescent wave fluorescence, up-converting phosphors, multi- photon fluorescence, etc.), chemiluminescence, electrochemiluminescence, light scattering, optical absorbance, radioactivity, magnetic fields, enzymatic activity (e.g., by measuring enzyme activity through enzymatic reactions that cause changes in optical absorbance or fluorescence or cause the emission of chemiluminescence). Alternatively, detection techniques may be used that do not require the use of labels, e.g., techniques based on measuring mass (e.g., surface acoustic wave measurements), refractive index (e.g., surface plasmon resonance measurements), or the inherent luminescence of an analyte.
The methods may involve the steps of isolating nucleic acids from the sample and/or an amplification step. Typically, a nucleic acid comprising a sequence of interest can be obtained from a biological sample, more particularly from a sample comprising DNA (e.g. gDNA or cDNA) or RNA (e.g. mRNA). Release, concentration and isolation of the nucleic acids from the sample can be done by any method known in the art.
Various commercial kits are available such as the High pure PCR Template Preparation Kit (Roche Diagnostics, Basel, Switzerland) or the DNA purification kits (PureGene, Gentra, Minneapolis, US). Other, well-known procedures for the isolation of DNA or RNA from a biological sample are also available (Sambrook et al., Cold Spring Harbor Laboratory Press 1989, Cold Spring Harbor, N.Y., USA; Ausubel et al., Current Protocols in Molecular Biology 2003, John Wiley & Sons).
When the quantity of the nucleic acid is low or insufficient for the assessment, the nucleic acid of interest may be amplified. Such amplification procedures can be accomplished by those methods known in the art, including, for example, the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, T7-polymerase amplification, and reverse transcription polymerase reaction (RT-PCR).
Polymerase chain reaction (PCR) technology is practiced routinely by those having ordinary skill in the art and its uses in diagnostics are well known and accepted. Methods for practicing PCR technology are disclosed in "PCR Protocols: A Guide to Methods and Applications", Innis, M. A., et al. Eds. Academic Press, Inc. San Diego,
Calif. (1990) which is incorporated herein by reference. Applications of PCR technology are disclosed in "Polymerase Chain Reaction" Erlich, H. A., et al., Eds. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) which is incorporated herein by reference. U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,965,188 and U.S. Pat. No. 5,075,216, which are each incorporated herein by reference describe methods of performing PCR. PCR technology allows for the rapid generation of multiple copies of DNA sequences by providing 5' and 3' primers that hybridize to sequences present in an RNA or DNA molecule, and further providing free nucleotides and an enzyme which fills in the complementary bases to the nucleotide sequence between the primers with the free nucleotides to produce a complementary strand of DNA.
PCR primers can be designed routinely by those having ordinary skill in the art using sequence information. The mRNA or cDNA is combined with the primers, free nucleotides and enzyme following standard PCR protocols. The mixture undergoes a series of temperature changes. If the test gene transcript or cDNA generated therefrom is present, that is, if both primers hybridize to sequences on the same molecule, the molecule comprising the primers and the intervening complementary sequences will be exponentially amplified. The amplified DNA can be easily detected by a variety of well known means. If no gene transcript or cDNA generated therefrom is present, no PCR product will be exponentially amplified.
PCR product may be detected by several well known means. One method for detecting the presence of amplified DNA is to separate the PCR reaction material by gel electrophoresis and stain the gel with ethidium bromide in order to visual the amplified DNA if present. A size standard of the expected size of the amplified DNA is preferably run on the gel as a control.
In some instances, such as when unusually small amounts of RNA are recovered and only small amounts of cDNA are generated therefrom, it is desirable to perform a PCR reaction on the first PCR reaction product. The second PCR can be performed to make multiple copies of DNA sequences of the first amplified DNA. A nested set of primers are used in the second PCR reaction. The nested set of primers hybridize to sequences downstream of the 5' primer and upstream of the 3' primer used in the first reaction.
Branched chain oligonucleotide hybridization may be performed as described in
U.S. Pat. No. 5,597,909, U.S. Pat. No. 5,437,977 and U.S. Pat. No. 5,430,138, which are each incorporated herein by reference. Northern blot analysis methods are well known by those having ordinary skill in the art and are described in Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Additionally, mRNA extraction, electrophoretic separation of the mRNA, blotting, probe preparation and hybridization are all well known techniques that can be routinely performed using readily available starting material.
Hybridization methods for nucleic acids are well known to those of ordinary skill in the art (see, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York). The nucleic acid molecules hybridize under stringent conditions to nucleic acid markers expressed in cancer cells. The nucleic acid markers disclosed herein are known genes and fragments thereof. Targets are nucleic acids selected from the group, including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and may be natural or synthetic. In all embodiments, nucleic acid molecules from human tissue are preferred. The tissue may be obtained from a subject or may be grown in culture. Binding assays for measuring starvation marker levels may use solid phase or homogenous formats. Suitable assay methods include sandwich or competitive binding assays. Examples of sandwich immunoassays are described in U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al., both of which are incorporated herein by reference. Examples of competitive immunoassays include those disclosed in U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler et al., all of which are incorporated herein by reference.
Multiple starvation markers may be measured using a multiplexed assay format, e.g., multiplexing through the use of binding reagent arrays, multiplexing using spectral discrimination of labels, multiplexing by flow cytometric analysis of binding assays carried out on particles (e.g., using the Luminex system
Detection of a protein in a test sample involves routine methods. The skilled artisan can detect the presence or absence of a protein using well known methods. One such method is an immunoassay. In general, immunoassays involve the binding of proteins in a sample to a solid phase support such as a plastic surface. Detectable antibodies are then added which selectively binding to the protein of interest. Detection of the antibody indicates the presence of the protein. The detectable antibody may be a labeled or an unlabeled antibody. Unlabeled antibody may be detected using a second, labeled antibody that specifically binds to the first antibody or a second, unlabeled antibody which can be detected using labeled protein A, a protein that complexes with antibodies. Various immunoassay procedures are described in Immunoassays for the 80's, A. Voller et al., Eds., University Park, 1981, which is incorporated herein by reference.
Simple immunoassays such as a dot blot and a Western blot involve the use of a solid phase support which is contacted with a test sample. Any proteins present in the test sample bind the solid phase support and can be detected by a specific, detectable antibody preparation. The intensity of the signal can be measured to obtain a quantitative readout. Other more complex immunoassays include forward assays for the detection of a protein in which a first anti-protein antibody bound to a solid phase support is contacted with the test sample. After a suitable incubation period, the solid phase support is washed to remove unbound protein. A second, distinct anti-protein antibody is then added which is specific for a portion of the specific protein not recognized by the first antibody. The second antibody is preferably detectable. After a second incubation period to permit the detectable antibody to complex with the specific protein bound to the solid phase support through the first antibody, the solid phase support is washed a second time to remove the unbound detectable antibody. Alternatively, in a forward sandwich assay a third detectable antibody, which binds the second antibody is added to the system. Other types of immunometric assays include simultaneous and reverse assays. A simultaneous assay involves a single incubation step wherein the first antibody bound to the solid phase support, the second, detectable antibody and the test sample are added at the same time. After the incubation is completed, the solid phase support is washed to remove unbound proteins. The presence of detectable antibody associated with the solid support is then determined as it would be in a conventional assays. A reverse assay involves the stepwise addition of a solution of detectable antibody to the test sample followed by an incubation period and the addition of antibody bound to a solid phase support after an additional incubation period. The solid phase support is washed in conventional fashion to remove unbound protein/antibody complexes and unreacted detectable antibody.
In some instances, commercially available antibodies or antibody fragments that bind starvation marker polypeptides may be used in the methods disclosed herein. For example, antibodies against LAMP are well known in the art and are commercially available from several companies including but not limited to the following LAMP-1 antibodies in the Santa Cruz Catalog 1D4B (sc- 19992, mouse), 4E151 ( sc-71489, rat), 5K53 (sc-71488, mouse), C-20 (sc-8098, m, r, h), E-5 (sc- 17768, h > m, r), H-228 (sc- 5570, h > m, r), H4A3 (sc-20011, human), H5G11 (sc-18821, human), LY1C6 (sc- 65236, rat), B-T47 (sc-65331, human), and N-19 (sc-8099, human); the following LAMP-2 antibodies in the Santa Cruz Catalog 4E152 (sc-71491, mouse), 5K54 (sc- 71490, mouse), 6A430 (sc-71492, mouse), ABL-93 (sc-20004, m, h), C-20 (sc-8100, m, r, h), D-20 (sc-34241, h > r), GL2A7 (sc-47749, mouse), H-207 (sc-5571, h > m, r), H4B4 (sc-18822, human), M3/84 (sc-19991, mouse), N-17 (sc-8101, human), ), M3/84.6.34 (sc-81729, mouse), and T-19 (sc-34245, rat); LAMPl (LylC6) and LAMP2 (GL2A7) Antibodies from STRESSMARQ BIOSCIENCES INC. Victoria, BC Canada; LAMP- 1 Antibody (cat # 3629) from ProSci Incorporated CA; Monoclonal Antibody to CD208/DC-LAMP (Clone 1010E1.01, Alexa 488 Conjugated) from IMGENEX; and Anti-LAMP-2 antibody produced in rabbit PRS3627 from Sigma, Rb pAb to LC3A/B (purified 50ug (0.2mg/mL) abeam cat# ab58610); Rat mAb to LAMP 1 (1D48) (purified lOOug (0.5mg/mL) abeam cat# ab25245); R-Phycoerythrin-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG (H+L) (minimal cross-reaction to Human, Mouse, and Rat Serum Proteins) (Jackson ImmunoResearch l.OmL cat# 111-116-144); Fluorescein (FITC)-conjugated AffiniPure F(ab')2 Fragment Donkey Anti-Rat IgG (H+L) (minimal cross-reactivity to Bovine, Chicken, Goat, Guinea Pig, Syrian Hamster, Horse, Human, Mouse, Rabbit, and Sheep Serum Proteins) (0.3mg/mL cat# 712-096- 153). In general human antibodies are preferred.
A number of methods are well known for the detection of antibodies. For instance, antibodies can be detectably labeled by linking the antibodies to an enzyme and subsequently using the antibodies in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), such as a capture ELISA. The enzyme, when
subsequently exposed to its substrate, reacts with the substrate and generates a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes which can be used to detectably label antibodies include, but are not limited to malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
A detectable label is a moiety, the presence of which can be ascertained directly or indirectly. Generally, detection of the label involves an emission of energy by the label. The label can be detected directly by its ability to emit and/or absorb photons or other atomic particles of a particular wavelength (e.g., radioactivity, luminescence, optical or electron density, etc.). A label can be detected indirectly by its ability to bind, recruit and, in some cases, cleave another moiety which itself may emit or absorb light of a particular wavelength (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, etc.). An example of indirect detection is the use of a first enzyme label which cleaves a substrate into visible products. The label may be of a chemical, peptide or nucleic acid molecule nature although it is not so limited. Other detectable labels include radioactive isotopes such as P 32 or H 3 , luminescent markers such as fluorochromes, optical or electron density markers, etc., or epitope tags such as the FLAG epitope or the HA epitope, biotin, avidin, and enzyme tags such as horseradish peroxidase, β-galactosidase, etc. The label may be bound to a peptide during or following its synthesis. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for the peptides described herein, or will be able to ascertain such, using routine experimentation. Furthermore, the coupling or conjugation of these labels to the peptides of the invention can be performed using standard techniques common to those of ordinary skill in the art.
Another labeling technique which may result in greater sensitivity consists of coupling the molecules described herein to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.
Conjugation of the peptides including antibodies or fragments thereof to a detectable label facilitates, among other things, the use of such agents in diagnostic assays. Another category of detectable labels includes diagnostic and imaging labels (generally referred to as in vivo detectable labels) such as for example magnetic resonance imaging (MRI): Gd(DOTA); for nuclear medicine: 201 Tl, gamma-emitting radionuclide 99mTc; for positron-emission tomography (PET): positron-emitting isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F- fluoride, copper-64, gadodiamide, and radioisotopes of Pb(II) such as 203Pb; 11 lln.
As used herein, "conjugated" means two entities stably bound to one another by any physiochemical means. It is important that the nature of the attachment is such that it does not impair substantially the effectiveness of either entity. Keeping these parameters in mind, any covalent or non-covalent linkage known to those of ordinary skill in the art may be employed. In some embodiments, covalent linkage is preferred. Noncovalent conjugation includes hydrophobic interactions, ionic interactions, high affinity interactions such as biotin-avidin and biotin- strep tavidin complexation and other affinity interactions. Such means and methods of attachment are well known to those of ordinary skill in the art.
The methods may also be accomplished using in situ hybridization or in vivo imaging methods. In situ hybridization technology involves the addition of detectable probes which contain a specific nucleotide sequence to fixed cells. If the cells contain complementary nucleotide sequences, the probes, which can be detected, will hybridize to them. In vivo Imaging involves the labeling of nucleic acids or proteins in vivo. The probes used in such methods are typically labeled for detection.
The methods of the invention are useful in determining the course of treatment of a variety of cancers, including but not limited to ovarian cancer, renal cancer, skin cancer, and breast cancer. Other cancers treatable according to the invention include but are not limited to glioblastoma multiforme, leukemias, lymphomas, Hodgkins and Non- Hodgkins, thymomas. Other cancers are well known to the skilled artisan.
The current standard of care for ovarian cancer is predominately surgical resection. This includes removal of the uterus, ovaries, fallopian tubes, cancer in the abdomen or pelvis, removal of lymph nodes Chemotherapeutic treatment is
recommended as an adjuvant therapy to treat either residual tumor cells or cancer recurrence. The most commonly used chemotherapeutic agents include paclitaxel, carboplatin or cisplatin. These treatment options cause many undesirable side effects; and unfortunately, between 50% to 70% of women who are treated for ovarian cancer with surgery and chemotherapy have cancer return within 5 years.
Ovarian cancer is highly vascularized, meaning that there is a critical dependence on blood supply for growth. Because ovarian cancer is highly vascularized, the rationale for using drugs that block blood vessel formation that, in turn, prevents required nutrients to feed the tumor cells, seems a rational proposition. Blood vessel formation results from stimulation of endothelial cells by vascular endothelial growth factor (VEG-F) and currently available treatment consists of an antibody, a protein complex that neutralizes the growth factor VEG-F. The antibody is known as AVASTIN® or bevacizumab.
The treatment for metastatic Renal cell Carinoma (RCC) has undergone extensive research and progression over the last twenty years. There is evidence that RCC is immunogenic based on antigens seen on RCC cells as well as treatment outcomes with immunotherapy. Yet, the response rates to immunotherapy are only about fifteen percent. The traditional treatment for RCC was high dose Interleukin-2. IL-2 is a cytokine that stimulates cell-mediated immunity. Newer treatment models have been recently approved by the FDA and include targeted immunotherapy.
RCC is also known as a very vascular tumor and newer treatment paradigms focus on the use of tyrosine kinase inhibitors. In analysis of hereditary clear cell RCC with Von Hippel-Lindau, evidence supports VEGF as the primary inducer of
angiogenesis in RCC. Thus, given the lack of systemic treatment for RCC and the vascular nature of the tumor, antiangiogenic targeted approaches have become increasingly available. This transition in the treatment of RCC has led to less treatment associated toxicity as well as increased response rates. Although bevacuzimab has been shown to have increased inhibition of VEGF and upregulation in the immunosuppressive cells, the response rates of patient' s have failed to be as high as initially hoped.
In March 2011, the FDA approved the drug Ipilumubab (VERVOX®) for the treatment of stage III unresectable melanoma and Stage IV melanoma. This agent is the first approved since IL-2 in 1996. Ipilumumab is expensive and has some serious immune-mediated side effects, such as endocrinopathy, hepatitis, and colitis.
Additionally, chemotherapy has been widely tried and has not proven to be effective in treating melanoma. Only 15 to 20 percent of melanoma patients respond to
chemotherapy, with IL-2 the only agent showing some benefit; and in those same patients, there is no net increase in survival time, in spite of shrinking tumor sizes and serious side effects. The goal for improving therapy recently has been to promote immunotherapy in combination with traditional chemotherapy to stimulate an anti-tumor immune response. Unfortunately, these efforts have not yet proven successful.
Because melanoma is also highly vascularized, recent work has been aimed at blocking blood vessel formation that, in turn, prevents required nutrients to feed the tumor cells. A treatment strategy to inhibit the growth factor effects of VEGF may provide an effective solution.
While much progress has been made in the field of cancer therapy, the leading cause of death from any cancer is the development of drug resistance. We have found that there exists a common metabolic strategy when drug resistance arises. Stressed or starved cells respond by a series of intracellular events, including use of selective survival strategies, such as autophagy, as described above. Fatty acid oxidation also provides fuel for a profound cellular survival strategy.
The assays described herein for identifying expression levels of starvation biomarkers assist in selecting a course of treatment for these difficult to treat cancers as well as other cancers. The expression of the starvation marker is indicative of an autophagic tumor cell. If the tumor cell is an autophagic tumor cell, then autophagy inhibitors and/or fatty acid metabolism inhibitors should be administered prior to the starvation signal compound. According to one set of embodiments, the cells are exposed to an autophagy inhibitor. An "autophagy modulator," as used herein, is a lysosomotropic agent, meaning that it accumulates preferentially in the lysosomes of cells in the body and blocks pathways involved in break down of cellular components. An autophagy inhibitor, as used herein, is any compound which blocks the collection or metabolism of lipids in the lysosome. The inhibitor is effective for killing cells by inhibiting autophagy in cells that depend on autophagy to survive. While no one knows exactly the mechanism by which autophagy inhibitors function, it may well be through the inhibition of the acidic hydrolases (enzymes in the lysosomes) that are necessary to break down proteins, lipids, etc. for processing and removal by increasing the pH to decrease the necessary acidity for the enzymes to work.
In some embodiments, the autophagy inhibitor is selected from the group consisting of: chloroquine compounds, 3-methyladenine, bafilomycin Al, 5-amino-4- imidazole carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1, analogues of cAMP, and drugs which elevate cAMP levels, adenosine, N6-mercaptopurine riboside, wortmannin, and vinblastine.
The autophagy inhibitor is preferably a chloroquine compound. Chloroquine is a synthetically manufactured drug containing a quinoline nucleus (The Merck Index, p. 2220, 1996). The chloroquine compounds useful according to the invention include chloroquine analogs and derivatives. A number of chloroquine analogs and derivatives are well known. For example, suitable compounds include but are not limited to chloroquine, chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate, hydroxychloroquine sulphate, quinacrine, primaquine, mefloquine, halofantrine, lumefantrine and tafenoquine or enantiomers, derivatives, analogs, metabolites, pharmaceutically acceptable salts, and mixtures thereof.
Chloroquine and hydroxychloroquine are generally racemic mixtures of (-)- and (-i-)-enantiomers. The (-)-enantiomers are also known as (R)-enantiomers (physical rotation) and 1 -enantiomers (optical rotation). The (-i-)-enantiomers are also known as (S)-enantiomers (physical rotation) and r-enantiomers (optical rotation). Preferably, the (-)-enantiomer of chloroquine is used. The enantiomers of chloroquine and
hydroxychloroquine can be prepared by procedures known to the art. The compounds of the invention, such as, chloroquine may exhibit the
phenomena of tautomerism, conformational isomerism, geometric isomerism, and/or optical isomerism. The invention covers any tautomeric, conformational isomeric, optical isomeric and/or geometric isomeric forms of the compounds described herein, as well as mixtures of these various different forms.
Thus in some embodiments the autophagy inhibitor useful in the invention is a 4- aminoquinoline. 4-aminoquinolines include compounds having the following structure:
Figure imgf000030_0001
or a pharmaceutically acceptable salt or prodrug thereof, wherein Riis defined herein;
each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration; and
Ri is 1 or 2 hydrogens, alkyl, cycloalkyl, aryl, substituted alkyl, substituted cycloalkyl or substituted aryl.
In other embodiments the 4- aminoquinoline has the following structure:
Figure imgf000030_0002
or a pharmaceutically acceptable salt or prodrug thereof, wherein R2 and R3 are defined herein;
each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration;
R2 and R3 is independently a hydroxalkyl, an alkyl, alkyloxy, alkylcarboxy, alkylene or alkenylene having from one to six carbon atoms.
Examples of 4-aminoquinolines useful according to the invention include but are not limited to chloroquine, 2-hydroxychloroquine, amodiaquine,
mondesethylchloroquine, quinoline phosphate, and chloroquine phosphate or mixtures thereof.
Choloroquine:
Figure imgf000031_0001
-hydroxychloroquine
Figure imgf000031_0002
Other examples of preferred chloroquine compounds that can be used in the invention include chloroquine diphosphate and hydroxychloroquine (Plaquenil™).
According to one set of embodiments, the cells are exposed to a fatty acid metabolism inhibitor. Metabolic disruption of fatty acids can be achieved using inhibitors of fatty acid metabolism. A "fatty acid metabolism inhibitor," as used herein, is a compound able to inhibit (e.g., prevent, or at least decrease or inhibit the activity by an order of magnitude or more) a reaction within the fatty acid metabolism pathway, such as an enzyme-catalyzed reaction within the pathway. The inhibitor may inhibit the enzyme, e.g., by binding to the enzyme or otherwise interfering with operation of the enzyme (for example, by blocking an active site or a docking site, altering the configuration of the enzyme, competing with an enzyme substrate for the active site of an enzyme, etc.), and/or by reacting with a coenzyme, cofactor, etc. necessary for the enzyme to react with a substrate. The fatty acid metabolism pathway is the pathway by which fatty acids are metabolized within a cell for energy (e.g., through the synthesis of ATP and the breakdown of fatty acids into simpler structures, such as C02, acyl groups, etc.) or to produce a carbohydrate source. For example inhibitors of fatty acid metabolism include inhibitors of fatty acid oxidation, fatty acid transporter inhibitors, reductase inhibitors, and isomerase inhibitors within the fatty acid metabolism pathway.
The fatty acid metabolism inhibitor in some embodiments is an inhibitor of fatty acid oxidation, a fatty acid transporter inhibitor, a reductase inhibitor, or an isomerase inhibitor within the fatty acid metabolism pathway. In one embodiment the reductase is 2,4-dienoyl-CoA reductase. In another embodiment the isomerase is 2,4-dienoyl-CoA isomerase. In yet other embodiments the inhibitor of fatty acid metabolism is an inhibitor of fatty acid oxidation and is any one or more of the following: oxirane carboxylic acid compound, such as etomoxir (2-(6-(4-chlorophenoxy)-hexyl)-oxirane-2- carboxylic acid ethyl ester), 2-(4-(3-chlorophenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(3,4- dichlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(4-fluorophenoxy)- hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-phenoxyhexyl)-oxirane-2-carboxylic acid ethyl ester, cerulenin, 5-(tetradecyloxy)-2-furoic acid, oxfenicine, methyl palmoxirate, metoprolol, amiodarone, perhexiline, aminocamitine, hydrazonopropionic acid, 4-bromocrotonic acid, trimetazidine, ranolazine, hypoglycin, dichloroacetate, methylene cyclopropyl acetic acid, beta-hydroxy butyrate, and a non-hydrolyzable analog of carnitine or pharmacologically acceptable salts thereof.
The fatty acid metabolism pathway includes several enzymatic reactions, which use various enzymes such as reductases or isomerases. Specific examples of enzymes within the fatty acid metabolism pathway include 2,4-dienoyl-CoA reductase, 2,4- dienoyl-CoA isomerase, butyryl dehydrogenase, etc, as further discussed below. In one embodiment, the fatty acid metabolism inhibitor is an inhibitor able to inhibit a beta- oxidation reaction in the fatty acid metabolism pathway. In another embodiment, the inhibitor is an inhibitor for a fatty acid transporter (e.g., a transporter that transports fatty acids into the cell, or from the cytoplasm into the mitochondria for metabolism). In yet another embodiment, the inhibitor may react or otherwise inhibit key steps within the fatty acid metabolism pathway. In still another embodiment, the inhibitor may be an inhibitor of fatty acids as a source of energy in the mitochondria. For example, the inhibitor may inhibit the breakdown of intermediates such as butyryl CoA, glutaryl CoA, or isovaleryl CoA.
2,4-dienoyl-CoA reductase is an enzyme within the fatty acid metabolism pathway that catalyzes reduction reactions involved in the metabolism of polyunsaturated fatty acids. Certain fatty acids are substrates for 2,4-dienoyl-CoA reductases located within the mitochondria. In some cases, fatty acids may be transported into the mitochondria through uncoupling proteins. The uncoupling protein may, in certain instances, increase the mitochondrial metabolism to increase the availability of fatty acids within the mitochondria and/or increase the throughput of beta- oxidation within the mitochondria.
The enzyme 2,4-dienoyl-CoA isomerase is an enzyme within the fatty acid metabolism pathway that catalyzes isomerization of certain fatty acids. One step in the metabolism of certain polyunsaturated fatty acids may be protective against reactive oxygen intermediates ("ROI"). Thus, by generating substrates and antagonists for the activity of 2,4-dienyol-CoA isomerase, the metabolic production of reactive oxygen intermediates may be enhanced and/or reduced. This, in turn, affects the levels of fatty acids in the cell.
Thus, it is to be understood that, as used herein, compounds useful for inhibiting fatty acid metabolism (i.e., "fatty acid metabolism inhibitors") are also useful for altering cellular production of reactive oxygen; compounds described in reference to fatty acid metabolism inhibition should also be understood herein to be able to alter reactive oxygen production within a cell. For example, by altering the ability of a cell to metabolize a fatty acid, the ability of the cell to produce reactive oxygen may also be affected, since one pathway for a cell to produce reactive oxygen intermediates is through the metabolism of fatty acids. Thus, in some cases, the production of reactive oxygen can be affected by exposing a cell to, or removing a cell from, a fatty acid metabolism inhibitor.
The inhibitor of fatty acid metabolism may be an inhibitory nucleic acid. The inhibitory nucleic acid may be, for instance, specific for an enzyme selected from the group consisting of 2,4-dienoyl-CoA reductase, 2,4-dienoyl-CoA isomerase, and butyryl dehydrogenase.
In other embodiments the inhibitor of fatty acid metabolism is oxamate. The oxamate may be, for instance an alkyl oxamate such as, ethyl oxamate or sodium oxamate.
In yet another embodiment, the inhibitor of fatty acid metabolism is a compound having the following structure:
Figure imgf000034_0001
or a pharmaceutically acceptable salt or prodrug thereof, wherein R4 is defined herein; wherein the dashed line is a double bond at one of the indicated positions and a single bond in the other; wherein R4 is O-C-CH3, -ONa, -OH, -0-(CH2)3-CH3, -CH2 - C(0)-C(0)-0- R8 or -CH=C(OH)-C(0)-0- R8> alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl, heterocycloalkyl, substituted alkyl, substituted cycloalkyl or substituted aryl, substituted aralkyl, substituted heteroaryl, substituted heteroaralkyl, substituted heterocyclyl, substituted heterocycloalkyl; wherein X is: =0, =N— O R2; and wherein R2 is independently selected from hydrogen, H2, alkyl, cycloalkyl, aryl, substituted alkyl, substituted cycloalkyl or substituted aryl.
In some preferred embodiments the fatty acid inhibitor is an oxamate including, for example, each of the following:
Ethyl oxamate
Figure imgf000034_0002
Sodium oxamate
Figure imgf000035_0001
Ethyl thiooxamate
o
Oxamic acid
Figure imgf000035_0002
Butyl oxamate
Figure imgf000035_0003
Pyruvate derivatives have been described in the art and are useful for inhibiting fatty acid production. For instance, US Patents, such as 5,395,822; 6,916,850;
6,086,789; 5,968,727; 5,047,427 and 5,256,697 (the specific pyruvate derivatives, salts etc are incorporated by reference), describe pyruvate derivatives, conjugates and salts.
The method involves the use of a fatty acid metabolism inhibitor that is an oxirane carboxylic acid compound capable of inhibiting fatty acid metabolism, or a pharmacologically acceptable salt thereof in some embodiments. The subject may not have an indication otherwise indicated for treatment with the compound. In some embodiments the oxirane carboxylic acid compound has the formula:
Figure imgf000036_0001
or a pharmaceutically acceptable salt or prodrug thereof, wherein R5> R6 and R7 are herein; wherein R5 represents a hydrogen atom, a halogen atom, a 1-4C alkyl group, a 1-4C alkoxy group, a nitro group or a trifluoromethyl group, R6 has one of the meanings of R5, R7 represents a hydrogen atom or a 1-4C alkyl group, Y represents the grouping— O— (CH2)m— , m is 0 or a whole number from 1 to 4, and n is a whole number from 2 to 8 wherein the sum of m and n is a whole number from 2 to 8. R5 in some embodiments is a halogen atom, R6 is a hydrogen atom, m is 0, and n is 6. In other embodiments R7 is an ethyl group. The oxirane carboxylic acid compound is etomoxir in some embodiments.
It is most particularly preferred to use etomoxir, i.e., 2-(6-(4-chlorophenoxy)- hexyl)-oxirane-2-carboxylic acid ethyl ester. Examples of other oxirane carboxylic acid compounds useful in the invention are 2-(4-(3-chlorophenoxy)-butyl)-oxirane-2- carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2-carboxylic acid ethyl ester, 2- (6-(3,4-dichlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(4- fluorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, and 2-(6-phenoxyhexyl)- oxirane-2-carboxylic acid ethyl ester, the corresponding oxirane carboxylic acids, and their pharmacologically acceptable salts.
The foregoing class of oxirane carboxylic acid compounds, including etomoxir, has been described by Horst Wolf and Klaus Eistetter in United States Patent 4,946,866 for the prevention and treatment of illnesses associated with increased cholesterol and/or triglyceride concentration, and by Horst Wolf in United States Patent 5,739,159 for treating heart insufficiency. The preparation of oxirane carboxylic acid compounds, and their use for blood glucose lowering effects as an ant diabetic agent, is described in Jew et al United States Patent 6,013,666. Etomoxir has been described as an inhibitor of mitochondrial carnitine palmitoyl transferase-I by Mannaerts, G. P., L. J. Debeer, J. Thomas, and P. J. De Schepper "Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats," J. Biol. Chem. 254:4585-4595, 1979.
The foregoing United States Patents 4,946,866, 5,739,159, and 6,013,666, United States Patent Application 20030036199, and the foregoing publication by Mannaerts, G. P., L. J. Debeer, J. Thomas, and P. J. De Schepper, are incorporated herein by reference. In addition, U.S. Patent Application Serial No. 10/272,432, filed October 15, 2002, entitled "Methods for Regulating Co-Stimulatory Molecule Expression with Reactive Oxygen," by M. K. Newell, et al. is incorporated herein by reference in its entirety.
Other, non-limiting examples of fatty acid metabolism inhibitors include fatty acid transporter inhibitors, beta- oxidation process inhibitors, reductase inhibitors, and/or isomerase inhibitors within the fatty acid metabolism pathway. Specific examples of other fatty acid metabolism inhibitors include, but are not limited to, cerulenin, 5-
(tetradecyloxy)-2-furoic acid, oxfenicine, methyl palmoxirate, metoprolol, amiodarone, perhexiline, aminocamitine, hydrazonopropionic acid, 4-bromocrotonic acid, trimetazidine, ranolazine, hypoglycin, dichloroacetate, methylene cyclopropyl acetic acid, and beta-hydroxy butyrate. As a another example, the inhibitor may be a non- hydrolyzable analog of carnitine.
In one embodiment, the fatty acid metabolism inhibitor is a carboxylic acid. In some cases, the carboxylic acid may have the structure:
Figure imgf000037_0001
where R14 comprises an organic moiety, as further described below. In some cases, R14 may include at least two nitrogen atoms, or R14 may include an aromatic moiety (as further described below), such as a benzene ring, a furan, etc.
In another embodiment, the fatty acid metabolism inhibitor has the structure:
Figure imgf000037_0002
where each of R 5 and R16 independently comprises organic moiety. In some instances, either or both of R15 and R16 may independently be an alkyl, such as a straight- chain alkyl, for instance, methyl, ethyl, propyl, etc. In certain cases, R16 may have at least 5 carbon atoms, at least 10 carbon atoms, or at least 15 or more carbon atoms. For example, in one embodiment, R16 may be a tetradecyl moiety. In other cases, R16 may include an aromatic moiety, for example, a benzene ring. In still other cases, R16 may have the structure:
Figure imgf000038_0001
where R 3 comprises an organic moiety and Ar 1 comprises an aromatic moiety. R17 may be a an alkyl, such as a straight-chain alkyl. In some instances, Ar1 may be a benzene ring or a derivative thereof, i.e., having the structure:
Figure imgf000038_0002
wherein each of R18, R19, R2o, R2i, and R22 is hydrogen, a halogen, an alkyl, an alkoxy, etc.
In yet another embodiment, the fatty acid metabolism inhibitor has the structure:
Figure imgf000038_0003
where each of R2 , R24, R25, R%, R27 R28 and R29 independently comprises hydrogen, a halogen, or an organic moiety, such as an alkyl, an alkoxy, etc. In some cases, R23 and R24 together may define an organic moiety, such as a cyclic group. For example, the fatty acid metabolism inhibitor may have the structure:
Figure imgf000039_0001
wherein R3o comprises an organic moiety, such as an alkyl, an alkoxy, an aromatic moiety, an amide, etc. An exam le, of R 0 is:
Figure imgf000039_0002
wherein Ar comprises an aromatic moiety, such as a benzene ring or a benzene derivative, as previously described.
In another set of embodiments, the cells may be exposed to an agent that inhibits the synthesis or production of one or more enzymes within the fatty acid metabolism pathway. Exposure of the cells to the agent thus inhibits fatty acid metabolism within the cell. For example, in one embodiment, an inhibitory oligonucleotide such as a RNAi or antisense oligonucleotide may be used that selectively binds to regions encoding enzymes present within the fatty acid metabolism pathway, such as 2,4-dienoyl-CoA reductase or 2,4-dienoyl-CoA isomerase.
Thus, agents that inhibit enzymes of the fatty acid metabolism pathway include enzymes of the fatty acid metabolism pathway expression inhibitors. A enzymes of the fatty acid metabolism pathway expression inhibitor as used herein is molecule that knocks down expression of an enzyme of the fatty acid metabolism pathway. Thus, the invention also features the use of small nucleic acid molecules, referred to as short interfering nucleic acid (siNA) that include, for example: microRNA (miRNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), and short hairpin RNA (shRNA) molecules to knockdown expression of proteins such as enzymes of the fatty acid metabolism pathway. An siNA of the invention can be unmodified or chemically- modified. An siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Furthermore, siNA having multiple chemical modifications may retain its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic applications.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065;
Perrault et al, 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International
Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; and Sproat, U.S. Pat. No. 5,334,711; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein). Modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.
There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996,
Biochemistry , 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565 568; Pieken et al. Science, 1991, 253, 314317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334 339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al.).
In one embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double- stranded siNA molecule comprises a nucleotide sequence identical to the nucleotide sequence or a portion thereof of the targeted RNA. In another embodiment, one of the strands of the double- stranded siNA molecule comprises a nucleotide sequence that is substantially complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double- stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the target RNA. In another embodiment, each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
In some embodiments an siNA is an shRNA, shRNA-mir, or microRNA molecule encoded by and expressed from a genomically integrated transgene or a plasmid-based expression vector. Thus, in some embodiments a molecule capable of inhibiting mRNA expression, or microRNA activity, is a transgene or plasmid-based expression vector that encodes a small-interfering nucleic acid. Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siRNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue-specific expression systems. In some embodiments, transgenes and expression vectors are controlled by tissue specific promoters. In other embodiments transgenes and expression vectors are controlled by inducible promoters, such as tetracycline inducible expression systems.
In another embodiment, a small interfering nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. The recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid- specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters.
Developmentally-regulated promoters are also encompassed, for example the murine hox promoters and the a-fetoprotein promoter.
Other inhibitor molecules that can be used include sense and antisense nucleic acids (single or double stranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, antibodies, and aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins. Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al.,
Leukemia. 6(11): 1786-94, 1993; Valera et al., J. Biol. Chem. 269(46):28543-6, 1994; Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng et al., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer Res. 55(l):90-5, 1995; Lewin et al., Nat Med. 4(8):967-71, 1998). For example, neoplastic reversion was obtained using a ribozyme targeted to an H-Ras mutation in bladder carcinoma cells (Feng et al., Cancer Res. 55(10):2024-8, 1995). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9,1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al., Nucleic Acids Symp Ser. (29): 121-2, 1993).
Anti-sense oligonucleotides may be designed to hybridize to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of an enzymes of the fatty acid metabolism pathway encoded by a given DNA sequence (e.g. either native polypeptide or a mutant form thereof), so that its expression is reduce or prevented altogether. Anti-sense techniques may be used to target a coding sequence; a control sequence of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with control sequences. Anti-sense oligonucleotides may be DNA or RNA and may be of around 14-23 nucleotides, particularly around 15-18 nucleotides, in length. The construction of antisense sequences and their use is described in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).
It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, though total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence.
The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective sense RNA molecules to hybridize. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene.
Triple helix approaches have also been investigated for sequence- specific gene suppression. Triple helix forming oligonucleotides have been found in some cases to bind in a sequence- specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A.
88(18):8227-31, 1991; Duval- Valentin et al., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A. 93(7):2811-6, 1996;
Porumb et al., Cancer Res. 56(3):515-22, 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Hanvey et al., Antisense Res. Dev. 1(4):307-17, 1991; Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor et al., Arch. Surg. 132(11): 1177-83, 1997). Minor-groove binding polyamides can bind in a sequence-specific manner to DNA targets and hence may represent useful small molecules for future suppression at the DNA level (Trauger et al., Chem. Biol. 3(5):369- 77, 1996). In addition, suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz Nature 329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6, 1989; Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989). In some cases suppression strategies have led to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein.
The diverse array of suppression strategies that can be employed includes the use of DNA and/or RNA aptamers that can be selected to target, for example, a protein of interest such as enzymes of the fatty acid metabolism pathway.
2,4-dienoyl-CoA reductase has been described in for instance Koivuranta et al Biochemical Journal 1994, 304, p. 787. It is also disclosed in NCBI gene ID 1666 (DECR1) as well as NCBI genbank Accession number U78302. The sequence of 2,4- dienoyl-CoA isomerase is disclosed in NCBI gene ID 1891 (ECH1).
Nucleic acid sequence for 2,4-dienoyl-CoA reductase is taagctttaa aaacatgtaa aaaggacatt aaattgacat cttttttgtg ttaggtcacc aaggagcagt gggacaccat agaagaactc atcaggaaga caaaaggttc ctaagaccac tttggccttc atcttggtta cagaaaaggg aatagaaatg aaacaaatta tctctcatct tttgactatt tcaagtctaa taaattctta attaacaaac attcattgaa tatgtattat gtgccaggcc agtgatagcc attgtatatt caaagataaa taaaatgaaa tatagtcttc aaaacattaa aaaaaaaagg agggcatggg gagagtaggt aaaggctcct ctttacctattt (SEQ ID NO. 13)
The amino acid sequence for 2,4-dienoyl-CoA reductase is
MKLPARVFFTLGSRLPCGLAPRRFFSYGTKILYQNTEALQSKFFSPLQKAMLPPN SFQGKVAFITGGGTGLGKGMTTLLSSLGAQCVIASRKMDVLKATAEQISSQTGN KVHAIQCDVRDPDMVQNTVSELIKVAGHPNIVINNAAGNFISPTERLSPNAWKTI TDIVLNGTAFVTLEIGKQLIKAQKGAAFLSITTIYAETGSGFVVPSASAKAGVEA MSKSLAAEWGKYGMRFNVIQPGPIKTKGAFSRLDPTGTFEKEMIGRIPCGRLGT VEELANLAAFLCSDYASWINGAVIKFDGGEEVLISGEFNDLRKVTKEQWDTIEEL IRKTKG (SEQ ID NO. 14).
In some embodiments the cells may be treated with both an autophagic inhibitor and an inhibitor of fatty acid metabolism. In some embodiments when both are administered it is preferred to administer the autophagic inhibitor first.
If the tumor cell is not autophagic, then the tumor can be directly treated with the starvation signal compounds. A starvation signal compound, as used herein, refers to a cancer therapy involving the blocking of a metabolic pathway or blood vessel formation. A starvation signal compound as used herein includes VEGF antagonists, rapimycin, and glycolytic inhibitors.
A VEGF antagonist is a compound that inhibits the activity of VEGF. In some embodiments a VEGF antagonists is an anti-VEGF antibody. Anti-VEGF antibodies, as used herein, refer to peptides that bind to VEGF with sufficient affinity and specificity to prevent VEGF from interacting with VEGF receptor and reduce VEGF signaling. The term "VEGF" refers to the vascular endothelial cell growth factor, as described by Leung et al. Science, 246: 1306 (1989), and Houck et al. Mol. Endocrin., 5: 1806 (1991), together with the naturally occurring allelic and processed forms thereof. The term "VEGF" is also used to refer to known truncated forms of the polypeptide. Preferably, the anti-VEGF antibody of the invention can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein the VEGF activity is involved. An anti-VEGF antibody will usually not bind to other VEGF homologues such as VEGF-B or VEGF-C, nor other growth factors such as PIGF, PDGF or bFGF. A preferred anti- VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599, including but not limited to the antibody known as bevacizumab (AVASTIN®).
Bevacizumab is a major drug developed for treating cancer, including metastatic cancer, and has the trade name AVASTIN ®, by Genentech/Roche. Bevacizumab is a humanized monoclonal antibody, and was the first commercially available angiogenesis inhibitor. It stops tumor growth by preventing the formation of new blood vessels (angiogenesis) by targeting and inhibiting the function of a natural protein called vascular endothelial growth factor that stimulates new blood vessel formation. The drug was first developed as a genetically engineered version of a mouse antibody that contains both human and mouse components, a monoclonal antibody against VEGF-A.
In some embodiments the VEGF antibody is not VEGFR-3 mAb disclosed by Imclone Systems Inc., New York, N.Y.
Numerous VEGF and VEGF receptor antibodies are available commercially for research purposes. Certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three or four CDRs or "hypervariable regions" in both in the light-chain and the heavy-chain variable domains, as discussed above.
The starvation signal compound of the invention may also be an mTOR
(mammalian target of rapamycin) inhibitor. mTOR inhibitors include but are not limited to rapimycin (sirolimus). Rapimycin is an immunosuppressant that has been shown to be useful in the treatment of cancer.
A glycolytic inhibitor may also be used in the methods of the invention.
Glycolytic inhibitors, oxamate and iodoacetate, for instance, are inhibitors of
gluconeogenesis. Preferred glycolytic inhibitors are 2-deoxyglucose compounds, defined herein as homologs, analogs, and/or derivatives of 2-deoxy-D-glucose. Glycolytic inhibitors particularly useful herein can have the formula:
Figure imgf000046_0001
or a pharmaceutically acceptable salt or prodrug thereof, wherein Rg, Rjo, Rn, R12, and R13 are herein; wherein X represents an O or S atom; R9 represents a hydrogen atom or a halogen atom; R10 represents a hydroxyl group, a halogen atom, a thiol group, or CO-R6; Rn, Ri2, and R13 each represent a hydroxyl group, a halogen atom, or CO- R14, R14 represents an alkyl group of from 1 to 20 carbon atoms, and at least two of R11; R12, and R1 are hydroxyl groups. In one embodiment the 2-deoxyglucose compound is 2-deoxy-D-glucose.
The invention also encompasses a method of treating cancer by administering a mTOR inhibitor such as rapamycin and an autophagy inhibitor and/or a fatty acid metabolism inhibitor. The compounds may be administered simultaneously, in one or separate administrations. Alternatively they may be administered at different times. For instance the autophagy inhibitor and/or a fatty acid metabolism inhibitor may be administered before or after the mTOR inhibitor. Based on the information concerning metabolic stress and survival strategies in ovarian cancer cells, RCC and melanoma and in pre-clinical studies in animals, a combined therapy of VEGF antagonist (i.e. AVASTIN® to block VEGF-mediated blood vessel formation) and autogphagy inhibitor (i.e. hydroxy-chloroquine) in a specific dose and timing of administration protocol is another aspect of the invention. A preferred treatment protocol involves AVASTIN, administered at 15 mg/Kg IV every 3 months; and Hydroxychloroquine administered at 200-400 mg per day. AVASTIN neutralizes VEGF and lead to decreased nutrition to the tumor, while Hydroxychloroquine blocks autophagy.
The molecules useful herein are isolated molecules. As used herein, the term
"isolated" means that the referenced material is removed from its native environment, e.g., a cell. Thus, an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material is occurs naturally (e.g., cytoplasmic or membrane component). The isolated molecules may be
substantially pure and essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. In particular, the molecules are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing. Because an isolated peptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the peptide may comprise only a small percentage by weight of the preparation. The peptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems. In some embodiments, the peptide is a synthetic peptide.
The term "purified" in reference to a protein or a nucleic acid, refers to the separation of the desired substance from contaminants to a degree sufficient to allow the practitioner to use the purified substance for the desired purpose. Preferably this means at least one order of magnitude of purification is achieved, more preferably two or three orders of magnitude, most preferably four or five orders of magnitude of purification of the starting material or of the natural material. In specific embodiments, a purified thymus derived peptide is at least 60%, at least 80%, or at least 90% of total protein or nucleic acid, as the case may be, by weight. In a specific embodiment, a purified thymus derived peptide is purified to homogeneity as assayed by, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis, or agarose gel electrophoresis.
The therapeutic compounds described herein can be administered in combination with other therapeutic agents and such administration may be simultaneous or sequential. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The administration of the other therapeutic agent, including chemotherapeutics and the chloroquine, hydroxychloroquine, or AVASTIN® can also be temporally separated, meaning that the therapeutic agents are administered at a different time, either before or after, the administration of the therapeutics described herein. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.
Thus, in some instances, the invention also involves administering another cancer treatment (e.g. , radiation therapy, chemotherapy or surgery) to a subject. Examples of conventional cancer therapies include treatment of the cancer with agents such as All- trans retinoic acid, Actinomycin D, Adriamycin, anastrozole, Azacitidine, Azathioprine, Alkeran, Ara-C, Arsenic Trioxide (Trisenox), BiCNU Bleomycin, , Busulfan, CCNU, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Cytoxan, DTIC, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, 5-flurouracil, Epirubicin, Epothilone, Etoposide, exemestane, Erlotinib, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Herceptin, Hydrea, Ifosfamide, Irinotecan, Idarubicin, Imatinib, letrozole, Lapatinib, Leustatin, 6-MP, Mithramycin, Mitomycin, Mitoxantrone, Mechlorethamine, megestrol, Mercaptopurine, Methotrexate, Mitoxantrone, Navelbine, Nitrogen Mustard, Oxaliplatin, Paclitaxel, pamidronate disodium, Pemetrexed, Rituxan, 6-TG, Taxol, Topotecan, tamoxifen, taxotere, Teniposide, Tioguanine, toremifene, trimetrexate, trastuzumab, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, Velban, VP- 16, and Xeloda.
Other therapeutics for cancer involve antibodies or other binding proteins conjugated to a cytotoxic agents. The conjugates include an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (i.e., a radioconjugate). Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296). Enzymatically active toxins and fragments thereof which can be used in the conjugates include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
For selective destruction of the cell, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, 1131, 1125, Y90, Re186, Re188, Sm153,
Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc 99 m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-I l l, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc99m or I123, .Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP) , succinimidyl-4- (N-maleimidomethyl)cyclohexane- 1 - carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon- 14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase- sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
When used in combination with the therapies of the invention the dosages of known therapies may be reduced in some instances, to avoid side effects.
Cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physician's Desk Reference (56th ed., 2002). In some embodiments, the therapeutic compounds of the invention are formulated into a pharmaceutical composition that further comprises one or more additional anticancer agents.
The active agents of the invention are administered to the subject in an effective amount for treating the subject. An "effective amount", for instance, is an amount necessary or sufficient to realize a desired biologic effect. For instance an effective amount is that amount sufficient to prevent or inhibit autophagy. An effective amount for treating precancerous tissue may be an amount sufficient to prevent, delay or inhibit the development of a tumor in the subject compared to the levels in the absence of treatment. According to some aspects of the invention, an effective amount is that amount of a compound of the invention alone or in combination with another
medicament, which when combined or co-administered or administered alone, results in a biological affect associated with treating the precancerous tissue. Prevention or inhibition as used in this context refers to any reduction or delay in tumor formation as a result of the treatment when compared to an untreated subject.
The effective amount of a compound of the invention in the treatment of a subject may vary depending upon the specific compound used, the mode of delivery of the compound, and whether it is used alone or in combination. The effective amount for any particular application can also vary depending on such factors as the type and/or amount of catastrophic trigger to which the subject is exposed, the particular compound being administered for treatment, the size of the subject, or the severity of the disorder. One of ordinary skill in the art can empirically determine the effective amount of a particular molecule of the invention without necessitating undue experimentation. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity in and of itself and yet is entirely effective to treat the particular subject.
Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays, animal studies and human studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In particular a number of the autophagy and fatty acid metabolism inhibitors described herein have been safely administered to humans. Safe doses for chronic or acute therapies of these compounds are known to the skilled artisan. For example chloroquine and hydroxychloroquine have been chronically administered to humans for the treatment of malaria infection as well as some forms of autoimmune disease.
Dichloroacetate (DCA) has been administered to subjects for the treatment of metabolic disorders. Chronic therapy with these compounds at doses effective for inhibiting autophagy have proven to be safe in long term administration protocols. Chloroquine typically is administered in a dosage of 300mg-600mg to adults for the treatment of malarial infection. DCA can be used, for example, in dosages of 1- 25 mg/kg of body weight per day, 1- 15 mg/kg of body weight per day, or 5- 10 mg/kg of body weight per day.
As used herein, the term treat, treated, or treating when used with respect to a disorder refers to a prophylactic treatment which increases the resistance of a subject to development of the disease or, in other words, decreases the likelihood that the subject will develop the disease as well as a treatment after the subject has developed the disease in order to fight the disease, prevent the disease from becoming worse, or slow the progression of the disease compared to in the absence of the therapy.
Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic
Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock,
Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
Certain compounds as described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. The compounds provided herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. In certain embodiments, the compounds as described herein are enantiopure compounds. In certain other embodiments, mixtures of stereoisomers are provided. Furthermore, certain compounds, as described herein may have one or more double bonds that can exist as either the cis or trans, or the E or Z isomer, unless otherwise indicated. The invention additionally encompasses the compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers, e.g., racemic mixtures of E/Z isomers or mixtures enriched in one E/Z isomer.
The terms "enantiomerically enriched," "enantiomerically pure" and "non- racemic," as used interchangeably herein, refer to compositions in which the percent by weight of one enantiomer is greater than the amount of that one enantiomer in a control mixture of the racemic composition (e.g., greater than 1: 1 by weight). For example, an enantiomerically enriched preparation of the (S)-enantiomer, means a preparation of the compound having greater than 50% by weight of the (S)-enantiomer relative to the (R)- enantiomer, more preferably at least 75% by weight, and even more preferably at least 80% by weight. In some embodiments, the enrichment can be much greater than 80% by weight, providing a "substantially enantiomerically enriched," "substantially
enantiomerically pure" or a "substantially non-racemic" preparation, which refers to preparations of compositions which have at least 85% by weight of one enantiomer relative to other enantiomer, more preferably at least 90% by weight, and even more preferably at least 95% by weight. In preferred embodiments, the enantiomerically enriched composition has a higher potency with respect to therapeutic utility per unit mass than does the racemic mixture of that composition. Enantiomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred enantiomers can be prepared by asymmetric syntheses. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L.
Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).
When a range of values is listed, it is intended to encompass each value and subrange within the range. For example "Cl-6 alkyl" is intended to encompass, CI, C2, C3, C4, C5, C6, Cl-6, Cl-5, Cl-4, Cl-3, Cl-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl. As used herein, alone or as part of another group, "alkyl" refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 6 carbon atoms ("Cl-6 alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("Cl-5 alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("CI— 4 alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("Cl-3 alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("Cl-2 alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("CI alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-6 alkyl"). Examples of Cl-6 alkyl groups include methyl (CI), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") are substituted with one or more substituents. In certain embodiments, the alkyl group is an unsubstituted Cl-6 alkyl (e.g., -CH3). In certain embodiments, the alkyl group is a substituted Cl-6 alkyl.
As used herein, "alkyloxy" refers to an alkyl group, as defined herein, substituted with an oxygen atom, wherein the point of attachment is the oxygen atom. In certain embodiments, the alkyl group has 1 to 6 carbon atoms ("Cl-6 alkyloxy"). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("Cl-4 alkyloxy"). Examples of Cl-4 alkyloxy groups include methoxy (CI), ethoxy (C2), propoxy (C3), isopropoxy (C3), butoxy (C4), tert-butoxy (C5) and the like. Examples of Cl-6 alkyloxy groups include the aforementioned Cl-4 alkyloxy groups as well as pentyloxy (C5), isopentyloxy (C5), neopentyloxy (C5), hexyloxy (C6) and the like. Unless otherwise specified, each instance of the alkyl moiety of the alkyloxy group is independently unsubstituted (an "unsubstituted alkyloxy") or substituted (a "substituted alkyloxy") with one or more substituents. In certain embodiments, the alkyloxy group is an unsubstituted Cl-6 alkyloxy. In certain embodiments, the alkyloxy group is a substituted Cl-6 alkyloxy.
As used herein, "alkylcarboxy" refers to a group of the formula -C(=0)ORa or - OC(=0)Ra, wherein Ra is an alkyl group as defined herein. In certain embodiments, the alkyl of the alkylcarboxy group has 1 to 6 carbon atoms ("Cl-6 alkylcarboxy"). In some embodiments, the alkyl of the alkylcarboxy group has 1 to 5 carbon atoms ("Cl-5 alkylcarboxy"). In some embodiments, the alkyl of the alkylcarboxy group has 1 to 4 carbon atoms ("CI— 4 alkylcarboxy"). In some embodiments, the alkyl of the
alkylcarboxy group has 1 to 3 carbon atoms ("Cl-3 alkylcarboxy"). In some
embodiments, the alkyl of the alkylcarboxy group has 1 to 2 carbon atoms ("Cl-2 alkylcarboxy"). Unless otherwise specified, each instance of the alkyl of the
alkylcarboxy group is independently unsubstituted (an "unsubstituted alkylcarboxy") or substituted (a "substituted alkylcarboxy") with one or more substituents. In certain embodiments, the alkylcarboxy group is an unsubstituted Cl-6 alkylcarboxy. In certain embodiments, the alkylcarboxy group is a substituted Cl-6 alkylcarboxy.
As used herein, alone or as part of another group, "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 6 carbon atoms and one or more carbon-carbon double bonds ("C2-6 alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C2-5 alkenyl"). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C2-4 alkenyl"). In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("C2-3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4) and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6) and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C2-6 alkenyl. In certain
embodiments, the alkenyl group is a substituted C2-6 alkenyl.
As used herein, alone or as part of another group, "alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 6 carbon atoms and one or more carbon-carbon triple bonds ("C2-6 alkynyl"). In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C2-5 alkynyl"). In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C2-4 alkynyl"). In some embodiments, an alkynyl group has 2 to 3 carbon atoms ("C2-3 alkynyl"). In some embodiments, an alkynyl group has 2 carbon atom ("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
Examples of C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4) and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6) and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C2-6 alkynyl. In certain embodiments, the alkynyl group is a substituted C2-6 alkynyl.
As used herein, a "saturated or unsaturated acyclic hydrocarbon" refers to radical of a saturated or unsaturated, straight-chain or branched, hydrocarbon group having from 1 to 20 carbon atoms and optionally one or more carbon-carbon double or triple bonds. In certain embodiments, the hydrocarbon group is saturated. In some embodiments, the hydrocarbon group is unsaturated, and contains one or more carbon-carbon double or triple bonds. In some embodiments, the hydrocarbon group contains 1-10 carbon atoms. In certain embodiments, the hydrocarbon group contains 1-5 carbon atoms. In some embodiments, the hydrocarbon group contains 1-4 carbon atoms. In some embodiments, the hydrocarbon group contains 1-3 carbon atoms. In some embodiments, the hydrocarbon group contains 1-2 carbon atoms.
As used herein, "hydroxyl" or "hydroxy" refers to the group -OH.
As used herein, "oxo" refers to the group =0.
Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, referred to without the suffix "-ene," describe a monoradical of alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, respectively, and as defined herein, wherein the monoradical is attached to another group by only one single bond. Groups referred to with the suffix "-ene", such as alkylene, alkenylene, alkynylene, carbocyclylene, heterocyclylene, arylene and heteroarylene groups, describe a diradical of alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl, respectively, and as defined herein, wherein the diradical is attached to one or two groups by two single bonds.
As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(Cl-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
As used herein, the term "prodrug" means a biologically active derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide the pharmacologically active compound. In this instance, the "prodrug" is a compound administered to a subject, and the pharmacologically active compound is the "active metabolite thereof." In certain cases, a prodrug has improved physical and/or delivery properties over the parent compound. Prodrugs are typically designed to enhance pharmaceutically and/or pharmacokinetically based properties associated with the parent compound. The advantage of a prodrug can lie in its physical properties, such as enhanced water solubility for parenteral administration at
physiological pH compared to the parent compound, or it enhances absorption from the digestive tract, or it may enhance drug stability for long-term storage. Multiple doses of the molecules of the invention are also contemplated. In some instances, when the molecules of the invention are administered with another therapeutic, for instance, an anti- cancer agent a sub-therapeutic dosage of either or both of the molecules may be used. A "sub-therapeutic dose" as used herein refers to a dosage which is less than that dosage which would produce a therapeutic result in the subject if administered in the absence of the other agent.
Pharmaceutical compositions of the present invention comprise an effective amount of one or more agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Moreover, for animal (e.g. , human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. The compounds are generally suitable for administration to humans. This term requires that a compound or
composition be nontoxic and sufficiently pure so that no further manipulation of the compound or composition is needed prior to administration to humans.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. , antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The compounds may be sterile or non-sterile.
The agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally,
intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. , liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). In a particular embodiment, intraperitoneal injection is contemplated.
In any case, the composition may comprise various antioxidants to retard oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g. , methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
The agent may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g. , those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g. , glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g. , triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.
The compounds of the invention may be administered directly to a tissue. Direct tissue administration may be achieved by direct injection. The compounds may be administered once, or alternatively they may be administered in a plurality of administrations. If administered multiple times, the compounds may be administered via different routes. For example, the first (or the first few) administrations may be made directly into the affected tissue while later administrations may be systemic.
The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
According to the methods of the invention, the compound may be administered in a pharmaceutical composition. In general, a pharmaceutical composition comprises the compound of the invention and a pharmaceutically-acceptable carrier. Pharmaceutically- acceptable carriers for peptides, monoclonal antibodies, and antibody fragments are well- known to those of ordinary skill in the art. As used herein, a pharmaceutically- acceptable carrier means a non-toxic material that does not interfere with the
effectiveness of the biological activity of the active ingredients.
Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Exemplary pharmaceutically acceptable carriers for peptides in particular are described in U.S. Patent No. 5,211,657. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically- acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
The compounds of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections, and usual ways for oral, parenteral or surgical administration. The invention also embraces pharmaceutical compositions which are formulated for local administration, such as by implants. Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as a syrup, an elixir or an emulsion.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Techniques for preparing aerosol delivery systems are well known to those of skill in the art.
Generally, such systems should utilize components which will not significantly impair the biological properties of the active agent (see, for example, Sciarra and Cutie,
"Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue
experimentation .
The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g. , by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. , in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer' s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
In yet other embodiments, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International Application No. PCT/US/03307 (Publication No. WO 95/24929, entitled "Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, 1994). PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing a biological macromolecule. The polymeric matrix may be used to achieve sustained release of the agent in a subject. In accordance with one aspect of the instant invention, the agent described herein may be encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agent is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the agent include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular, pulmonary, or other surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the agents of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
In general, the agents of the invention may be delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly- vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate),
poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.
Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters,
polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compound, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non- polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the platelet reducing agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Therapeutic formulations of the peptides or antibodies or other therapeutic may be prepared for storage by mixing a peptide or antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
The peptide or other therapeutic may be administered directly to a cell or a subject, such as a human subject alone or with a suitable carrier. Alternatively, a peptide may be delivered to a cell in vitro or in vivo by delivering a nucleic acid that expresses the peptide to a cell. Various techniques may be employed for introducing nucleic acid molecules of the invention into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid molecule-calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the foregoing viruses including the nucleic acid molecule of interest, liposome-mediated transfection, and the like. For certain uses, it is preferred to target the nucleic acid molecule to particular cells. In such instances, a vehicle used for delivering a nucleic acid molecule of the invention into a cell (e.g., a retrovirus, or other virus; a liposome) can have a targeting molecule attached thereto. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid molecule delivery vehicle.
Especially preferred are monoclonal antibodies. Where liposomes are employed to deliver the nucleic acid molecules of the invention, proteins that bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake. Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acid molecules into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acid molecules.
A peptide of the invention may also be expressed directly in mammalian cells using a mammalian expression vector. Such a vector can be delivered to the cell or subject and the peptide expressed within the cell or subject. The recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid- specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters.
Developmentally-regulated promoters are also encompassed, for example the murine hox promoters and the a-fetoprotein promoter.
As used herein, a "vector" may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
The invention also includes articles, which refers to any one or collection of components. In some embodiments the articles are kits. The articles include
pharmaceutical or diagnostic grade compounds of the invention in one or more containers. The article may include instructions or labels promoting or describing the use of the compounds of the invention.
In one embodiment, a kit comprises antibodies against the starvation markers being measured in a method of the invention. The kit may further comprise assay diluents, standards, controls and/or detectable labels. The assay diluents, standards and/or controls may be optimized for a particular sample matrix.
As used herein, "promoted" includes all methods of doing business including methods of education, hospital and other clinical instruction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with compositions of the invention in connection with treatment of infections, cancer, and autoimmune disease.
"Instructions" can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.
Thus the agents described herein may, in some embodiments, be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. A kit may include one or more containers housing the components of the invention and instructions for use. Specifically, such kits may include one or more agents described herein, along with instructions describing the intended therapeutic application and the proper administration of these agents. In certain embodiments agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents. The kit may be designed to facilitate use of the methods described herein by physicians and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the invention. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflects approval by the agency of manufacture, use or sale for human administration.
The kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject. The kit may include a container housing agents described herein. The agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely. Alternatively the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.
The following examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.
EXAMPLES
Example 1: Figure 1 example shows bar graphs of FSC vs SSC dot plots for WM35 human melanoma cells treated with Etomoxir, Chloroquine, 2-deoxyglucose, and Chloroquine + Etomoxir. The dot plots demonstrate sensitivity to chloroquine and etomoxir treatment. The cells were treated with chloroquine at O.lmM, and Etomoxir at 0.5mM. The expression of LC3 is higher in the dead population of cells when treated with chloroquine and 2-deoxyglucose, but is decreased with etomoxir and the
combinations of compounds. LC3 expression is relatively lower in the treated cells when compared to the no treatment in the live population of cells. LAMP expression is increased with Chloroquine and Etomoxir in the live population. The bar graph shows the relative numbers of live and dead cells in response to the treatments.
Example 2: B16F1 mouse melanoma cells were used to conduct similar experiments to those shown in Example 1. Figure 2 is FSC vs SSC dot plots showing the relative size and granularity of the B16F1 mouse melanoma cell line. The dot plots include: no treatment control, chloroquine, chloroquine + 2-deoxyglucose, 2- deoxyglucose. The addition of chloroquine demonstrates death in the B16F1 cells as measured by FSC vs SSC. In addition the trypan blue counts show the same result as the FSC vs. SSC dot plots. There is 88% cell death in chloroquine treated cells and 89% cell death in chloroquine + 2-deoxyglucose, as compared to 25% cell death in the no treatment control. Chloroquine was treated at 0.05mM and 2-deoxyglucose was treated at 0.4mM.
B16F1 cells express a relatively large amount of LAMP and LC3 before treatment. The graphs show that the addition of the compounds chloroquine and 2- deoxyglucose decreased the expression of both LAMP and LC3.
Example 3: Similar experiments were performed on an HTB-77 cell line as were performed in Examples 1 and 2. The data is shown in Figure 3. The HTB-77 cells were treated with chloroquine at O.lmM and etomoxir at 0.5mM. Figure 3 shows the results of the FSC vs SSC dot plots, along with the percent death graph demonstrating that etomoxir and chloroquine (and the combination) cause more death in the HTB-77s when compared to the no treatment control. The MFI of LC3 in the live cell population is decreased when treated with etomoxir, chloroquine, and the combination of both compounds. The dead population of HTB-77 shows an increase in LC3 expression with the cells treated with etomoxir and chloroquine.
Example 4: Similar experiments were performed on an HTB-77 cell line as were performed in Examples 1 and 2. The data is shown in Figure 4. The HTB-77 cells were treated with chloroquine at O.lmM and etomoxir at 0.5mM. This data shows an increase in death when treated with chloroquine first, followed by etomoxir treatment. The LC3 expression on the live cells is higher when treated with etomoxir first and the LC3 expression on the dead cells is decreased when treated with chloroquine first. The
LAMP expression is increased in the live population of cells when they are treated with chloroquine first. The dead population demonstrates a relative higher expression of LAMP when treated with etomoxir first.
Example 5: Similar experiments were performed on ACHN renal carcinoma cells as were performed in the Examples above. The data is shown in Figure 5. The FSC vs SSC dot plots show ACHN renal carcinoma cells treated with Etomoxir at 0.5mM for 24 hours followed by Chloroquine at O. lmM for an additional 24 hours, and vice versa (shown in bar graph format in Figure 5). The renal cells treated with chloroquine first followed by etomoxir show more death. The LAMP expression in the dead population is higher in the cells treated with etomoxir first, then followed by chloroquine. The LC3 expression in both the dead and live cell populations is higher in the etomoxir treated first followed by chloroquine treatment cells. Example 6: T24 bladder tumor cells treated with etomoxir at 0.5mM and chloroquine at 0. ImM. The data is shown in Figure 6. These trypan blue counts indicated that the T24 cells are sensitive to chloroquine and etomoxir. These cells were treated with etomoxir, chloroquine and etomoxir + chloroquine for 24 hours, and counted at 24 hours. The counts are shown in the first 4 bars of the graph starting from the left. The last two bars show the cells that were treated with etomoxir first for 24 hours, followed by chloroquine for an additional 24 hours and vice versa. At this point, they were counted for percent cell death. The cells that were first treated with chloroquine followed by etomoxir showed an increased rate in cell death.
Example 7: WM35 Melanoma, L1210, and L1210 DDP cells were treated according to the methods described below and levels of starvation markers were analyzed. L1210 and L1210DDP cells were grown in T25 flasks at 1.0X106 cells/mL in lOmL of 5% FBS complete RPMI. Cells were treated with Rapamycin (LC
Laboratories) at a final concentration of 10μΜ. WM35 cells were grown in a 6 well plate at 1.0X106 cells/mL in 5mL of 10% FBS complete RPMI. Cells were treated with Rapamycin (LC Laboratories) at a final concentration of 10μΜ. After 24 hours, cells were harvested and counted using trypan blue on a hemocytometer. Cells were stained for the following: MitoTracker Red; LAMP-1; and LC3A/B.
MitoTracker Red
Mitochondrial membrane potential was assessed using Mitotracker Red (CM-
H2XROS, Invitrogen). The cells were resuspended in warm (37o C PBS containing a final concentration of 0.5μΜ dye. The cells were incubated for 20 minutes, pelleted, and resuspended in PBS for analysis.
LAMP-1 and LC3A/B
Cells were fist fixed using the Cytoperm/Cytofix Kit (BDBioscience) According to the manufacturer's directions. Next non-specific binding was blocked using FC:Block (BDBioscience) for 10 minutes on ice. Cells were fist stained using monoclonal antibodies to either LAMP-1 or LC3A/B (Abeam) for 20minutes on ice. Cells are then pelleted and responded in cold staining buffer (BDBioscience). Next cells were stained with their respective fluorochrome conjugated second step antibodies and incubated for 20 minutes on ice. Cells were finally pelleted and resuspended in cold staining buffer (BDBioscience) for analysis. Cells were analyzed on a BD FACS Canto II flow cytometer. Data was analyzed using FlowJo software and the raw data is shown in Figure 14 as dot plots.
The data was presented as bar graphs in Figure 4, showing the changes in LAMP and LC3 expression levels.
Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.
What is claimed is:

Claims

1. A method for selecting a course of treatment of a subject having cancer, comprising: obtaining from the subject a tumor cell, determining the expression of one or more starvation markers which are differentially expressed in cancers under distinct metabolic conditions, and selecting a course of treatment appropriate to the cancer of the subject depending on the expression of the one or more starvation markers.
2. A method for identifying a property of a tumor, comprising: selecting a tumor cell and determining whether the tumor cell expresses a starvation marker selected from the group consisting of LAMP and LC3, wherein expression of the starvation marker is indicative of an autophagic tumor cell.
3. The method of claim 1 or 2, wherein the starvation marker is LAMP1.
4. The method of claim 1 or 2, wherein the starvation marker is LAMP2.
5. The method of claim 1 or 2, wherein the starvation marker is LC3.
6. The method of claim 2, further comprising selecting a course of treatment for a subject based on the expression of the starvation marker.
7. The method of claim 1 or 6, wherein the course of treatment involves administering a starvation signal compound to the subject when the tumor cell is starvation marker negative.
8. The method of claim 1 or 6, wherein the course of treatment involves administering an autophagy inhibitor to the subject when the tumor cell is starvation marker positive.
9. A method for treating a subject, comprising: administering to a subject who has been identified as having a starvation marker negative tumor, a therapeutically acceptable amount of a starvation signal compound to treat the tumor.
10. A method for treating a subject, comprising: administering to a subject who has been identified as having a starvation marker positive tumor a therapeutically acceptable amount of an autophagy inhibitor and/or a fatty acid metabolism inhibitor.
11. The method of claim 10, further comprising determining the expression of one or more starvation markers in a tumor cell, following the autophagy inhibitor and/or a fatty acid metabolism inhibitor treatment to determine if the tumor cell is starvation marker inhibitor negative.
12. The method of claim 10 or 11, further comprising administering to the subject a therapeutically acceptable amount of a starvation signal compound to treat the tumor after the autophagy inhibitor and/or a fatty acid metabolism inhibitor treatment.
13. The method of claim 9 or 12, wherein the starvation signal compound is an anti-VEGF antagonist, optionally an anti-VEGF antibody.
14. The method of claim 9 or 12, wherein the starvation signal compound is bevacizumab.
15. The method of claim 9 or 12, wherein the starvation signal compound is rapamycin.
16. The method of claim 9 or 12, wherein the anti-cancer therapy is a glycolysis inhibitor.
17. The method of any one of claims 1-16, wherein the tumor is selected from the group consisting of a melanoma, an ovarian tumor, a glioblastoma, a breast cancer, and a renal tumor.
18. The method of any one of claims 9-17, further comprising administering a chemotherapeutic agent to the subject.
19. The method of any one of claims 8 and 10-18, wherein the autophagy inhibitor is a 4- aminoquinoline.
20. The method of claim 19, wherein the 4- aminoquinoline has the structure:
Figure imgf000075_0001
or a pharmaceutically acceptable salt or prodrug thereof;
each instance of the dotted line independently represents a single bond or a double bond which can be in the cis or trans configuration;
wherein each of R2 and R3 is independently a hydroxalkyl, an alkyl, alkyloxy, alkylcarboxy, alkylene or alkenylene having from one to six carbon atoms.
21. The method of any one of claims 8 and 10-18, wherein the autophagy inhibitor is chloroquine or hydroxychloroquine.
22. The method of any one of claims 9-21, further comprising determining the expression of one or more starvation markers in a tumor cell of the subject before the treatment is administered.
23. The method of any one of claims 1-8, 11, and 22, wherein the step of determining the expression of the starvation marker is performed by detecting the expression of starvation marker nucleic acid molecules.
24. The method of claim 23, wherein the expression of the starvation marker nucleic acid molecules is determined by a method selected from the group consisting of nucleic acid hybridization and nucleic acid amplification.
25. The method of claim 24, wherein the nucleic acid hybridization is performed using a solid-phase nucleic acid molecule array.
26. The method of claim 24, wherein the nucleic acid amplification method is real-time PCR.
27. The method of any one of claims 1-8, 11, and 22, wherein the step of determining the expression of the starvation marker is performed by detecting the expression of starvation marker peptides.
28. The method of claim 27, wherein the expression levels are determined by an immunological method.
29. The method of claim 28, wherein the immunological method is performed using a solid-phase antibody array.
30. The method of claim 28, wherein the immunological method is an ELISA or ELISPOT assay.
31. The method of claim 10 or 11, wherein the fatty acid metabolism inhibitor is an inhibitor of fatty acid oxidation, a fatty acid transporter inhibitor, a reductase inhibitor, or an isomerase inhibitor within the fatty acid metabolism pathway.
32. The method of claim 31, wherein the inhibitor of fatty acid metabolism is an inhibitor of fatty acid oxidation and is selected from the group consisting of an oxirane carboxylic acid compound, such as etomoxir (2-(6-(4-chlorophenoxy)-hexyl)-oxirane-2- carboxylic acid ethyl ester), 2-(4-(3-chlorophenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(4-(3-trifluoromethylphenoxy)-butyl)-oxirane-2-carboxylic acid ethyl ester, 2-(5(4-chlorophenoxy)-pentyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(3,4- dichlorophenoxy)-hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-(4-fluorophenoxy)- hexyl)-oxirane-2-carboxylic acid ethyl ester, 2-(6-phenoxyhexyl)-oxirane-2-carboxylic acid ethyl ester, cerulenin, 5-(tetradecyloxy)-2-furoic acid, oxfenicine, methyl palmoxirate, metoprolol, amiodarone, perhexiline, aminocamitine, hydrazonopropionic acid, 4-bromocrotonic acid, trimetazidine, ranolazine, hypoglycin, dichloroacetate, methylene cyclopropyl acetic acid, beta-hydroxy butyrate, and a non-hydrolyzable analog of carnitine or pharmacologically acceptable salts thereof.
33. The method of claim 31, wherein the inhibitor of fatty acid metabolism is an inhibitory nucleic acid.
34. The method of claim 33, wherein the inhibitory nucleic acid is specific for an enzyme selected from the group consisting of 2,4-dienoyl-CoA reductase, 2,4-dienoyl- CoA isomerase, and butyryl dehydrogenase.
35. The method of claim 31, wherein the inhibitor of fatty acid metabolism is oxamate.
36. The method of claim 31 wherein the fatty acid metabolism inhibitor is an oxirane carboxylic acid compound capable of inhibiting fatty acid metabolism, or a pharmacologically acceptable salt thereof.
37. The method of claim 36 wherein the oxirane carboxylic acid compound is etomoxir.
38. The method of claim 16, wherein the glycolytic inhibitor is a 2-deoxyglucose compound.
39. The method of claim 38 wherein the 2-deoxyglucose compound is 2-deoxy- D-glucose.
40. A method comprising
providing a starvation marker from a tumor tissue sample,
determining the expression level of the starvation marker, comparing the expression level of the starvation marker in the tissue sample with threshold value of a starvation marker, wherein a lower level of expression in the tumor cell is indicative of the tumor cells susceptibility to starvation signal compound.
41. The method of claim 40, wherein the threshold value is an expression level value of a starvation marker from a control cell.
42. The method of claim 41, wherein the control cell is a non-cancerous cell of the same type of tissue as the tumor cell.
43. The method of claim 40, wherein there is at least a 2-fold difference in mean expression levels between the at starvation marker and the threshold level.
44. A method of treating melanoma, comprising administering daily to a subject having melanoma a therapeutically effective dose of an autophagy inhibitor and administering once every 2-4 months to the subject a therapeutically effective amount of bevacizumab.
45. A method of treating ovarian cancer, comprising administering daily to a subject having ovarian cancer a therapeutically effective dose of an autophagy inhibitor and administering once every 2-4 months to the subject a therapeutically effective amount of bevacizumab.
46. A method of treating renal cancer, comprising administering daily to a subject having renal cancer a therapeutically effective dose of an autophagy inhibitor and administering once every 2-4 months to the subject a therapeutically effective amount of bevacizumab.
47. The method of any one of claims 44-46, wherein the bevacizumab is administered in a dose range of 12-16 mg/Kg IV.
48. The method of any one of claims 44-46, wherein the bevacizumab is administered in a dose of 15 mg/Kg IV.
49. A method of treating cancer, comprising administering to a subject having cancer a therapeutically effective dose of an autophagy inhibitor and administering to the subject a therapeutically effective amount of rapimycin.
50. The method of claim 49, wherein the autophagy inhibitor is administered daily.
51. The method of any one of claims 44-50, wherein the autophagy inhibitor is hydroxychloroquine .
52. The method of any one of claims 44-50, wherein the autophagy inhibitor is chloroquine.
53. The method of any one of claims 44-53, wherein the autophagy inhibitor is administered in a dose range of 100-500 mg per day.
54. The method of any one of claims 44-53, wherein the autophagy inhibitor is administered in a dose range of 200-400 mg per day.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110227159A (en) * 2018-03-05 2019-09-13 义慧科技(深圳)有限公司 The drug and CPT1a inhibitor for improving entity tumor confrontation blood vessel drug eluting drug resistance are in application wherein
CN112255334A (en) * 2020-09-28 2021-01-22 复旦大学 Small molecule marker for distinguishing junctional ovarian tumor from malignant ovarian tumor and application thereof
JP2021020926A (en) * 2014-03-06 2021-02-18 ユニヴァーシティ オブ サザン カリフォルニアＵｎｉｖｅｒｓｉｔｙ ｏｆ Ｓｏｕｔｈｅｒｎ Ｃａｌｉｆｏｒｎｉａ Use of short term starvation regimen in combination with kinase inhibitors to enhance conventional chemotherapeutics efficacy and feasibility and reverse side effects of kinases in normal cells and tissues

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010008554A2 (en) * 2008-07-14 2010-01-21 The Regents Of The University Of Colorado Methods and products for treating proliferative diseases
CN101920015A (en) * 2010-05-25 2010-12-22 中国人民解放军第二军医大学 Pharmaceutical composition for cancer treatment
WO2012007783A1 (en) * 2010-07-13 2012-01-19 Institut Gustave Roussy Kits and methods for detecting the ability to induce an immunogenic cancer cell death in a subject
US20120115153A1 (en) * 2009-04-17 2012-05-10 Cbs Bioscience, Co., Ltd. Marker for prognosis of liver cancer
WO2012064671A1 (en) * 2010-11-08 2012-05-18 The Ohio State University Research Foundation Compositions and methods for increasing drug efficacy in cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010008554A2 (en) * 2008-07-14 2010-01-21 The Regents Of The University Of Colorado Methods and products for treating proliferative diseases
US20120115153A1 (en) * 2009-04-17 2012-05-10 Cbs Bioscience, Co., Ltd. Marker for prognosis of liver cancer
CN101920015A (en) * 2010-05-25 2010-12-22 中国人民解放军第二军医大学 Pharmaceutical composition for cancer treatment
WO2012007783A1 (en) * 2010-07-13 2012-01-19 Institut Gustave Roussy Kits and methods for detecting the ability to induce an immunogenic cancer cell death in a subject
WO2012064671A1 (en) * 2010-11-08 2012-05-18 The Ohio State University Research Foundation Compositions and methods for increasing drug efficacy in cancer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUO, G.-F. ET AL.: "Autophagy-related proteins Beclin-1 and LC3 predict cetuximab efficacy in advanced colorectal cancer", WORLD JOURNAL OF GASTROENTEROLOGY, vol. 17, 2011, pages 4779 - 4786 *
MAYCOTTE, P. ET AL.: "Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy", AUTOPHAGY, vol. 8, February 2012 (2012-02-01), pages 200 - 212 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021020926A (en) * 2014-03-06 2021-02-18 ユニヴァーシティ オブ サザン カリフォルニアＵｎｉｖｅｒｓｉｔｙ ｏｆ Ｓｏｕｔｈｅｒｎ Ｃａｌｉｆｏｒｎｉａ Use of short term starvation regimen in combination with kinase inhibitors to enhance conventional chemotherapeutics efficacy and feasibility and reverse side effects of kinases in normal cells and tissues
JP7038432B2 (en) 2014-03-06 2022-03-18 ユニヴァーシティ オブ サザン カリフォルニア Use of short-term fasting regimens in combination with kinase inhibitors to enhance the efficacy and feasibility of conventional chemotherapeutic agents and to reverse the side effects of kinases in normal cells and tissues.
CN110227159A (en) * 2018-03-05 2019-09-13 义慧科技(深圳)有限公司 The drug and CPT1a inhibitor for improving entity tumor confrontation blood vessel drug eluting drug resistance are in application wherein
CN110227159B (en) * 2018-03-05 2021-09-21 义慧科技(深圳)有限公司 Medicine for improving drug resistance of solid tumor to anti-vascular drugs and application of CPT1a inhibitor in medicine
CN112255334A (en) * 2020-09-28 2021-01-22 复旦大学 Small molecule marker for distinguishing junctional ovarian tumor from malignant ovarian tumor and application thereof

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