WO2006012419A2 - Inhibition de nf-kb - Google Patents

Inhibition de nf-kb Download PDF

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Publication number
WO2006012419A2
WO2006012419A2 PCT/US2005/025884 US2005025884W WO2006012419A2 WO 2006012419 A2 WO2006012419 A2 WO 2006012419A2 US 2005025884 W US2005025884 W US 2005025884W WO 2006012419 A2 WO2006012419 A2 WO 2006012419A2
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cells
cell
activity
cancer
aminoacridines
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PCT/US2005/025884
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English (en)
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WO2006012419A3 (fr
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Andrei V. Gudkov
Katerina V. Gurova
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Cleveland Clinic Foundation
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Priority to EP05791579A priority Critical patent/EP1771203A4/fr
Priority to JP2007522754A priority patent/JP2008507545A/ja
Priority to AU2005267117A priority patent/AU2005267117C1/en
Publication of WO2006012419A2 publication Critical patent/WO2006012419A2/fr
Priority to US11/624,828 priority patent/US20070270455A1/en
Publication of WO2006012419A3 publication Critical patent/WO2006012419A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • the present invention is generally related to the modulation of cell growth or apoptosis. More specifically, the present invention is related to compositions for modulating cell growth or apoptosis, methods of use thereof, and methods of identification thereof.
  • p53 controls genetic stability and reduces the risk of cancer through induction of growth arrest or apoptosis in response to DNA damage or deregulation of proto-oncogenes.
  • the efficacy of p53 as a tumor-preventing factor is reflected by the frequency of p53 loss in at least 50% of human tumors due to inactivating mutations.
  • Mdm2 is considered an attractive target for suppression by small molecules or other approaches in order to selectively kill tumor cells by restoring p53 function.
  • Renal cell carcinomas maintain wild type but functionally inactive p53.
  • the mechanism of p53 repression in RCC is dominant, which indicates the existence of a so far unknown molecular target for restoration of p53 function in cancer.
  • a condition associated with NF- ⁇ B activity may be treated by administering to a patient in need thereof a composition comprising an inhibitor of NF- ⁇ B.
  • the NF- ⁇ B activity may be constitutive, induced or at a basal level.
  • the inhibition of NF- ⁇ B may activate p53.
  • the inhibition of NF- ⁇ B may activate functionally silent p53.
  • the condition treated may be cancer, inflammation, autoimmune disease, graft versus host disease, a condition associated with HIV infection, or pre-cancerous cells which have acquired dependence on constitutively active NF- KB.
  • NF- ⁇ B may be an aminoacridine of the formula:
  • Ri is H or halogen
  • R 2 is H or optionally substituted alkoxy
  • R 3 is H or optionally substituted alkoxy
  • R 4 is H or optionally substituted aliphatic, aryl, or heterocycle.
  • the aminoacridine may be 9-aminoacridine or quinacrine.
  • the composition may further comprise an activator of a death receptor of a TNF family polypeptide.
  • the activator may be a TNF polypeptide, such as NGF, CD40L, CD137L/4-1BBL, TNF- ⁇ , CD134L/OX40L, CD27L/CD70, FasL/CD95, CD30L, TNF- ⁇ /LT- ⁇ , LT- ⁇ , or TRAIL.
  • An agent that modulates functionally silent p53 may be identified by adding a candidate agent to a cell comprising a p53-responsive reporter and measuring the level of signal of the p53- responsive reporter. The agent may be identified by a difference in the signal compared to a control. The agent may increase or decrease the activity of p53. The cell may comprise a functionally silent p53.
  • An agent that modulates NF- ⁇ B may be identified by adding a candidate agent to a cell comprising a p53 -responsive reporter and measuring the level of signal of the p53-responsive reporter. The agent may be identified by a difference in the signal compared to a control. The agent may increase or decrease the activity of NF- ⁇ B.
  • the cell may comprise a functionally silent p53.
  • the cell may also comprise an NF- ⁇ B transactivation complex.
  • Figure 1 indicates that the restoration of p53 -mediated transactivation in RCC cells is accompanied by death of RCC cells.
  • Figure IA p53-responsive reporter activity in RCC45ConALacZ cells transduced with different concentration of p53 or GFP expressing lentiviral vectors, ⁇ -galactosidase activity (ONPG staining) was measured 48 hours after lentiviral transduction and normalized by protein concentration.
  • Figure IB Cell survival was measured at 96 hours after lentiviral transduction by methylene blue staining and presented as a percentage of intensity of methylene blue staining of cell transduced with p53 -virus to the same cells transduced with the same concentration of GFP-virus.
  • Figure 2 indicates the p53 restoration activity of agents of the formula of compound 1.
  • Figure 2A Choice of readout cells and setting of selection criterion. MCF7, ACHN, RCC26b and RCC45 cells all containing ConALacZ reporter were plated into 96 well plates and incubated in the medium containing different concentrations of doxorubicin for 24 hours. Then ⁇ -galactosidase activity was measured by ONPG staining and normalized by protein concentration.
  • Figure 2B indicates that 9AA causes the strongest activation of p53 -dependent reporter in RCC45 cells.
  • Agents of the formula of compound 1 were tested in dose dependent assay on p53 transactivation in RCC45ConALacZ cells. Bars represent relative activity of each compound calculated as a fold of p53 activation, induced by a compound, over the effect of 2 ⁇ M of doxorubicin (results of three experiments).
  • Figure 3 indicates that 9AA induces p53 transcriptional activity in different tumor cells.
  • Figure 3A indicates that 9AA induces p53 -responsive reporters in a dose-dependent manner.
  • RCC45ConALacZ and MCF7ConALacZ cells were incubated in media containing indicated concentrations of doxorubicin (dox) or 9AA (all concentrations are presented in ⁇ M) for 24 hours and then ⁇ -galactosidase activity was measured by ONPG staining, normalized by protein content and presented as a fold of reporter induction compared with untreated cells.
  • Figure 3B indicates that 9AA induces expression of endogenous p53 -dependent targets.
  • FIG. 3D Data presented as a fold of reporter induction over untreated controls by the most effective dose of 9AA over effect of dox.
  • Figure 3D indicates that 9AA induces ⁇ -galactosidase activity in a p53-dependent manner.
  • HT1080ConALuC transduced with anti- p53 or anti-GFP siRNA expressing constructs were treated with 5 ⁇ M of 9AA for 24 hours.
  • Bars represent folds of p53 responsive reporter induction over untreated controls.
  • Box represents western blot analysis of p53 expression in total protein lysates of both cells variants in basal and doxorubicin treated conditions (to evaluate basal and DNA-damage induced level of p53).
  • Figure 3E shows a dose-response curve of p53-responsive reporter activity in HT1080ConALuc cells treated with 9AA for 24 hours (no normalization for protein concentration was done). Fold induction presented as fold of reporter activation over untreated control.
  • Figure 3F shows the time dependence of the p53-inducing effect of 9AA. HT1080ConALuC cells were treated for 1 hour with 20 ⁇ M of 9AA and then ⁇ -galactosidase activity was measured at the indicated time points. Fold induction presented as fold of reporter activation over untreated control. [0013]
  • Figure 4 indicates that 9AA-associated cytotoxicity is p53 -dependent.
  • Figure 4A shows the survival of HT1080-sip53 and HT1080-siGFP cells treated with indicated concentrations of 9AA (see Material and Methods) with results presented as a percentage of cells compared with an untreated control.
  • Figure 4B shows other pairs of cells with different levels of p53 tested in the manner described for Figure 4A.
  • the upper panel shows a western blot analysis of p53 protein level in the generated pairs of cells.
  • the lower panel shows the relative number of cells after treatment with 9aa treatment (2 ⁇ M) compared to an untreated control (100%).
  • Figure 4C shows a cell cycle analysis of HT1080 sip53 or HT1080 siGFP cells treated with 3 or 20 ⁇ M of 9AA during the indicated periods of time or 2 ⁇ M of dox during 24 hours.
  • Figure 4D shows the p53 -dependence of the cytotoxicity of different drugs.
  • the same experiment as described in Figure 4A was done using 30d9 (primary hit, analogue of 9aa, 1-10 ⁇ M) doxorubicin (dox, 0.1-1 ⁇ M), campothecin (camp, 0.16-1.6 ⁇ M), vinblastin (vinbl, 0.1-1 ⁇ M) and taxol (tax, 0.06-0.6 ⁇ M). Bars are plotted for the dose of drugs, demonstrating the highest difference in sensitivity between p53 "plus” and "minus” cells.
  • Figure 4E indicates that 9AA is more toxic for RCC cells than for normal kidney epithelial cells (NKE).
  • Figure 4F indicates that 9AA is more toxic for RCC cells at low concentrations.
  • Several cell types (NKE, - normal kidney epithelial cells, RCC45, ACHN - RCC cell lines, HCTl 16 - colon carcinoma, p53 wild type, SK-N-SH - neuroblastoma, p53 wild type, LNCaP prostate adenocarcinoma, p53 wild type, DU145, PC3 - prostate adenocarcinoma, p53 deficient, Mel7, Mel29 melanomas, 041 - fibroblasts from patient with Li-Fraumeni syndrome, p53-null, WI38 - normal human diploid fibroblasts) were treated with 2 ⁇ M of 9AA as described in Figure 4A and cell survival was compared with the corresponding untreated cells.
  • Figure 5 shows that agents of the formula of compound 1 are toxic for tumor cells with active p53.
  • Figure 5A shows the p53 inducing effects of agents in vivo.
  • HT1080ConALuC cells were inoculated into two flanks of nude mice. When tumors reach 5mm in diameter, mice were injected intraperitoneally with indicated concentrations of the drugs (mg/kg, 3 mice per group). After 24 hours mice were sacrificed, tumors were isolated, lysed in Reporter Lysis Reagent (Promega) and luciferase activity was measured in 10 mg of tumor proteins. Bars represent fold of induction of luciferase activity in tumors, treated with drugs over luciferase activity in tumors treated with vehicle.
  • FIG. 5B HT1080sip53 or HT1080siGFP cells were inoculated in the left and right flank of nude mice respectively. When tumors reached 5mm in diameter, mice were injected intraperitoneally with vehicle (50%DMSO in PBS), quinacrine (QC, 50mg/kg) and 5- fluorouracil (5FU, 35 mg/kg) every 24 hours (5 mice per group). Results are presented as medians of relative tumor volume for each tumor comparing with the volume of tumor in the first day of treatment.
  • vehicle 50%DMSO in PBS
  • quinacrine QC, 50mg/kg
  • 5- fluorouracil 5FU, 35 mg/kg
  • Figure 6 shows the testing of the potential mechanism of 9AA activity.
  • Figure 6A shows the measurement of DNA - topoisomerase II complex formation in cells treated with 9-AA.
  • Figure 6B shows the p53 phosphorylation status in RCC45 cells treated with 9AA (5 ⁇ M) or dox (1 ⁇ M) for 16 hours.
  • Western blot analysis of total protein lysates was performed using antibodies against p53 (DOl) and against specific sites of phosphorylation in p53.
  • Figure 6C shows the effect of 9AA on proteasome activity.
  • HCTl 16 cells were treated for 30 minutes with 1 ⁇ M PS-341. PS-341 was washed off after 30 minutes and cells were re-incubated in drug free medium prior to analysis at 3 hours or 16 hours post-treatment.
  • Figure 7 shows the effects of 9AA on the NF- ⁇ B pathway.
  • Figure 7A shows that 9AA inhibits NF- ⁇ B-dependent transcription.
  • H1299-NF-kBLuc cells were treated with different concentrations of 9 AA and quinacrine (QC) two hours before (TNF after 9 AA or QC) or simultaneously (TNF and 9AA or QC) with TNFa (10 ng/ml). 6 hours after addition of TNFa luciferase activity was measured in cell lysates.
  • Figure 7B shows that 9AA inhibits reconstitution of IKB levels stimulated by TNF.
  • HT 1080 cells were treated with TNF (10 ng/ml) in the presence or absence of 9AA (10 ⁇ M).
  • FIG. 7C H1299-NF- kBLuc cells were treated with indicated concentrations of 9AA and TNF (10 ng/ml). After 6 hours, cytoplasmic and nuclear extracts were isolated and used for luciferase or gel-shift assay, respectively.
  • Figure 7D shows that 9AA causes accumulation of p65/p50 and p50/p50 NF- ⁇ B complexes.
  • Gel-shift assays were performed with nuclear extracts of H 1299 cells, treated with 9AA (10 ⁇ M) and TNF (10 ng/ml) for 30 minutes.
  • Figure 7E shows that 9AA retards exit of p65 complexes from the nuclei.
  • Figure 7G shows that 9AA causes increase in p50 protein level.
  • Figure 7H shows that 9AA does not inhibit NF- ⁇ B transactivation induced by trichostatin A (TSA).
  • TSA trichostatin A
  • H 1299 cells with integrated NF- ⁇ B-dependent luciferase reporter were treated with 100 nM of TSA in the presence or absence of 9AA (20 niM) for 4 or 16 hours.
  • TNFa treatment was used as a control of reporter activity.
  • Figure 8 shows that 9AA activates p53-dependent transcription through inhibition of NF- KB.
  • Figure 8A shows that IKB SuperSupressor (ss) activates p53 in RCC cells.
  • ACHN cells were cotransfected with p21-ConALuc and indicated plasmids, containing IkB SuperSuppressor, p53 and Arf cDNA or anti-Hdm2 siRNA. Forty-eight hours later, luciferase activity was measured in cell lysates. Normalization was done by cotransfection of the pCMV-LacZ plasmid.
  • Figure 8B shows that IKB SuperSupressor inhibits NF- ⁇ B transcriptional activity.
  • ACHN cells were cotransfected with the NF- ⁇ B-responsive reporter pNF- ⁇ BLuc and IKB Super Suppressor (SS). Forty-eight hours later luciferase activity was measured in cell lysates.
  • Figure 8C shows that 9AA cannot activate p53 in cells with inhibited NF- ⁇ B.
  • ACHN cells were cotransfected with either pConALuc or pNF- ⁇ BLuc and IKB Super Supressor (SS) or empty vector. Twenty-four hours post-transfection all cells were split and treated with 9AA (10 ⁇ M). NF- ⁇ B reporter activity was measured 6 hours post-treatment and p53 responsive reporter activity was measured 24 hours after treatment. Normalization was done by cotransfection of pCMV-LacZ plasmid (for cells, transfected by different set of plasmids) and by protein concentrations (for 9AA treated and untreated cells).
  • Figure 9 shows the synergistic effect of 9-aminoacridine with death ligands.
  • Figure 10 shows that 9AA and QC are anti-RCC agents.
  • Fig 1OA shows a comparison of IC50% doses of 9AA, QC and several anti-cancer agents of different RCC and non-RCC cells. IC50% for each cell line and each drug was determined.
  • Each point represents IC50% of particular cell line, which are grouped as follows: (i) black circles - RCC cell lines (ACHN, RCC9, RCC13, RCC29, RCC45, RCC54), (ii) red triangles - non-RCC cell lines (MCF7, HT1080, H1299, U20S, LNCaP, HCTl 16), (iii) green squares - normal kidney cells (NKE).
  • Fig 1OB shows that quinacrine activates p53 -responsive reporter in ex vivo-cultured RCC tumors. X-gal staining of tumor and normal kidney pieces transduced with p53-responsive reporter lentivirus ex vivo and treated with quinacrine or doxorubicin.
  • Fig 1OC shows that quinacrine sensitizes RCC45 and RCC54 but not normal kidney epithelium (NKE) to TRAIL.
  • Cells pleated in 96-well plates were incubated 24 hours in the presence of indicated concentrations of TRAIL and quinacrine; cell numbers were estimated using methylene blue assay.
  • Fig 1OD shows the anti-tumor activity of quinacrine (QC).
  • 10 7 of ACHN cells were inoculated under the skin of nude mice. At the moment tumors achieved 5mm in diameter QC administrations were started intraperitoneally, 50mg/kg. 5FU (35mg/kg) was used as a control. Tumor size was measured every other day and presented as a fold increase in tumor volume.
  • branched refers to a group containing from 1 to 24 backbone atoms wherein the backbone chain of the group contains one or more subordinate branches from the main chain.
  • Preferred branched groups herein contain from 1 to 12 backbone atoms.
  • branched groups include, but are not limited to, isobutyl, t-butyl, isopropyl,
  • unbranched refers to a group containing from 1 to 24 backbone atoms wherein the backbone chain of the group extends in a direct line.
  • Preferred unbranched groups herein contain from 1 to 12 backbone atoms.
  • cyclic or "cyclo” as used herein alone or in combination refers to a group having one or more closed rings, whether unsaturated or saturated, possessing rings of from 3 to
  • backbone atoms preferably 3 to 7 backbone atoms.
  • the term "lower” as used herein refers to a group with 1 to 6 backbone atoms.
  • saturated refers to a group where all available valence bonds of the backbone atoms are attached to other atoms. Representative examples of saturated groups include, but are not limited to, butyl, cyclohexyl, piperidine and the like.
  • aliphatic refers to an unbranched, branched or cyclic hydrocarbon group, which may be substituted or unsubstituted, and which may be saturated or unsaturated, but which is not aromatic.
  • aliphatic further includes aliphatic groups, which comprise oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
  • aromatic refers to an unsaturated cyclic hydrocarbon group having 4n+2 delocalized ⁇ (pi) electrons, which may be substituted or unsubstituted.
  • aromatic further includes aromatic groups, which comprise a nitrogen atom replacing one or more carbons of the hydrocarbon backbone. Examples of aromatic groups include, but are not limited to, phenyl, naphthyl, thienyl, furanyl, pyridinyl, (is)oxazoyl and the like.
  • substituted refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group.
  • Preferred substituted groups herein are substituted with one to five, most preferably one to three substituents.
  • An atom with two substituents is denoted with "di,” whereas an atom with more than two substituents is denoted by "poly.”
  • substituents include, but are not limited to aliphatic groups, aromatic groups, alkyl, alkenyl, alkynyl, aryl, alkoxy, halo, aryloxy, carbonyl, acryl, cyano, amino, nitro, phosphate-containing groups, sulfur- containing groups, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, acylamino, amidino, imino, alkyl
  • alkyl refers to a branched or unbranched, saturated aliphatic group.
  • Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.
  • alkenyl refers to a branched or unbranched, unsaturated aliphatic group containing at least one carbon-carbon double bond which may occur at any stable point along the chain.
  • alkenyl groups include, but are not limited to, ethenyl, E- and Z-pentenyl, decenyl and the like.
  • alkynyl refers to a branched or unbranched, unsaturated aliphatic group containing at least one carbon-carbon triple bond which may occur at any stable point along the chain.
  • alkynyl groups include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, hexynyl, decynyl and the like.
  • aryl refers to a substituted or unsubstituted aromatic group, which may be optionally fused to other aromatic or non-aromatic cyclic groups.
  • Representative examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, benzylidine, xylyl, styrene, styryl, phenethyl, phenylene, benzenetriyl and the like.
  • alkoxy refers to an alkyl, alkenyl or alkynyl group bound through a single terminal ether linkage.
  • alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, 2-butoxy, tert- butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3- hexoxy, 3-methylpentoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, and trichloromethoxy.
  • aryloxy refers to an aryl group bound through a single terminal ether linkage.
  • halogen refers to fluorine "F”, chlorine “Cl”, bromine “Br”, iodine "I", and astatine "At”.
  • Representative examples of halo groups include, but are not limited to, chloroacetamido, bromoacetamido, idoacetamido and the like.
  • hetero refers to a group that includes one or more atoms of any element other than carbon or hydrogen.
  • Representative examples of hetero groups include, but are not limited to, those groups that contain heteroatoms including, but not limited to, nitrogen, oxygen, sulfur and phosphorus.
  • heterocycle refers to a cyclic group containing a heteroatom.
  • heterocycles include, but are not limited to, pyridine, piperadine, pyrimidine, pyridazine, piperazine, pyrrole, pyrrolidinone, pyrrolidine, morpholine, thiomorpholine, indole, isoindole, imidazole, triazole, tetrazole, furan, benzofuran, dibenzofuran, thiophene, thiazole, benzothiazole, benzoxazole, benzothiophene, quinoline, isoquinoline, azapine, naphthopyran, furanobenzopyranone and the like.
  • carbonyl or “carboxy” as used herein alone or in combination refers to a group that contains a carbon-oxygen double bond.
  • groups which contain a carbonyl include, but are not limited to, aldehydes (i.e., formyls), ketones (i.e., acyls), carboxylic acids (i.e., carboxyls), amides (i.e., amidos), imides (i.e., imidos), esters, anhydrides and the like.
  • CH 2 C(Q)C(O)O- where Q is an aliphatic or aromatic group.
  • cyano refers to a carbon-nitrogren double bond.
  • Representative examples of cyano groups include, but are not limited to, isocyanate, isothiocyanate and the like.
  • amino refers to a group containing a backbone nitrogen atom.
  • Representative examples of amino groups include, but are not limited to, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido and the like.
  • phosphate-containing group refers to a group containing at least one phosphorous atom in an oxidized state.
  • Representative examples include, but are not limited to, phosphonic acids, phosphinic acids, phosphate esters, phosphinidenes, phosphinos, phosphinyls, phosphinylidenes, phosphos, phosphonos, phosphoranyls, phosphoranylidenes, phosphorosos and the like.
  • sulfur-containing group refers to a group containing a sulfur atom. Representative examples include, but are not limited to, sulfhydryls, sulfenos, sulfinos, sulfinyls, sulfos, sulfonyls, thios, thioxos and the like.
  • optionally substituted alkyl means that the alkyl group may or may not be substituted and that the description includes both unsubstituted alkyl and alkyl where there is a substitution.
  • the term "effective amount,” when used in reference to a compound, product, or composition as provided herein, means a sufficient amount of the compound, product or composition to provide the desired result.
  • the exact amount required will vary depending on the particular compound, product or composition used, its mode of administration and the like. Thus, it is not always possible to specify an exact "effective amount.” However, an appropriate effective amount may be determined by one of ordinary skill in the art informed by the instant disclosure using only routine experimentation.
  • suitable refers to a group that is compatible with the compounds, products, or compositions as provided herein for the stated purpose. Suitability for the stated purpose may be determined by one of ordinary skill in the art using only routine experimentation.
  • administer when used to describe the dosage of a compound, means a single dose or multiple doses of the compound.
  • apoptosis refers to a form of cell death that includes progressive contraction of cell volume with the preservation of the integrity of cytoplasmic organelles; condensation of chromatin (i.e., nuclear condensation), as viewed by light or electron microscopy; and/or DNA cleavage into nucleosome-sized fragments, as determined by centrifuged sedimentation assays.
  • Cell death occurs when the membrane integrity of the cell is lost (e.g., membrane blebbing) with engulfment of intact cell fragments ("apoptotic bodies") by phagocytic cells.
  • the term "cancer” means any condition characterized by resistance to apoptotic stimuli.
  • cancer treatment means any treatment for cancer known in the art including, but not limited to, chemotherapy and radiation therapy.
  • the term "combination with” when used to describe administration of an aminoacridine and an additional treatment means that the aminoacridine may be administered prior to, together with, or after the additional treatment, or a combination thereof.
  • the term "treat” or “treating” when referring to protection of a mammal from a condition means preventing, suppressing, repressing, or eliminating the condition.
  • Preventing the condition involves treating the mammal prior to onset of the condition.
  • Suppressing the condition involves treating the mammal after induction of the condition but before its clinical appearance.
  • Repressing the condition involves treating the mammal after clinical appearance of the condition such that the condition is reduced or maintained.
  • Elimination the condition involves treating the mammal after clinical appearance of the condition such that the mammal no longer suffers the condition.
  • tumor cell means any cell characterized by resistance to apoptotic stimuli.
  • the present invention is related to the discovery that p53 may be activated in those cancer cells that have functionally blocked p53 by inhibiting NF- ⁇ B activity. Inactivation of p53 pathway in tumors is a much broader phenomenon than p53 mutations. Even if a tumor maintains wild type p53, its function is usually either completely or partially lost. These cases are especially interesting from the therapeutic standpoint since p53 in such cancers can be viewed as a target for a pharmacological reactivation. There are some types of tumors in which p53 activity is blocked by tissue-specific mechanisms.
  • Hdm2 overexpression is especially frequent in sarcomas, while E6 of human papilloma virus inactivates p53 in the majority of cervical carcinomas.
  • RCC provides another example of that kind of tumor, which is especially interesting for the analysis since wild type p53 in RCC, as we recently reported, is repressed by an unknown dominant mechanism that is likely to be tissue specific.
  • p53 reactivation seems to be an attractive strategy for treatment of this, so far, incurable form of cancer as well as other cancers with similar mechanisms for inactivating p53.
  • NF- ⁇ B activity is linked with the suppression of apoptosis in vitro and in vivo.
  • NF- ⁇ B NF- ⁇ B Activation of NF- ⁇ B in tumor cells presumably contributes to their malignant phenotype by providing resistance to both natural (e.g., TNF, Fas or TRAIL) and pharmacological (chemotherapeutic drugs) death stimuli. While constitutively active NF- ⁇ B has been described in many tumor types, the connection between activation of NFKB and the inhibition of p53 has failed to be fully appreciated.
  • Cancers such as those with functional or wild-type p53, may be treated by inhibiting NF- ⁇ B activity, which may lead to restoration of wild-type p53 activity and its activation.
  • Inhibitors of NF- ⁇ B activity may also be used to sensitize cancers to p53-dependent and p53- independent apoptosis by treatments such as chemotherapeutics, radiotherapy or natural death ligands, such as TNF polypeptides. Regardless of their p53 status, the majority of human cancers have constitutively hyperactivated NF- ⁇ B.
  • inhibitors of NF- ⁇ B may be used for treatment of any tumor regardless of their p53 status due to the reprogramming of transactivation NF- ⁇ B complexes into transrepression complexes.
  • Aminoacridines are representative examples of agents which may be used to inhibit NF- KB activity.
  • the aminoacridine may be of the following formula:
  • Ri is H or halogen
  • R 2 is H or optionally substituted alkoxy
  • R 3 is H or optionally substituted alkoxy
  • R4 is H or optionally substituted aliphatic, aryl, or heterocycle.
  • aminoacridines include, but are not limited to, 9- aminoacridine or Mepacrine, which is otherwise known as Quinacrine, as well as those aminoacridines described in Example 2. The use of aminoacridines to sensitize tumor cells is attractive because many aminoacridines have limited side effects.
  • 9AA has been used as therapeutic agent since 1942. Certain 9AA derivatives have been believed to be intercalating capable of DNA damaging activity; however, we found that 9AA and quinacrine did not show DNA damaging activity. Both 9aa and quinacrine were found to be more toxic to tumor than to normal cells in vitro and in vivo. Moreover, both compounds were shown to be capable of p53 activation and p53-dependent killing of a variety of tumor cell types, besides RCC. p53 dependence of their anti-tumor activity clearly distinguishes the aminoacridines from conventional chemotherapeutic drugs based on their targeting of tumors with wild type or functional p53.
  • Aminoacridines do not fit any known category of p53 activating agents. Although they may cause accumulation of p53, they do not induce p53 phosphorylation, unlike DNA damaging drugs. Moreover, aminoacridines do not cause DNA damage. Instead, the primary effect of aminoacridines appeared to be not p53 activation but repression of NF- ⁇ B, which later leads to p53 induction. Importantly, inhibition of NF- ⁇ B activates p53 function in a cell in which it cannot be "waked up" by any of the direct approaches to p53 activation, including introduction of Arf, knockdown of Hdm2 or ectopic overexpression of p53.
  • Inhibition of NF- ⁇ B is usually achieved through stabilization of the main negative regulator of NF- ⁇ B, IKB. Genetically, it can be done by mutating regulatory phosphorylation sites of this protein and pharmacologically - through inhibition of upstream kinases leading to a block of IKB phosphorylation. Many known chemical inhibitors of NF- ⁇ B act through this mechanisms. Stabilization of IKB results in cytoplasmic sequestration and functional inactivation of NF- ⁇ B complexes as transcription factors. [0064] The activity of aminoacridines may be superior to previous drugs since they promotes strong accumulation of NF- ⁇ B complexes in the nuclei in response to activating stimuli accompanied with a complete repression of transactivation.
  • aminoacridines may inhibit NF- ⁇ B by a mechanism acting downstream of IKB and involving conversion of NF- ⁇ B into an inactive complex.
  • the lack of NF- ⁇ B-dependent transcription may lead to the depletion of the pool of IKB (that is a direct transcription target of NF- ⁇ B) and retention of NF- ⁇ B in the nucleus due to the lack of nuclear export, normally exerted by IKB.
  • the knockout of any of the cellular factors involved in NF- ⁇ B activation IKK ⁇ , IKK ⁇ , TBKl, PKC-zeta
  • the aminoacridines may be effective not only against the IKB phosphorylation arm of NF- ⁇ B signaling ("canonical" NF- ⁇ B activation pathway), but also through alternative mechanisms of NF- ⁇ B activation. This is supported by the ability of aminoacridines, such as 9AA, to block stimulated NF- ⁇ B activity and also effectively reduce basal levels of constitutive NF- ⁇ B activity in tumor cells. By contrast, IKK2 inhibitors are only able to block stimulated NF- ⁇ B activity. 4. Compositions
  • the present invention relates to a composition comprising an aminoacridine and optionally a chemotherapeutic.
  • the present invention also relates to a composition comprising an aminoacridine and optionally a TNF polypeptide. a. Chemotherapeutic
  • the chemotherapeutic may be any pharmacological agent or compound that induces apoptosis.
  • the pharmacological agent or compound may be, for example, a small orgnanic molecule, peptide, polypeptide, nucleic acid, or antibody.
  • the chemotherapeutic may be a cytotoxic agent or cytostatic agent, or combination thereof.
  • Cytotoxic agents prevent cancer cells from multiplying by: (1) interfering with the cell's ability to replicate DNA and (2) inducing cell death and/or apoptosis in the cancer cells.
  • Cytostatic agents act via modulating, interfering or inhibiting the processes of cellular signal transduction which regulate cell proliferation and sometimes at low continuous levels.
  • Classes of compounds that may be used as cytotoxic agents include the following: alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard, chlormethine, cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide; antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphat
  • proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
  • Microtubule affecting agents interfere with cellular mitosis and are well known in the art for their cytotoxic activity.
  • Microtubule affecting agents useful in the invention include, but are not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives (e.g., NSC 608832), thiocolchicine NSC 361792), trityl cysteine (NSC 83265), vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574), natural and synthetic epothilones including but not limited to
  • cytotoxic agents such as epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes such as cis-platin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; and haematopoietic growth factors.
  • Cytostatic agents that may be used include, but are not limited to, hormones and steroids (including synthetic analogs): 17. alpha.
  • cytostatic agents are antiangiogenics such as matrix metalloproteinase inhibitors, and other VEGF inhibitors, such as anti-VEGF antibodies and small molecules such as ZD6474 and SU6668 are also included.
  • VEGF inhibitors such as anti-VEGF antibodies and small molecules such as ZD6474 and SU6668 are also included.
  • Anti-Her2 antibodies from Genetech may also be utilized.
  • a suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Also included are Imclone antibody C225 immunospecific for the EGFR, and src inhibitors.
  • cytostatic agent also suitable for use as an cytostatic agent is Casodex® (bicalutamide, Astra Zeneca) which renders androgen-dependent carcinomas non-proliferative.
  • Casodex® (bicalutamide, Astra Zeneca) which renders androgen-dependent carcinomas non-proliferative.
  • antiestrogen Tamoxifen® which inhibits the proliferation or growth of estrogen dependent breast cancer.
  • Inhibitors of the transduction of cellular proliferative signals are cytostatic agents. Representative examples include epidermal growth factor inhibitors, Her-2 inhibitors, MEK-I kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Src kinase inhibitors, and PDGF inhibitors.
  • TNF Polypeptides include epidermal growth factor inhibitors, Her-2 inhibitors, MEK-I kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Src kina
  • the TNF polypeptide may be a member of the TNF superfamily of ligands.
  • Representative examples of TNF polypeptides include, but are not limited to, NGF, CD40L, CD137L/4-1BBL, TNF- ⁇ , CD134L/OX40L, CD27L/CD70, FasL/CD95, CD30L, TNF- ⁇ /LT- ⁇ , LT- ⁇ , and TRAIL.
  • TNF polypeptide may be TRAIL, which induces apoptosis mainly in tumor but not in normal cells.
  • TNF-R receptors bound by the TNF polypeptides include, but are not limited to, LNGFR/p75, CD40, CD137/4-1BB/ILA, TNFRI/p55/CD120a, TNFRII/p75/CD120b, CD134/OX40/ACT35, CD27, Fas/CD95/APO-l, CD30/K.-1, LT- ⁇ R, DR3, DR4, DR5, DcRl/TRID, TR2, GITR and osteoprotegerin.
  • TNF family members Due to their unique ability to induce apoptosis in tumor cells, TNF family members are considered to be potential anticancer pharmaceuticals. However, many tumor cells escape pro- apoptotic action of death ligands, thereby reducing the use of these agents to death ligand- sensitive cancers and allowing the tumor to escape host immune response.
  • the use of an inhibitor of NF-kB may be used to sensitize tumor cells to the killing of a death ligand, such as a TNF polypeptide.
  • TNF polypeptides may be used in the place of the TNF polypeptide.
  • an antibody may be used that mimics the activity of a TNF polypeptide.
  • Representative examples of such antibodies include, but are not limited to, an agonist antibody to FAS, TRAIL receptor or TNFR.
  • aptamers and other synthetic ligands capable to activate the corresponding receptors may be used. c. Salts
  • compositions may be useful in various pharmaceutically acceptable salt forms.
  • pharmaceutically acceptable salt refers to those salt forms which would be apparent to the pharmaceutical chemist, i.e., those which are substantially non ⁇ toxic and which provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion. Other factors, more practical in nature, which are also important in the selection, are cost of the raw materials, ease of crystallization, yield, stability, hygroscopicity and flowability of the resulting bulk drug.
  • pharmaceutical compositions may be prepared from the active ingredients or their pharmaceutically acceptable salts in combination with pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable salts of the active agents include, but are not limited to, salts formed with a variety of organic and inorganic acids such as hydrogen chloride, hydroxymethane sulfonic acid, hydrogen bromide, methanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid, sulfamic acid, glycolic acid, stearic acid, lactic acid, malic acid, pamoic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethonic acid, and include various other pharmaceutically acceptable salts, such as, e.g., nitrates, phosphates, borates, tartrates, citrates, succinates, benzoates
  • compositions such as quaternary ammonium ions are contemplated as pharmaceutically acceptable counterions for anionic moieties.
  • pharmaceutically acceptable salts of the compounds of the present invention may be formed with alkali metals such as sodium, potassium and lithium; alkaline earth metals such as calcium and magnesium; organic bases such as dicyclohexylamine, tributylamine, and pyridine; and amino acids such as arginine, lysine and the like.
  • the pharmaceutically acceptable salts may be synthesized by conventional chemical methods. Generally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid or base, in a suitable solvent or solvent combination.
  • the counterions of the salts may be determined by the reactants used to synthesized the compounds. There may be a mixture of counterions of the salts, depending on the reactants. For example, where NaI is added to facilitate the reaction the counterion may be a mixture of Cl and I counter anions. Furthermore preparatory HPLC may cause the original counterion to be exchanged by acetate anions when acetic acid is present in the eluent. The counterions of the salts may be exchanged to a different counterion. The counterions are preferably exchanged for a pharmaceutically acceptable counterion to form the salts described above. Procedures for exchanging counterions are described in WO 2002/042265, WO 2002/042276 and S.D.
  • composition may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.
  • additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.
  • the composition may be in the form of tablets or lozenges formulated in a conventional manner.
  • tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents.
  • Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone.
  • Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol.
  • Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica.
  • Disintegrants include, but are not limited to, potato starch and sodium starch glycollate.
  • Wetting agents include, but are not limited to, sodium lauryl sulfate). Tablets may be coated according to methods well known in the art.
  • the composition may also be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs.
  • the composition may also be formulated as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, nonaqueous vehicles and preservatives.
  • Suspending agent include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats.
  • Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia.
  • Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol.
  • Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid.
  • the composition may also be formulated as suppositories, which may contain suppository bases including, but not limited to, cocoa butter or glycerides.
  • the composition may also be formulated for inhalation, which may be in a form including, but not limited to, a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane.
  • the composition may also be formulated transdermal formulations comprising aqueous or nonaqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
  • composition may also be formulated for parenteral administration including, but not limited to, by injection or continuous infusion.
  • Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents.
  • the composition may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.
  • the composition may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.
  • the composition may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
  • the composition may also be formulated as a liposome preparation.
  • the liposome preparation can comprise liposomes which penetrate the cells of interest or the stratum corneum, and fuse with the cell membrane, resulting in delivery of the contents of the liposome into the cell.
  • liposomes may be used such as those described in U.S. Patent No. 5,077,21 1, U.S. Patent No. 4,621,023 or U.S. Patent No. 4,508,703, which are incorporated herein by reference.
  • a composition intended to target skin conditions can be administered before, during, or after exposure of the skin of the mammal to UV or agents causing oxidative damage.
  • Other suitable formulations can employ niosomes.
  • Niosomes are lipid vesicles similar to liposomes, with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum. 5.
  • the composition may be used for treating a condition associated with NF-kB activity in vivo by administering to a patient in need thereof an aminoacridine.
  • the NF- ⁇ B activity may be at any level, the reduction of which would lead to treatment of the condition.
  • the NF -KB activity may also be at a basal level.
  • the NF- ⁇ B activity may also be at a constitutive level.
  • the NF- ⁇ B activity may also be at an induced constitutive level.
  • the condition associated with NF-kB activity may be cancer.
  • a variety of cancers may be treated including, but not limited to, the following: carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, renal, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histi
  • Transformation induced by tax of HTLV may share the same molecular targets involved in RCC.
  • NF-kB is constitutively active in tax-transformed cells. Similar to RCC, p53 activity is inhibited through activation of NF-kB in tax-transformed cells and p53 inhibition does not involve sequestering of p300. Based on the shared mechanism of p53 inactivation, the compositions may also be used to treat HTLV-induced leukemia. Regardless of their p53 status, the majority of human cancers have constitutively hyperactivated NF-kB.
  • the composition may also be capable of inhibiting NF-kB by reprogramming transactivation NF-kB complexes into transrepression complexes, which may also be used for treatment of any tumor regardless of their p53 status.
  • the compositions may also be used for treating HIV infections since HIV LTRs are strongly dependent on NF-kB activity.
  • composition may also be used as an adjuvant therapy to overcome anti-cancer drug resistance that may be caused by constitutive NF-kB activation.
  • the anti-cancer drug may be a chemotherapeutic described herein. a. Administration
  • the composition may be administered simultaneously or metronomically with other anti ⁇ cancer treatments such as chemotherapy and radiation therapy.
  • anti ⁇ cancer treatments such as chemotherapy and radiation therapy.
  • the term “metronomically” as used herein means the administration of the composition at times different from the chemotherapy and at certain frequency relative to repeat administration and/or the chemotherapy regiment.
  • the composition may be administered in any manner including, but not limited to, orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, or combinations thereof.
  • Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, and intraarticular.
  • the composition may also be administered in the form of an implant, which allows slow release of the composition as well as a slow controlled i.v. infusion. b. Dosage
  • a therapeutically effective amount of an agent required for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the patient, and is ultimately determined by the attendant physician.
  • the desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as one, two, three, four or more subdoses per day. Multiple doses often are desired, or required.
  • the composition When given in combination with other therapeutics, the composition may be given at relatively lower dosages. In addition, the use of targeting agents may allow the necessary dosage to be relatively low. Certain compositions may be administered at relatively high dosages due to factors including, but not limited to, low toxicity, high clearance, low rates of cleavage of the tertiary amine. As a result, the dosage of a composition may be from about 1 ng/kg to about 200 mg/kg, about 1 ⁇ g/kg to about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg.
  • the dosage of a composition may be at any dosage including, but not limited to, about 1 ⁇ g/kg, 25 ⁇ g/kg, 50 ⁇ g/kg, 75 ⁇ g/kg, 100 ⁇ g/kg, 125 ⁇ g/kg, 150 ⁇ g/kg, 175 ⁇ g/kg, 200 ⁇ g/kg, 225 ⁇ g/kg, 250 ⁇ g/kg, 275 ⁇ g/kg, 300 ⁇ g/kg, 325 ⁇ g/kg, 350 ⁇ g/kg, 375 ⁇ g/kg, 400 ⁇ g/kg, 425 ⁇ g/kg, 450 ⁇ g/kg, 475 ⁇ g/kg, 500 ⁇ g/kg, 525 ⁇ g/kg, 550 ⁇ g/kg, 575 ⁇ g/kg, 600 ⁇ g/kg, 625 ⁇ g/kg, 650 ⁇ g/kg, 675 ⁇ g/kg, 700 ⁇ g/kg, 725 ⁇ g/kg, 750 ⁇ g/kg, 7
  • the composition may also be used to diagnose whether a tumor of a patient is capable of being treated by the composition.
  • a sample of the tumor may be obtained from the patient.
  • Cells of the tumor may then be transduced with a p53 reporter system, such as a p53-responstive lacZ reporter.
  • the transduced cells may then be incubated with the composition.
  • the production of a p53-mediated signal above controls indicates that the tumor may be treated by the composition. 7. Screening Methods
  • the present invention also relates to methods of identifying agents that modulate NF- ⁇ B activity.
  • An agent that modulates NF- ⁇ B activity may be identified by a method comprising adding a candidate modulator of NF- ⁇ B activity to a cell-based NF- ⁇ B activated expression system, whereby a modulator of NF- ⁇ B activity is identified by the ability to alter the level of NF- ⁇ B activated expression.
  • An agent that modulates NF- ⁇ B activity may also be identified by a method comprising adding a candidate modulator of NF- ⁇ B activity to a cell-based p53 activated expression system, whereby a modulator of NF- ⁇ B activity is identified by the ability to alter the level of p53 activated expression.
  • An agent that modulates NF- ⁇ B activity may also be identified by a method comprising adding an aminoacridine and a candidate modulator of NF- KB activity to an NF- ⁇ B or p53 activated expression system, comparing the level of NF- ⁇ B or p53 activated expression to a control, whereby a modulator of NF- ⁇ B activity is identified by the ability to alter the level of NF- ⁇ B or p53 activated expression system compared to the control.
  • the cell may comprise a functionally silent p53.
  • the cell may also comprise an NF- ⁇ B transactivation complex.
  • the p53 activated expression system may be in a renal carcinoma cell line.
  • the cell line may also be a sarcoma cell line.
  • the cell line may also be a cell line with amplified mdm2.
  • the cell line may also be a cell line that expresses HPV-E6 or is capable thereof..
  • Candidate agents may be present within a library (i.e., a collection of compounds). Such agents may, for example, be encoded by DNA molecules within an expression library. Candidate agent be present in conditioned media or in cell extracts. Other such agents include compounds known in the art as "small molecules," which have molecular weights less than 10 daltons, preferably less than 10 4 daltons and still more preferably less than 10 3 daltons. Such candidate agents may be provided as members of a combinatorial library, which includes synthetic agents (e.g., peptides) prepared according to multiple predetermined chemical reactions.
  • synthetic agents e.g., peptides
  • the screening methods may be performed in a variety of formats, including in vitro, cell- based and in vivo assays. Any cells may be used with cell-based assays.
  • cells for use with the present invention include mammalian cells, more preferably human and non-human primate cells.
  • Cell-base screening may be performed using genetically modified tumor cells expressing surrogate markers for activation of NF- ⁇ B and/or p53.
  • Such markers include, but are not limited to, bacterial ⁇ -galactosidase, luciferase and enhanced green fluorescent protein (EGFP).
  • the amount of expression of the surrogate marker may be measured using techniques standard in the art including, but not limited to, colorimetery, luminometery and fluorimetery.
  • Representative examples of cells that may be used in cell-based assays include, but are not limited to, renal cell carcinoma cells.
  • the conditions under which a suspected modulator is added to a cell are conditions in which the cell can undergo apoptosis or signaling if essentially no other regulatory compounds are present that would interfere with apoptosis or signaling.
  • Effective conditions include, but are not limited to, appropriate medium, temperature, pH and oxygen conditions that permit cell growth.
  • An appropriate medium is typically a solid or liquid medium comprising growth factors and assimilable carbon, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins, and includes an effective medium in which the cell can be cultured such that the cell can exhibit apoptosis or signaling.
  • the media may comprise Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
  • Cells may be cultured in a variety of containers including, but not limited to tissue culture flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and carbon dioxide content appropriate for the cell. Such culturing conditions are also within the skill in the art.
  • Methods for adding a suspected modulator to the cell include, but are not limited to, electroporation, microinjection, cellular expression (i.e., using an expression system including naked nucleic acid molecules, recombinant virus, retrovirus expression vectors and adenovirus expression), use of ion pairing agents and use of detergents for cell permeabilization.
  • the present invention has multiple aspects, illustrated by the following non-limiting examples. Examples - Materials and Methods
  • Renal cell carcinoma cell lines used, RCC45, RCC54 and ACHN are described in Gurova, et al. (2004). Cancer Res 64, 1951-1958. H1299, HT1080, MCF7, LNCaP, PC3, DU145, HCTl 16, SK-N-SH, WI38 cells were obtained from ATCC.
  • the primary culture of normal kidney epithelial cells (NKE) was provided by J. Didonato (Cleveland Clinic Foundation, OH). 041 fibroblast cell line from Li-Fraumeni patient was provided by G. Stark.
  • Mel7 and Mel29 cells are melanoma cell lines, described in Kichina, et al. (2003). Oncogene 22, 4911- 4917.
  • Reporter cell lines with p53 responsive ⁇ -galactosidase were described in Gurova, et al. (2004). Cancer Res 64, 1951-1958. Reporter cell lines with p53 responsive luciferase was generated by transfection of p21-ConALuc plasmid with following selection on G418.
  • Reporter cell lines with NF- ⁇ B-dependent luciferase were obtained by cotransfection of pNF- ⁇ BLuc and pEGFP-mito (Clontech) plasmids followed by selection on G418 (marker provided by pEGFP- mito plasmid). Reporter cell lines with myc, or Clock/Bmal responsive reporters were kindly provided by C. Burkhart and M. Antoch (Cleveland Clinic Foundation, OH). [0109] Cells with inhibited p53 expression were generated by retroviral transduction of pBabeHl-sip53 or pBabeHl-siGFP vectors for siRNA expression followed by selection on puromycin.
  • p53, Arf expression vectors, pBabeHl-siHdm2, p21-ConALuc reporter plasmid are described in Gurova, et al. (2004). Cancer Res 64, 1951-1958.
  • pNF- ⁇ BLuc plasmid was provided by N. Neznanov (Cleveland Clinic Foundation, ref. 59).
  • pCDNA3 vector expressing pss-I ⁇ B was provided by I. Budunova (Northwestern University).
  • pBabeHl-sip53 and pBabeHl-siGFP vectors for siRNA expression were generated by insertion of Hl promoter and 64 oligonucleotide loop template for siRNA expression into left LTR of pBabeHl-puro vector analogously to pBabeHl-siHDM2 vector, described in Gurova, et al. (2004). Cancer Res 64, 1951-1958. Sequences for siRNA against p53 and GFP are described in Brummelkamp, et al. (2002). Science 296, 550-553. Lentiviral plasmids for p53 or GFP expression are described in Gurova, et al. (2004). Cancer Res 64, 1951-1958.
  • Packaging cells (A293 from Clontech) plated in 60mm plates were transfected with 2 ⁇ g of retroviral vector DNA using Lipofectamin Plus (Invitrogen) according to manufacturers recommendations. The medium was changed after 8 hours. Virus-containing media supplied with 8 ⁇ g/ml of Polybrene (Sigma) was collected at 24 and 48 hours post-transfection and used for infection. Virus-transduced cells were selected for the resistance to an appropriate selective agent (G418, hygromycin or puromycin, depending on the vector) up to a complete death of non- infected cells.
  • an appropriate selective agent G418, hygromycin or puromycin, depending on the vector
  • lentiviruses carrying p53 or EGFP were prepared using 293 cell line transfected with pLV-CMV-p53 and pLV-CMV-EGFP plasmids along with packaging plasmids encoding viral structural proteins and G-protein of vesicular stomatitis virus using lipofectamine reagent (Invitrogen).
  • Virus-containing media from 293T cells was collected 48 hours later and transferred to target cells in the presence of 4 ⁇ g/ml of polybrene and virus was concentrated 50-100 times by ultracentrifugation.
  • Virus titers typically 10 8 IU/ml
  • Ratla cells that are known to be resistant to ectopic expression of p53
  • Luciferase activity and ⁇ -galactosidase activity was measured in lysates prepared 48 hours after transfection with Cell Lysis Buffer (Promega) by luciferase assay system (Promega) or ⁇ -galactosidase enzyme system (Promega). Luminometric and colorimetric reactions were read on the Wallack 1420 plate reader (Perkin Elmer). Integrated reporter set-up. 2x10 4 of cells with integrated reporter were plated in 96 well plates. After overnights incubation chemical compounds or media from lentivirus producing cells were added. At different time points cell lysates were prepared using Reporter Lysis Buffer (Promega).
  • Luciferase or ⁇ -galactosidase activity and protein concentration were measured in aliquots of cell lysates using standard kits (Promega, Luciferase and ⁇ -galactosidase assay systems, Biorad Protein Assay Kit).
  • anti-p53 - monoclonal mouse DOl Sura-Cruz
  • anti-p21 - monoclonal mouse F-5 Sura- Cruz
  • anti-mdm2 - monoclonal mouse SMP 14 Anti-Cruz
  • p53 phosphorylation status was analyzed using phospho-p53 sampler kit from Cell signaling according to manufacturer's recommendations, anti-p65 - (C20, Santa Cruz), anti-phospho-p65 - (ser536, Cell Signaling), anti- I ⁇ Ba - (C21, Santa Cruz), anti-p50 - (NLS, Santa Cruz).
  • HRP-conjugated secondary antibodies were purchased from Santa-Cruz. Quantitation of the data was performed using Quantity One (BioRad).
  • HTl 080 cells were labeled for 24 hours with 0.02 to 0.04 mCi/mL of [ 14 C] thymidine, specific activity 53 mCi/mmol (Amersham).
  • the labeled HTl 080 cells were treated with different concentrations of etoposide (VP- 16), amsacrine (m-AMSA), or 9-aminoacridine for Ih.
  • the induction of topo II-mediated DNA scission was determined by measuring precipitation of the protein DNA complex using a modification of the SDS-KCl technique. Proteasome inhibitor assay
  • proteasome assay kit was purchased from Boston Biochem, Inc. and used according to the manufacturer's recommendations.
  • Nuclear extracts were prepared as described in Chernov, et al. (1997). Oncogene 14, 2503-2510. Annealed oligonucleotide, corresponding to NF- ⁇ B binding site (Santa-Cruz), was radio-labeled by [ ⁇ - 32 P]dCTP by Klenow polymerase and then by [ ⁇ - 32 P]dATP by T4 polynucleotide kinase. 10 7 cpm of labeled oligonucleotide was affinity purified on Probe Quant columns (Amersham).
  • Radio-labeled oligonucleotide was added to 10 ⁇ g of protein nuclear extract together with 1 ⁇ g of poly-dldC (Amersham) to prevent nonspecific binding and incubated for 30 minutes at room temperature.
  • poly-dldC Poly-dldC
  • 200ng of anti-p65, anti-p50 or anti- antibodies were added to the reaction (all antibodies are from Santa Cruz).
  • the entire reaction mixtures were loaded into 4% polyacrilamide gel in 0.5xTBE buffer and run for 2 hours at 200V. Dried gels were exposed to X-ray films for 30 minutes- 1 hour.
  • NIH Swiss athymic nude male mice, 5-6 weeks old were purchased from Harlan. 5x10 of tumor cells were inoculated into the flank of mice in lOO ⁇ L of PBS. When tumors reached 5mm diameter, intraperitoneal injections of drugs were started in 100 ⁇ l solution of 50% DMSO in PBS (except quinacrine, which was dissolved in PBS). As vehicle, 50% DMSO solution in PBS was used. Tumor size was measured in three dimensions every other day.
  • dormant p53 in RCC may be reactivated, and that reactivation leads to tumor cell death.
  • p53 became simultaneously cytotoxic and active in inducing the reporter in RCC45 cells (Fig. Ia and b).
  • cells such as RCC cells
  • reactivation of p53 in tumor cells may be cytotoxic.
  • the most active compound caused 22-fold induction of the reporter in RCC45 cells acting 7 times stronger than doxorubicin.
  • the library of structural analogues built around compound 30d9 and consisting of 40 chemicals was screened using the same cell-based reporter assay. Two agents of the formula of compound 1 were found to be active. [0127] A library of 59 derivatives of compound 30d9 were then screened, including the anti ⁇ cancer agent amsacrine (amsa) and anti-malaria agent quinacrine.
  • 9AA activated p53 at concentrations as low as 1 ⁇ M (Fig. 3e).
  • the kinetics of activation is unusually slow as compared with DNA-damaging stimuli: 9AA-induced p53-dependent transactivation becomes detectable only 12 hours and reaches maximum around 36 hours after treatment with the drug (Fig. 3f).
  • 9AA-induced p53-dependent transactivation becomes detectable only 12 hours and reaches maximum around 36 hours after treatment with the drug (Fig. 3f).
  • One-hour incubation with 9AA was enough to initiate p53 activation being detectable several hours later (Fig. 3f).
  • chemotherapeutic drugs acting through a DNA- damaging mechanism e.g., camptothecin, doxorubicin
  • microtubule network e.g., taxol, vinblastine
  • normal cells also possess active p53 we tested the toxicity of 9AA to normal kidney epithelial cells and human diploid fibroblasts WI38. Both normal cell types were more resistant to 9AA as compared with RCC and other tumor cells (Fig. 4e and f).
  • mice were inoculated with HT1080sip53 (left flank) and HT1080siGFP (right flank) cells to exploit p53 dependence of the quinacrine effect. After tumors reached 5mm in diameter, mice were injected i.p. daily with 50mg/kg of quinacrine, with 5-fluorouracil (5FU, 35mg/kg) being used for comparison. As presented in Figure 5b, quinacrine inhibited growth of p53 -expressing tumors to the same extend as 5FU and had no effect on the growth of p53-deficient tumors. This indicates that aminoacridines inhibit the in vivo growth of xenograft tumors in a p53 dependent manner (Fig. 5b).
  • mice were also inoculated with 2XlU" human prostate cancer cells using PC3 p53- negative or with DU 145 cells comprising mutant p53.
  • the mice were treated with quinacrine at a dose of 100 mg/kg by oral gavage or with sterile water, as a control. Tumor growth was reduced by -50% in quinacrine-treated mice. Interestingly, this indicates that aminoacridines also inhibit in vivo growth of xenograft tumors in a p53 -independent manner.
  • Proteasome inhibitors form another class of p53 -activating agents causing accumulation of unmodified p53 protein. Accumulation of non-phosphorylated p53 in response to 9AA treatment and predominant localization of the drug in the cytoplasm (monitored by fluorescent microscopy) suggested that aminoacridines could act as an inhibitor of proteasomal degradation. This hypothesis was ruled out using a direct in vitro assay (Fig. 6c) and by monitoring the effect of 9AA on the level of IKB, another target of proteasomal degradation [21], which was used as an independent indicator of proteasomal activity.
  • aminoacridines are a potent inhibitor of NF- ⁇ B transactivation that acts through an unusual mechanism. As opposed to previously described inhibitors that act by stabilizing IKB, aminoacridines may act by converting NF- ⁇ B complex into a transcriptionally inactive state that becomes trapped in the nucleus due to lack of induction of its shuttling factor IKB.
  • NF- ⁇ B complex Among the mechanisms that could be responsible for functional inactivation of NF- ⁇ B complex could be a lack of phosphorylation of p65 (reported as essential for the NF- ⁇ B activity) resulting in recruitment of histone deacetylases (HDAC) into the complex and conversion of chromatin in the transcription initiation sites into inactive form.
  • HDAC histone deacetylases
  • Inactive NF- ⁇ B is present in the nuclei of unstimulated cells in a complex with HDACs.
  • TSA trichostatin A
  • Blocking phosphorylation of p65 may not be the only mechanism of anti-NF- ⁇ B activity of aminoacridines.
  • treatment with 9AA caused an increase in cytoplasmic and nuclear pools of p50 (Fig. 7g).
  • p50/p50 homodimers, the proportion of which increased with 9AA-mediated accumulation of this NF- ⁇ B subunit (Fig. 7d) might also contribute to the formation of inactive NF- ⁇ B complexes.
  • Aminoacridines cause two strong effects in RCC cells. They activate p53 in an unusual way that does not involve DNA damage or inhibition of proteasomal degradation. It also inhibits NF- ⁇ B, also in an unusual way, by converting transcriptionally active NF- ⁇ B complexes into transcriptionally inactive ones, presumably due to the inhibition of p65/RelA phosphorylation.
  • the effects of aminoacridines on p53 and NF- ⁇ B are quite specific since they have no effect on transcription regulated by other tested factors, such as c-Myc or N-Myc, androgen receptor or CLOCK/BMALl (data not shown).
  • the most upregulated genes included p21, mdm2, as well as several other p53 targets (Table 1), while IKB alpha, IL-8 and several chemo- and cytokines (Table 2), all encoded by NF-kB-responsive genes, were strongly suppressed as a result of treatment. These results confirmed a dual effect of aminoacridines as an inducer of p53 and an inhibitor of NF-kB transcription.

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Abstract

Les aminoacridines sont des inhibiteurs de NF-?B. L'inhibition de NF-?B entraîne la réactivation de p53 dans les cellules cancéreuses dans lesquelles p53 est fonctionnellement bloquée.
PCT/US2005/025884 2004-07-20 2005-07-20 Inhibition de nf-kb WO2006012419A2 (fr)

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Cited By (6)

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US20090099191A1 (en) * 2006-02-02 2009-04-16 Gudkov Andrei V Inhibition of nf-kb
WO2010097641A1 (fr) * 2009-02-26 2010-09-02 Alexandre Vamvakides Ligands des récepteurs sigma (σ) possédant des propriétés anti-apoptotiques et/ou pro-apoptotiques sur les mécanismes cellulaires, et exhibant une activité prototypique cytoprotectrice et anti-cancer
WO2011031974A1 (fr) * 2009-09-10 2011-03-17 Southern Research Institute Analogues d'acridine à utiliser dans le traitement de gliomes
WO2018048195A1 (fr) * 2016-09-07 2018-03-15 부산대학교 산학협력단 Composition pour la prévention ou le traitement d'un cancer réfractaire contenant de la quinacrine en tant que principe actif
EP2294186B1 (fr) * 2008-05-20 2019-10-09 Incuron, Inc. Induction de mort cellulaire par inhibition d'une réponse de choc thermique adaptative
CN115212209A (zh) * 2021-04-19 2022-10-21 中国科学院分子细胞科学卓越创新中心 一种具有免疫佐剂活性和促进免疫疗法的抗肿瘤药物

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UA107652C2 (en) * 2008-10-06 2015-02-10 Incuron Llc Carbazole compounds and therapeutic uses of the compounds

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JP2001131066A (ja) 1999-08-25 2001-05-15 Nippon Kayaku Co Ltd アポトーシス増強剤
JP4204980B2 (ja) * 2002-03-07 2009-01-07 サムジン ファーマシューティカル カンパニー リミテッド ９−アミノアクリジン誘導体及びその製造方法
US20100112012A1 (en) * 2005-11-14 2010-05-06 Cleveland Clinic Foundation Modulation of immune responses

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090099191A1 (en) * 2006-02-02 2009-04-16 Gudkov Andrei V Inhibition of nf-kb
EP2294186B1 (fr) * 2008-05-20 2019-10-09 Incuron, Inc. Induction de mort cellulaire par inhibition d'une réponse de choc thermique adaptative
WO2010097641A1 (fr) * 2009-02-26 2010-09-02 Alexandre Vamvakides Ligands des récepteurs sigma (σ) possédant des propriétés anti-apoptotiques et/ou pro-apoptotiques sur les mécanismes cellulaires, et exhibant une activité prototypique cytoprotectrice et anti-cancer
US9180106B2 (en) 2009-02-26 2015-11-10 Anavex Life Sciences Corp. Sigma receptors ligands with anti-apoptotic and/or pro-apoptotic properties, over cellular mechanisms, exhibiting prototypical cytoprotective and also anti-cancer activity
WO2011031974A1 (fr) * 2009-09-10 2011-03-17 Southern Research Institute Analogues d'acridine à utiliser dans le traitement de gliomes
WO2018048195A1 (fr) * 2016-09-07 2018-03-15 부산대학교 산학협력단 Composition pour la prévention ou le traitement d'un cancer réfractaire contenant de la quinacrine en tant que principe actif
CN115212209A (zh) * 2021-04-19 2022-10-21 中国科学院分子细胞科学卓越创新中心 一种具有免疫佐剂活性和促进免疫疗法的抗肿瘤药物
CN115212209B (zh) * 2021-04-19 2023-11-28 中国科学院分子细胞科学卓越创新中心 一种具有免疫佐剂活性和促进免疫疗法的抗肿瘤药物

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