JP2014074056A - INHIBITION OF NF-κB - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify agents that are restorable of wild type p53 activity in tumor cells.SOLUTION: Aminoacridines are inhibitors of NF-κB. Inhibiting NF-κB leads to reactivation of p53 in cancer cells with functionally blocked p53. A method for treating a condition associated with NF-κB activity comprising the step of administering to a patient in need thereof a composition comprising an inhibitor of NF-κB, in which the NF-κB activity is constitutive or induced, and may be at a basal level.

Description

(Citation of related application)
This application claims the benefit of US Provisional Patent Application No. 60 / 589,637, filed July 20, 2004, the contents of which are incorporated herein by reference.

(Background of the Invention)
(1. Field of the Invention)
The present invention relates broadly to the regulation of cell proliferation or apoptosis. More specifically, the present invention relates to compositions, methods of use, and methods of identification thereof for modulating cell proliferation or apoptosis.

(2. Explanation of related technology)
Cancer frequency in humans increases as the population ages in the developed world. Despite extensive research on several types of cancer and stage at diagnosis, morbidity and mortality have not improved significantly in recent years. Programmed cell death, ie, induction of apoptosis, is one of the most interesting cancer treatment methods.

  p53 controls gene stability and reduces cancer risk through growth arrest or induction of apoptosis in response to DNA damage or proto-oncogene deregulation. The efficacy of p53 as a tumor prevention factor is reflected by the frequency of p53 loss in at least 50% of human tumors due to inactivating mutations. Several mechanisms for inactivation of wild-type p53 function have been described for human tumors, including excessive degradation of p53, usually through the proteasome and mediated by Mdm2. Mdm2 is considered an interesting target for small molecule suppression or other approaches to selectively kill tumor cells by restoring p53 function.

  Renal cell carcinoma (RCC) maintains p53, which is wild type but functionally inactive. The mechanism of repression of p53 in RCC is prevalent, indicating the presence of a molecular target so far unknown for restoration of p53 function in cancer. There is a great need to identify substances that can restore wild-type p53 activity in tumor cells.

(Summary of the Invention)
Conditions associated with NF-κB activity can be treated by administering to a patient in need thereof a composition comprising an inhibitor of NF-κB. NF-κB activity can be constitutive, induced or basal. Inhibition of NF-κB may activate p53. In some cases, inhibition of NF-κB activates functionally silent p53. The condition to be treated can be cancer, inflammation, autoimmune disease, graft versus host disease, conditions associated with HIV infection, or precancerous cells acquired in dependence on constitutively active NF-κB. . The forms of cancer that can be treated include renal cell carcinoma, sarcoma, prostate cancer, breast cancer, pancreatic cancer, myeloma, myeloid leukemia and lymphoblastic leukemia, neuroblastoma, glioblastoma, HTLV infection Include but are not limited to cancer caused by

  The inhibitor of NF-κB can be an aminoacridine of the following formula:

ここで、
Ｒ_１は、Ｈまたはハロゲンであり；
Ｒ_２は、Ｈまたは必要に応じて置換されたアルコキシであり；
Ｒ_３は、Ｈまたは必要に応じて置換されたアルコキシであり；そして
Ｒ_４は、Ｈまたは必要に応じて置換された脂肪族、アリール、もしくは複素環である。

here,
R ₁ is H or halogen;
R ₂ is H or optionally substituted alkoxy;
R ₃ is H or optionally substituted alkoxy; and R ₄ is H or optionally substituted aliphatic, aryl, or heterocycle.

  The aminoacridine can be 9-aminoacridine or quinacrine. The composition may further comprise a death receptor activator of a TNF family polypeptide. Activators include TNF polypeptides (eg, NGF, CD40L, CD137L / 4-1BBL, TNF-α, CD134L / OX40L, CD27L / CD70, FasL / CD95, CD30L, TNF-β / LT-α, LT-β Or TRAIL).

  Agents that modulate functionally silent p53 can be identified by adding candidate agents to cells containing the p53-responsive reporter and measuring the level of the signal of the p53-responsive reporter. This substance can be identified by the difference in signal compared to the control. This substance may increase or decrease the activity of p53. The cell may contain functionally silent p53.

のアミノアクリジンであり、ここで、
Ｒ_１は、Ｈまたはハロゲンであり；
Ｒ_２は、Ｈまたは必要に応じて置換されたアルコキシであり；
Ｒ_３は、Ｈまたは必要に応じて置換されたアルコキシであり；そして
Ｒ_４は、Ｈまたは必要に応じて置換された脂肪族、アリール、もしくは複素環である、項目１に記載の方法。
（項目１１）
前記アミノアクリジンが、９−アミノアクリジンおよびキナクリンからなる群より選択される、項目１０に記載の方法。
（項目１２）
前記組成物はさらに、ＴＮＦファミリーのポリペプチドの死受容体の活性化因子を含む、項目１０に記載の方法。
（項目１３）
前記活性化因子が、ＮＧＦ、ＣＤ４０Ｌ、ＣＤ１３７Ｌ／４−１ＢＢＬ、ＴＮＦ−α、ＣＤ１３４Ｌ／ＯＸ４０Ｌ、ＣＤ２７Ｌ／ＣＤ７０、ＦａｓＬ／ＣＤ９５、ＣＤ３０Ｌ、ＴＮＦ−β／ＬＴ−α、ＬＴ−β、およびＴＲＡＩＬからなる群より選択されるＴＮＦファミリーのポリペプチドである、項目１２に記載の方法。
（項目１４）
機能的にサイレントなｐ５３を活性化させる物質をスクリーニングする方法であって：
（ａ）候補の物質を、ｐ５３応答性レポーターを含む細胞に対して加える工程；
（ｂ）該ｐ５３応答性レポーターのシグナルのレベルを測定する工程
を包含し、それによって、（ｂ）での対照を上回るシグナルによって物質が同定される、方法。
（項目１５）
前記細胞が、機能的にサイレントなｐ５３を含む、項目１４に記載の方法。
（項目１６）
ＮＦ−κＢを阻害する物質をスクリーニングする方法であって：
（ａ）候補の物質を、ｐ５３応答性レポーターを含む細胞に対して加える工程；
（ｂ）該ｐ５３応答性レポーターのシグナルのレベルを測定する工程を包含し、それによって、（ｂ）での対照を上回るシグナルによって物質が同定される、方法。
（項目１７）
前記細胞が機能的にサイレントなｐ５３を含む、項目１６に記載の方法。
（項目１８）
前記細胞がＮＦ−κＢトランス活性化複合体を含む、項目１６に記載の方法。 Agents that modulate NF-κB can be identified by adding candidate agents to cells containing a p53-responsive reporter and measuring the level of the signal of the p53-responsive reporter. This substance can be identified by the difference in signal compared to the control. This substance may increase or decrease the activity of NF-κB. The cell may contain functionally silent p53. The cell may also contain an NF-κB transactivation complex.
For example, the present invention provides the following items.
(Item 1)
A method of treating a condition associated with NF-κB activity comprising administering to a patient in need thereof a composition comprising an inhibitor of NF-κB.
(Item 2)
Item 2. The method according to Item 1, wherein the NF-κB activity is constitutive or induced.
(Item 3)
Item 2. The method according to Item 1, wherein the NF-κB activity is at a basal level.
(Item 4)
The method according to item 1, wherein p53 is activated by inhibition of NF-κB.
(Item 5)
Item 2. The method according to Item 1, wherein the condition is cancer.
(Item 6)
Item 6. The method according to Item 5, wherein inhibition of NF-κB induces activation of wild-type p53 with impaired function.
(Item 7)
The cancer comprises renal cell cancer, sarcoma, prostate cancer, breast cancer, pancreatic cancer, myeloma, myeloid leukemia and lymphoblastic leukemia, neuroblastoma, glioblastoma, and cancer caused by HTLV infection 6. The method according to item 5, which is selected from the group.
(Item 8)
2. The method of item 1, wherein the condition is a condition associated with inflammation, autoimmune disease, graft versus host disease, or HIV infection.
(Item 9)
Item 2. The method according to Item 1, wherein the condition is a precancerous cell acquired in dependence on constitutively active NF-κB.
(Item 10)
An inhibitor of NF-κB has the following formula:

Of aminoacridine, where
R ₁ is H or halogen;
R ₂ is H or optionally substituted alkoxy;
2. The method of item 1, wherein R ₃ is H or optionally substituted alkoxy; and R ₄ is H or optionally substituted aliphatic, aryl, or heterocycle.
(Item 11)
Item 11. The method according to Item 10, wherein the aminoacridine is selected from the group consisting of 9-aminoacridine and quinacrine.
(Item 12)
11. The method of item 10, wherein the composition further comprises a death receptor activator of a TNF family polypeptide.
(Item 13)
The activator comprises NGF, CD40L, CD137L / 4-1BBL, TNF-α, CD134L / OX40L, CD27L / CD70, FasL / CD95, CD30L, TNF-β / LT-α, LT-β, and TRAIL. 13. The method according to item 12, which is a TNF family polypeptide selected from the group.
(Item 14)
A method of screening for a substance that activates functionally silent p53, comprising:
(A) adding a candidate substance to a cell containing a p53-responsive reporter;
(B) measuring the level of the signal of the p53-responsive reporter, whereby the substance is identified by a signal that exceeds the control in (b).
(Item 15)
15. The method of item 14, wherein the cell comprises functionally silent p53.
(Item 16)
A method of screening for a substance that inhibits NF-κB, comprising:
(A) adding a candidate substance to a cell containing a p53-responsive reporter;
(B) measuring the level of the signal of the p53-responsive reporter, whereby the substance is identified by a signal that exceeds the control in (b).
(Item 17)
The method of item 16, wherein the cell comprises functionally silent p53.
(Item 18)
The method of item 16, wherein the cell comprises an NF-κB transactivation complex.

FIG. 1 shows that restoration of transactivation mediated by p53 in RCC cells is effected by death of RCC cells. FIG. 1A: Activity of p-53 responsive reporters in RCC45ConALacZ cells transduced with lentiviral vectors expressing various concentrations of p53 or GFP. β-galactosidase activity (ONPG staining) was measured 48 hours after lentiviral transduction and normalized by protein concentration. FIG. 1B: Cell viability was measured by methylene blue staining 96 hours after lentiviral transduction and the intensity of methylene blue staining of cells transduced with p53 virus versus the same cells transduced with the same concentration of GFP virus. Shown as a percentage. FIG. 2 shows the p53 recovery activity of the compound of formula 1 FIG. 2A: Selection of readout cells and setting of selection criteria. MCF7, ACHN, RCC26b, and RCC45 cells (all containing ConALacZ reporter) were plated in 96-well plates and incubated for 24 hours in medium containing various concentrations of doxorubicin. Subsequently, β-galactosidase activity was measured by ONPG staining and normalized by protein concentration. FIG. 2B shows that 9AA causes the strongest activity of the p53-dependent reporter in RCC45 cells. Compounds of formula 1 were tested in a dose-dependent assay for p53 transactivation in RCC45ConALacZ cells. Bars indicate the relative activity of each compound calculated as a fold increase in p53 activation induced by the compound over the effect of 2 μM doxorubicin (results of 3 experiments). FIG. 3 shows that 9AA induces p53 transcriptional activity in various tumor cells. FIG. 3A shows that 9AA induces a p53-responsive reporter in a dose-dependent manner. RCC45ConALacZ cells and MCF7ConALacZ cells were incubated for 24 hours in medium containing the indicated concentrations of doxorubicin (dox) or 9AA (all concentrations shown in μM), after which β-galactosidase activity was measured by ONPG staining, Normalized by protein content and expressed as fold induction of reporter compared to untreated cells. FIG. 3B shows that 9AA induces expression of endogenous p53-dependent targets. Western blot analysis of total cell lysates of RCC45 and MCF7 cells treated with 10 μM 9AA or 2 μM doxorubicin for the indicated times (hours) and probed with anti-p53, anti-Hdm2, or anti-p21 antibodies. FIG. 3C shows that 9AA activates p53 more strongly than doxorubicin in the majority of tumor cells tested. The indicated cells incorporating the p53 responsive reporter were treated with various concentrations of 9AA (1-10 μM) and doxorubicin (dox, 0.2-2 μM) for 24 hours, after which β-galactosidase activity was stained with ONPG. And normalized by protein content. The data was presented as the fold of induction of the reporter over the untreated control with the most effective dose of 9AA over the dox effect. FIG. 3D shows that 9AA induces β-galactosidase activity in a p53-dependent manner. HT1080ConALuC transduced with constructs expressing anti-p53 or anti-GFP siRNA were treated with 5 μM 9AA for 24 hours. Bars show fold induction of p53-responsive reporter over untreated controls. Boxes show Western blots for expression of p53 in total protein lysates of cell mutants under both basic conditions and conditions treated with doxorubicin (basal levels of p53 and levels induced by DNA damage). To evaluate). FIG. 3E shows a dose response curve of p53 responsive reporter activity in HT1080ConALuc cells treated with 9AA for 24 hours (no normalization to protein concentration was performed). Fold induction was shown as fold reporter activation over untreated controls. FIG. 3F shows the time dependence of the p53 induction effect of 9AA. HT1080ConALuC cells were treated with 20 μM 9AA for 1 hour and then measured when they showed β-galactosidase activity. Fold induction was shown as fold reporter activation over untreated controls. FIG. 4 shows that the cytotoxicity associated with 9AA is p53 dependent. FIG. 4A shows the viability of HT1080-sip53 and HT1080-siGFP cells treated with the indicated concentrations of 9AA (see “Materials and Methods” section) as a percentage of cells compared to untreated controls. Together with the results. FIG. 4B shows another pair of cells with various levels of p53 tested in the manner described for FIG. 4A. The upper panel shows a Western blot analysis of p53 protein levels in the pair of cells generated. The lower panel shows the relative number of cells after treatment with 9aa (2 μM) compared to the untreated control (100%). FIG. 4C shows cell cycle analysis of HT1080 sip53 cells or HT1080 siGFP cells treated with 3 or 20 μM 9AA for the time indicated, or treated with 2 μM dox for 24 hours. FIG. 4D shows the p53 dependence of the cytotoxicity of various drugs. The same experiment described in FIG. 4A was performed using 30d9 (first hit, 9aa analog, 1-10 μM), doxorubicin (dox, 0.1-1 μM), campotethecin (camp, 0.16-1. 6 μM), vinblastine (vinbl, 0.1-1 μM), and taxol (tax, 0.06-0.6 μM). Bars are plotted for drug dose, indicating the greatest difference in sensitivity between p53 “+” and p53 “−” cells. FIG. 4E shows that 9AA is more toxic to RCC than normal kidney epithelial cells (NKE). The same experiment described in FIG. 4A was performed using NKE, RCC45, RCC54, and ACHN cells. FIG. 4F shows that 9AA is more toxic to RCC at low concentrations. Several cell types (NKE-normal kidney epithelial cells, RCC45, ACHN-RCC cell line, HCT116-colon cancer, p53 wild type, SK-N-SH-neuroblastoma, p53 wild type, LNCaP prostate line cancer , P53 wild type, DU145, PC3-prostate cancer, p53 deficient, Mel7, Mel29 melanoma, 041-fibroblasts from patients with Lee-Fraumeni cancer syndrome, p53 null, WI38—normal human diploid fibroblasts ) Were treated with 2 μM 9AA as described in FIG. 4A and cell viability was compared to corresponding untreated cells. FIG. 5 shows that the compound of formula 1 is toxic to tumor cells with active p53. FIG. 5A shows the in vivo p53-inducing effect of multiple substances. HT1080ConALuC cells were inoculated on both flank of nude mice. When tumors reached a diameter of 5 mm, mice were injected intraperitoneally with the indicated concentration of drug (mg / kg, 3 mice per group). Mice were sacrificed 24 hours later, tumors were isolated, lysed in Reporter Lysis Reagent (Promega), and luciferase activity was measured in 10 mg of tumor protein. Bars indicate the fold induction of luciferase activity in drug-treated tumors over luciferase activity in vehicle-treated tumors. FIG. 5B: HT1080 sip53 cells or HT1080 siGFP cells were inoculated into the left and right flank of nude mice, respectively. When tumors reached a diameter of 5 mm, mice were given a vehicle (50% DMSO in PBS), quinacrine (QC, 50 mg / kg), and 5-fluorouracil (5FU, 35 mg / kg) abdominal cavity every 24 hours. Internal injection (5 mice per group). Results are shown as the average of the relative tumor volumes of the individual tumors compared to the tumor volume on the first day of treatment. FIG. 6 shows a possible mechanistic test for 9AA activity. FIG. 6A shows the measurement of DNA-topoisomerase II complex formation in cells treated with 9AA. FIG. 6B shows the phosphorylation status of p53 in RCC45 cells treated with 9AA (5 μM) or dox (1 μM) for 16 hours. Western blot analysis of total protein lysates was performed using an antibody against p53 (DO1) and an antibody against a specific phosphorylation site in p53. FIG. 6C shows the effect of 9AA on proteasome activity. HCT116 cells were treated with 1 μM PS-341 for 30 minutes. PS-341 was washed 30 minutes later and cells were re-incubated in medium without drug before analysis at 3 or 16 hours after treatment. In the case of 9AA, cells were treated with 2 μM 9-aminoacridine for 3 or 16 hours continuously prior to analysis. Proteasome activity was determined by measuring the absorbance of free AMC resulting from cleavage of the AMC-fluorescent peptide. The proteasome activity of untreated cells was set at 100%. FIG. 6D shows the state of IκB-α phosphorylation after treatment with 9AA. PC-3 cells were treated with 10 μM 9-aminoacridine for 1, 2, 4, and 8 hours, or 10 μM MG-132 for 8 hours. Cell lysates were isolated at the indicated times after treatment and used for Western blotting. The blot was probed with both an antibody specific for the phosphorylated form of IκB-α and an antibody specific for the unphosphorylated form. A β-actin specific antibody was used as a loading control. FIG. 6E shows that treatment with 9AA leads to a decrease in IκB-α protein levels. Western blot analysis shows MCF7 cells incubated for 8 hours in 1 μM doxorubicin (dox) and 10 μM 9AA with anti-IκB antibody. FIG. 7 shows the effect of 9AA on the NF-κB pathway. FIG. 7A shows that 9AA inhibits NF-κB-dependent transcription. H1299-NF-κBLuc cells at various concentrations of 9AA and quinacrine (QC), before TNFa (10 ng / ml) (TNF after 9AA or QC) or simultaneously with TNFa (TNF and 9AA or QC) for 2 hours Processed. Luciferase activity in the cell lysate was measured 6 hours after the addition of TNFa. FIG. 7B shows that 9AA inhibits reconstitution of IκB levels stimulated by TNF. HT1080 cells were treated with TNF (10 ng / ml) in the presence of 9AA (10 μM) or in the absence of 9AA. Whole cell lysates were prepared at the indicated time points and analyzed by Western blotting with antibodies against IκB. FIG. 7C: H1299-NF-κBLuc cells were treated with the indicated concentrations of 9AA and TNF (10 ng / ml). After 6 hours, cytoplasmic and nuclear extracts were isolated and used for luciferase assay or gel shift assay, respectively. FIG. 7D shows that 9AA results in the accumulation of p65 / p50 and p50 / p50 NFκB complexes. Gel shift assays were performed using nuclear extracts of H1299 cells treated with 9AA (10 μM) and TNF (10 ng / ml) for 30 minutes. FIG. 7E shows that 9AA delays the elimination of nuclear-derived p65 complexes. Immunofluorescent staining of HT1080 cells treated with 9AA (10 mM) and TNF (10 ng / mL) for the indicated times with antibodies to p65. FIG. 7F shows that 9AA reduces phosphorylation of p65 in response to TNF. Upper panel: Western blot analysis of total cell lysates of HT1080 cells treated with TNF (10 ng / ml) in the presence of 9AA (10 μM) or in the absence of 9AA for the indicated times. The same membrane was probed with an antibody against whole p65 and an antibody against phospho-p65Ser536. Lower panel: quantification of the experiment shown in the upper panel using BioRad QuantityOne software. Results are shown as a multiple of the change in band intensity compared to the untreated control. FIG. 7G shows that 9AA causes an increase in p50 protein levels. Western blot analysis of nuclear and cytoplasmic fractions of HT1080 cells (processed as described in FIG. 7A). FIG. 7H shows that 9AA does not inhibit NF-κB transactivation induced by trichostatin A (TSA). H1299 cells incorporating the NF-κB-dependent luciferase reporter were treated with 100 nM TSA for 4 or 16 hours in the presence of 9AA (20 mM) or in the absence of 9AA. Treatment with TNFa was used as a control for reporter activity. FIG. 8 shows that 9AA activates p53-dependent transcription through inhibition of NF-κB. FIG. 8A shows that IκB SuperSuppresson (ss) activates p53 in RCC cells. ACHN cells were co-transfected with p21-ConALuc and the indicated plasmids containing IκB SuperSuppressor, p53, and Arf cDNA or anti-Hdm2 siRNA. After 48 hours, luciferase activity in the cell lysate was measured. Normalization was performed by co-transfection of pCMV-LacZ plasmid. FIG. 8B shows that IκB SuperSuppressor inhibits NF-κB transcriptional activity. ACHN cells were co-transfected with NF-κB responsive reporters pNF-κBLuc and IκB SuperSuppressor (SS). After 48 hours, luciferase activity in the cell lysate was measured. FIG. 8C shows that 9AA is unable to activate p53 in cells in which NF-κB is inhibited. ACHN cells were co-transfected with either pConALuc or pNF-κBLuc, and IκB SuperSuppressor (SS) or empty vector. Twenty-four hours after transfection, all cells were disassembled and treated with 9AA (10 μM). NF-κB reporter activity was measured 6 hours after treatment and p53-responsive reporter activity was measured 24 hours after treatment. Normalization was performed by co-transfection of pCMV-LacZ plasmid (for cells transfected with various plasmid sets) and by protein concentration (for cells treated with 9AA and untreated cells). FIG. 9 shows the synergistic effect of 9-aminoacridine with the death ligand. FIG. 10 shows that 9AA and QC are anti-RCC substances. FIG. 10A shows a comparison of IC 50% doses of 9AA, QC, and several anti-cancer agents for various RCC and non-RCC cells. IC50% was determined for individual cell lines and individual drugs. Each point represents the IC50% of a particular cell line and these are grouped as follows: (i) Kuromaru-RCC cell lines (ACHN, RCC9, RCC13, RCC29, RCC45, RCC54), (Ii) Red triangle-non-RCC cell lines (MCF7, HT1080, H1299, U20S, LNCaP, HCT116), (iii) Green squares-normal kidney cells (NKE). FIG. 10B shows that quinacrine activates a p53-responsive reporter in RCC tumors cultured ex vivo. X-gal staining of tumor and normal kidney fragments transduced with p53-responsive reporter lentivirus ex vivo and treated with quinacrine or doxorubicin. FIG. 10C shows that quinacrine sensitizes RCC45 and RCC54 to TRAIL but not normal kidney epithelial cells (NKE). Cells plated in 96-well plates were incubated for 24 hours in the presence of the indicated concentrations of TRAIL and quinacrine, and cell number was predicted using the methylene blue assay. FIG. 10D shows the antitumor activity of quinacrine (QC). 10 ⁷ ACHN cells were inoculated subcutaneously into nude mice. When the tumor reached a diameter of 5 mm, intraperitoneal administration of 50 mg / kg QC was started. 5FU (35 mg / kg) was used as a control. Tumor size was measured every other day and presented as a fold increase in tumor volume.

(Detailed explanation)
Prior to disclosing and describing the compounds, products and compositions and methods of the present invention, the terminology used herein is for the purpose of describing particular embodiments only. It is understood that it is not intended to be limiting. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” refer to the plural unless the context clearly dictates otherwise. It should be noted that references to are also included.

(1. Definition)
The term “branched” as used herein is a group containing from 1 to 24 skeletal atoms, where the skeletal chain of this group is one from the main chain. Means a group containing one or more associated branches. Preferred branched groups herein include 1 to 12 skeletal atoms. Examples of branched groups include isobutyl, t-butyl, isopropyl, —CH ₂ CH ₂ CH (CH ₃ ) CH ₂ CH ₃ , —CH ₂ CH (CH ₂ CH ₃ ) CH ₂ CH ₃ , —CH _2. Examples include, but are not limited to, CH ₂ C (CH ₃ ) ₂ CH ₃ , —CH ₂ CH ₂ C (CH ₃ ) _{3, and the} like.

  The term “unbranched” as used herein is a group containing from 1 to 24 skeletal atoms, where the skeletal chain of the group extends in a straight line. Means group. Preferred unbranched groups herein include 1 to 12 skeletal atoms.

  The term “cyclic” or “cyclo” as used herein, alone or in combination, has 3 to 12 skeletal atoms, preferably 3 to 7 skeletal atoms. Means a group having one or more closed rings (whether unsaturated or saturated) carrying the ring

  The term “lower” as used herein means a group having from 1 to 6 skeletal atoms.

  The term “saturated”, as used herein, means a group in which all available valence bonds of the backbone atoms are bonded to other molecules. Representative examples of saturated groups include, but are not limited to, butyl, cyclohexyl, piperidine and the like.

The term “unsaturated”, as used herein, means a group in which at least one available valence bond of two adjacent skeletal atoms is not bound to another atom. . Representative examples of unsaturated groups include, but are not limited to, —CH ₂ CH ₂ CH═CH ₂ , phenyl, pyrrole, and the like.

  The term “aliphatic” as used herein means an unbranched group, a branched group, or a cyclic hydrocarbon group, which may be substituted or It may be unsubstituted and may be saturated or unsaturated, but is not aromatic. The term “aliphatic” further includes aliphatic groups containing oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.

  The term “aromatic” as used herein means an unsaturated cyclic hydrocarbon group having 4n + 2 delocalized π (pi) electrons, which is substituted May or may not be substituted. The term “aromatic” further includes aromatic groups that contain 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, (iso) oxazolyl, and the like.

  The term “substituted” as used herein is a group in which one or more hydrogens or other atoms have been removed from carbon or a suitable heteroatom and replaced with another group. Means. Preferred substituted groups herein are substituted with 1 to 5, most preferably 1 to 3 substituents. An atom having two substituents is described as “di”, while an atom having two or more substituents is described by “poly”. Representative examples of such substituents include aliphatic groups, aromatic groups, alkyl, alkenyl, alkynyl, aryl, alkoxy, halo, aryloxy, carbonyl, acrylic, cyano, amino, nitro, phosphorus-containing groups, Sulfur-containing groups, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, acylamino, amidino, Imino, alkylthio, arylthio, thiocarboxylate, alkylsulfinyl, trifluoromethyl, azide, heterocyclyl, alkylaryl, heteroary , Semicarbazide, thiosemicarbazide, maleimide, oximino, imidate, cycloalkyl, cycloalkylcarbonyl, dialkylamino, arylcycloalkyl, arylcarbonyl, arylalkylcarbonyl, arylcycloalkylcarbonyl, arylphosphinyl, arylalkylphosphinyl, Arylcycloalkylphosphinyl, arylphosphonyl, arylalkylphosphonyl, arylcycloalkylphosphonyl, arylsulfonyl, arylalkylsulfonyl, arylcycloalkylsulfonyl, combinations thereof, and substituents thereof include, but are not limited to Not done.

  The term “unsubstituted”, as used herein, means a group to which no group is further attached or substituted.

  The term “alkyl” as used herein, alone or in combination, means a saturated aliphatic group that is branched or unbranched. Typical examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. These are not limited.

  The term “alkenyl” as used herein, alone or in combination, includes a branch containing at least one carbon-carbon double bond that can occur at any stable site along the chain. Means an unsaturated aliphatic group which is either branched or unbranched. Representative examples of alkenyl groups include, but are not limited to, ethenyl, E- and Z-pentenyl, decenyl, and the like.

  The term “alkynyl” as used herein, alone or in combination, includes a branched, containing at least one carbon-carbon triple bond that can occur at any stable site along the chain. Means an unsaturated aliphatic group which is branched or unbranched. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, hexynyl, decynyl, and the like.

  The term “aryl”, as used herein, alone or in combination, is substituted, which may be optionally fused to other aromatic or non-aromatic cyclic groups. Means an aromatic group which is substituted or unsubstituted. Representative examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, benzidine, xylyl, styrene, styryl, phenethyl, phenylene, benzenetriyl, and the like.

  The term “alkoxy” as used herein, alone or in combination, means an alkyl, alkenyl, or alkynyl group attached by one terminal ether linkage. Examples of alkoxy groups include 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.

  The term “aryloxy” as used herein, alone or in combination, refers to an aryl group attached by one terminal ether linkage.

  The terms “halogen”, “halide”, or “halo”, as used herein, alone or in combination, include fluorine “F”, chlorine “Cl”, bromine “Br”, iodine “ I "and astatine" At ". Representative examples of halo groups include, but are not limited to, chloroacetamide, bromoacetamide, iodoacetamide, and the like.

  The term “hetero”, as used herein, means a group that, in combination, contains one or more atoms of any element other than carbon or hydrogen. Representative examples of hetero groups include, but are not limited to, groups containing heteroatoms including but not limited to nitrogen, oxygen, sulfur, and phosphorus.

  The term “heterocycle” as used herein means a cyclic group containing a heteroatom. Representative examples of the heterocyclic ring include pyridine, piperazine, pyrimidine, pyridazine, piperazine, pyrrole, pyrrolidinone, pyrrolidine, morpholine, thiomorpholine, indole, isoindole, imidazole, triazole, tetrazole, furan, benzofuran, dibenzofuran, thiophene, Examples include, but are not limited to, thiazole, benzothiazole, benzoxazole, benzothiophene, quinoline, isoquinoline, azapine, naphthopyran, and furanobenzopyranone.

  The term “carbonyl” or “carboxy”, as used herein, alone or in combination, means a group containing a carbon-oxygen double bond. Representative examples of groups containing carbonyl include aldehydes (ie, formyl), ketones (ie, acyl), carboxylic acids (ie, carboxyl), amides (ie, amide), imides (ie, imide), esters, Examples of the anhydride include, but are not limited to, anhydride.

The term “acryl” as used herein, alone or in combination, means a group represented by CH ₂ ═C (Q) C (O) O—. Here, Q is an aliphatic or aromatic group.

  The terms “cyano”, “cyanate”, or “cyanide” as used herein, alone or in combination, mean a carbon-nitrogen double bond. Representative examples of the cyano group include, but are not limited to, isocyanate and isothiocyanate.

  The term “amino”, as used herein, alone or in combination, means a group that contains a skeletal 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.

  The term “phosphorus-containing group” as used herein means a group containing at least one phosphorus atom in an oxidized state. Representative examples include, but are not limited to, phosphoric acid, phosphinic acid, phosphate esters, phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho, phosphone, phosphoranyl, phosphoranilidene, phosphoroso and the like.

  The term “sulfur-containing group” as used herein means a group containing a sulfur atom. Representative examples include, but are not limited to, sulfhydryl, sulfeno, sulfino, sulfinyl, sulfo, sulfonyl, thio, thioxo, and the like.

  The terms “as needed” or “as needed”, as used herein, may or may not occur in subsequent events or situations, and this The description is meant to include examples where the above event or situation occurs and examples where it does not occur. For example, the expression “optionally substituted alkyl” may be substituted or unsubstituted in the alkyl group, and the description is substituted with unsubstituted alkyl. It means that both alkyls are included.

  The term “effective amount” when used with respect to a compound, product, or composition provided herein, is an amount of the compound, product, or composition sufficient to provide the desired result. Means a thing. The amount of extract 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 can be determined by one of ordinary skill in the art based on the information provided by the present disclosure by using only routine experimentation.

  The term “suitable”, as used herein, means a group that is compatible with the compound, product, or composition provided herein for the purpose described. Suitability for the purpose described can be readily determined by one skilled in the art using only routine experimentation.

  As used herein, the term “administering”, when used to describe the dosage of a compound, means a single dose or multiple doses of the compound.

  As used herein, “apoptosis” is a gradual contraction of cell volume that preserves the integrity of organelles; the condensation of chromatin that can be viewed with light or electron microscopy (ie, , Nuclear condensation); and / or one form of cell death that involves the cleavage of DNA into nucleosome-sized fragments as determined by a centrifuged sedimentation assay. Cell death occurs when cell membrane integrity is lost (eg, membrane blebbing), with engulfment of complete cell fragments (“apoptotic bodies”) by phagocytic cells.

  As used herein, the term “cancer” means any condition characterized by resistance to apoptotic stimuli.

  As used herein, the term “cancer treatment” means any treatment for cancer known in the art, including but not limited to chemotherapy and radiation therapy.

  As used herein, the term “in combination with” when used to describe administration of aminoacridine and another means of treatment, refers to aminoacridine prior to another treatment. Means that it can be administered together with, after, or in combination with another treatment.

  As used herein, the term “treatment” or “treating”, when referring to the protection of a mammal from a condition, prevents, suppresses, suppresses or eliminates this condition. Means that. Prevention of the condition includes treating the mammal prior to the onset of this condition. Suppression of the condition includes treating the mammal after induction of the condition but before its clinical findings. Suppressing the condition includes treating the mammal after a clinical finding of the condition so that the condition is alleviated or maintained. Elimination of the condition includes treating the mammal after the clinical observation of the condition so that the mammal is not subjected to any further conditions.

  As used herein, the term “tumor cell” means any cell characterized by resistance to apoptotic stimuli.

2. Mechanism mediated by NF-κB for suppression of p53 in tumors
The present invention relates to the discovery that by inhibiting NF-κB activity, p53 may be activated in cancer cells with functionally blocked p53. Inactivation of the p53 pathway in tumors is a much broader phenomenon than p53 mutations. Even though the tumor maintains wild-type p53, its function is usually either complete or partially lost. In these cases, p53 in such cancers is particularly interesting from a therapeutic point of view because it can be viewed as a target for pharmacological reactivation. There are several types of tumors in which p53 activity is blocked by tissue-specific mechanisms. Thus, overexpression of Hdm2 is particularly frequent in sarcomas, while human papillomavirus E6 inactivates p53 in most cervical cancers. RCC provides another example of this type of tumor, which, as recently reported by the inventors, is an unknown dominant mechanism by which wild-type p53 in RCC is probably tissue specific. It is a particularly interesting analysis subject. Thus, reactivation of p53 is responsible for the treatment of this so far untreatable form of cancer as well as the treatment of other cancers that have a similar mechanism for inactivating p53. It is considered an interesting policy about.

  NF-κB activity has been implicated in the inhibition of apoptosis in vitro and in vivo. At any time, many apoptotic resistant tumors acquire constitutive activation of NF-κB. Activation of NF-κB in tumor cells is probably both against natural death stimuli (eg, TNF, Fas, or TRAIL) and pharmacological death stimuli (chemotherapeutic agents). Providing resistance probably leads to its malignant phenotype. Although constitutively active NF-κB has been described in many tumor types, the relationship between NF-κB activation and p53 inhibition is not fully understood.

  Cancer (eg, cancer that has functional p53 or wild-type p53) may be treated by inhibiting NF-κB activity. This can lead to the recovery and activation of wild type p53 activity. Inhibitors of NF-κB activity are also against p53-dependent or p53-independent apoptosis by treatments such as chemotherapy, radiation therapy, naturally occurring death ligands (eg, TNF polypeptides). It may also be used to sensitize cancer. Regardless of their p53 status, most human cancers constitutively overactivate NF-κB. As a result, inhibitors of NF-κB can be used to treat any tumor, regardless of their p53 status, because of the reprogramming of the transactivated NF-κB complex into a transcriptional repression complex. it can.

(3. Aminoacridine)
Aminoacridine is a representative example of a substance that can be used to inhibit NF-κB activity. The aminoacridine can be of the following formula:

ここで、
Ｒ_１は、Ｈまたはハロゲンであり；
Ｒ_２は、Ｈまたは必要に応じて置換されたアルコキシであり；
Ｒ_３は、Ｈまたは必要に応じて置換されたアルコキシであり；そして
Ｒ_４は、Ｈまたは必要に応じて置換された脂肪族、アリール、もしくは複素環である。

here,
R ₁ is H or halogen;
R ₂ is H or optionally substituted alkoxy;
R ₃ is H or optionally substituted alkoxy; and R ₄ is H or optionally substituted aliphatic, aryl, or heterocycle.

  Representative examples of aminoacridine include 9-aminoacridine or mepacrine (also known as quinacrine), as well as aminoacridine described in Example 2. There is no limitation. The use of aminoacridine to sensitize tumor cells is attractive because many aminoacridine have only limited side effects.

  9AA has been used as a therapeutic since 1942. Certain 9AA derivatives are believed to be capable of mediating DNA damaging activity. However, the present inventors have found that 9AA and quinacrine do not show DNA damaging activity. Both 9aa and quinacrine have been shown to be more toxic to tumors than normal cells in vitro and in vivo. Furthermore, it has been shown that any compound can activate p53 for a variety of tumor cell types except RCC and can cause p53-dependent killing. Their p53-dependent anti-tumor activity clearly distinguishes aminoacridine from conventional chemotherapeutic agents based on targeting of tumors with their wild-type p53 or functional p53.

  Aminoacridine does not fit into any known category of p53 activator. Although they can cause accumulation of p53, they cannot induce phosphorylation of p53, unlike DNA damaging agents. Furthermore, aminoacridine does not cause DNA damage. Instead, the first effect of aminoacridine does not appear on p53 activation but on NF-κB suppression. This later leads to the induction of p53. Importantly, inhibition of NF-κB activates p53 function in cells that cannot be “wakened” by any of the direct approaches to p53 activation. These include Arf introduction, Hdm2 knockdown, or ectopic overexpression of p53.

  Inhibition of NF-κB is usually done through stabilization of IκB, the main negative regulator of NF-κB. Genetically, this can be done pharmacologically by mutating the regulatory phosphorylation sites of this protein and through inhibition of upstream kinases that lead to a block of IκB phosphorylation. Many known chemical inhibitors of NF-κB act through this mechanism. Stabilization of IκB results in cytoplasmic sequestration and functional inactivation of the NF-κB complex as a transcription factor.

  The activities of aminoacridine are more potent than previous drugs because they strongly promote the accumulation of NF-κB complexes in the nucleus in response to stimulation of activation with complete suppression of transactivation. May also be better. Thus, aminoacridine can act downstream of IκB and inhibit NF-κB by a mechanism that involves conversion of NF-κB to an inactive complex. NF-κB-dependent transcription deficiency is due to depletion of the pool of IκB (which is a direct transcriptional target of NF-κB) and the loss of nuclear excretion normally exerted by IκB NF-κB may be guided in the nucleus. Interestingly, the knockout of any of the cellular factors (IKKα, IKKβ, TBK1, PKC-ζ) involved in NF-κB activation does not mimic the effects of aminoacridine, This suggests that not all are targets of aminoacridine. Although it has recently been reported that nuclear accumulation of inactive NF-κB complexes containing p65 occurs after treatment of cells with UV, doxorubicin, and daunorubicin, both of these treatments are probably NF-κB. Due to the weak inhibitory activity, activating p53 is not comparable to aminoacridine.

  Aminoacridine is effective not only against the IκB phosphorylated arm of NF-κB signaling (the “standard” NF-κB activation pathway) but also through another mechanism of NF-κB activation. possible. This is supported by the ability of aminoacridine (eg, 9AA) to block stimulated NF-κB activity and also effectively reduces the basal level of constitutive NF-κB activity in tumor cells. In contrast, IKK2 inhibitors can only block stimulated NF-κB activity.

(4. Composition)
The present invention relates to compositions comprising aminoacridine and optionally a chemotherapeutic agent. The invention also relates to compositions comprising aminoacridine and optionally a TNF polypeptide.

(A. Chemotherapeutic agent)
A chemotherapeutic agent can be any pharmacological agent or compound that induces apoptosis. The pharmacological substance or compound may be, for example, a small organic molecule, peptide, polypeptide, nucleic acid, or antibody.

  The chemotherapeutic agent can be a cytotoxic agent or a cytostatic agent, or a combination thereof. Cytotoxic agents prevent cancer cells from growing by (1) interfering with the ability of the cell to replicate DNA and (2) inducing cell death and / or apoptosis in the cancer cell. Cytostatics act through the modulation, interference or inhibition of cellular signaling processes that regulate cell proliferation and are sometimes present at low continuous levels.

  Classes of compounds that can be used as cytotoxic agents include: alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazines). ); Uracil mustard, chloromethine, cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil, piperobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, And temozolomide; antimetabolites (including but not limited to folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): Totrexate, 5-fluorouracil, furoxiuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine; naturally occurring products and their derivatives (eg, vinca alkaloids, antitumor properties Antibiotics, enzymes, lymphokines, and epipodophyllotoxins: vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c, paclitaxel (paclitaxel is taxol (Taxol) ), Mitramycin, deoxyco-formycin, mitomycin-c, l-asparaginase, interfer Down (preferably, IFN-alpha), etoposide, and teniposide. Other proliferative cytotoxic agents are navelben, CPT-11, anastrazol, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

  Substances that affect microtubules interfere with cell mitosis, and they are well known in the art for cytotoxic activity. Substances that affect microtubules useful in the present invention include allocolchicine (NSC406604), halichondrin B (NSC609395), colchicine (NSC757), colchicine derivatives (eg, NEC33410), dolastatin 10 (NSC376128), maytansine (NSC153858) ), Lysoxin (NSC332598), paclitaxel (Taxol®, NSC125973), Taxol® derivative (eg, derivative (eg, NSC6088832), thiocolchicine (NSC361792), tritylcysteine (NSC83265), vinblastine sulfate (NSC49842) ), Vincristine sulfate (NSC 67574), naturally occurring epothilone and synthetic epothilone (epothi Including, but not limited to, A, epothilone B, and discodermolide (see Service, (1996) Science, 274: 2009), estramtin, nocodazole, MAP4, and the like. Examples of such materials are also described in Bulinski (1997) J. Cell Sci. 110: 3055-3064; Panda (1997) Proc.Natl.Acad.Sci.USA 94: 10560-10564; Muhlradt (1997) Cancer Res. 57: 3344-3346; Nicolaou (1997) Nature 387: 268-272; Vasquez (1997) Mol.Biol.Cell.8: 973-985; and Panda (19 96) J. Biol. Chem. 271: 29807-29812.

  Cytotoxic substances such as epidophilotoxins; anti-neoplastic enzymes; topoisomerase inhibitors; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitors; Also suitable are hormone therapeutics; leucovorin; tegafur; and hematopoietic growth factors.

  Cytostatics that can be used include, but are not limited to, hormones and steroids (including synthetic analogs): 17α-ethynylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone , Drostanolone propionate, test lactone, megestrol acetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Examples include, but are not limited to, flutamide, toremifene, and zoladex.

  Other cytostatics are anti-angiogenic agents 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. An anti-Her2 antibody from Genetech can also be utilized. A suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Also included are Imclone antibody C225 immunospecific for EGFR, and a src inhibitor.

  Casodex (R) (bicalutamide, Astra Zeneca) is also suitable for use as a cytostatic agent. This renders androgen-dependent carcinomas non-proliferative. Yet another example of a cytostatic agent is tamoxifen (Tamoxifen®), an anti-estrogen, which inhibits the growth or growth of estrogen-dependent breast cancer. Inhibitors of cell proliferation signal transduction are cytostatic agents. Representative examples include epidermal growth factor inhibitors, Her-2 inhibitors, MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Src kinase inhibitors, and PDGF inhibitors.

(B. TNF polypeptide)
The TNF polypeptide can be a member of the TNF superfamily of ligands. Representative examples of TNF polypeptides include NGF, CD40L, CD137L / 4-1BBL, TNF-α, CD134L / OX40L, CD27L / CD70, FasL / CD95, CD30L, TNF-β / LT-α, LT-β , And TRAIL, but are not limited thereto. Members of the TNF superfamily are naturally occurring proteins that are involved in the maintenance and function of the immune system and can induce apoptosis. The TNF polypeptide may be TRAIL, which induces apoptosis primarily in tumor cells but not in normal cells.

  The activity of these so-called “death ligands” is believed to be mediated by binding to members of the TNF receptor family. The TNF receptor family includes death domains that are similar in structure to their intracellular parts. Linkage of these receptors specific for individual death ligands triggers the activation of a cascade of events that result in caspase activation. Representative examples of TNF-R receptors bound by TNF polypeptides include LNGFR / p75, CD40, CD137 / 4- 1BB / ILA, TNFRI / p55 / CD120a, TNFRII / p75 / CD120b, CD134 / OX40 / Examples include, but are not limited to, ACT35, CD27, Fas / CD95 / APO-1, CD30 / Ki-1, LT-βR, DR3, DR4, DR5, DcR1 / TRID, TR2, GITR, and osteoprotegerin.

  Because of their unique ability to induce apoptosis in tumor cells, it is believed that members of the TNF family may be anticancer agents. However, many tumor cells avoid the pro-apoptotic effects of death ligands, thereby reducing the use of these substances against cancers that are sensitive to death ligands, allowing tumors to avoid host immune responses. The use of inhibitors of NF-κB can be used to sensitize tumor cells to death of death ligands (eg, TNF polypeptides).

  It is also envisioned that other materials can be used in place of the TNF polypeptide. For example, antibodies that mimic the activity of TNF polypeptides can be used. Representative examples of such antibodies include, but are not limited to, agonist antibodies to FAS, TRAIL receptor, or TNFR. In addition, aptamers and other synthetic ligands that can activate the corresponding receptors may be used.

(C. Salt)
The active ingredients of the composition can be useful in the form of various pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to the salt form that is apparent to the pharmacist, ie, substantially non-toxic, desired pharmacokinetic properties, taste, absorption, distribution, metabolism, or excretion. Means what provides the property. More practically, other factors that are also important in the selection are raw material costs, ease of crystallization, yield, stability, hygroscopicity, and fluidity of the resulting bulk drug. Conveniently, the pharmaceutical composition may be prepared from the active ingredient in combination with a pharmaceutically acceptable carrier or from a pharmaceutically acceptable salt thereof.

  Pharmaceutically acceptable salts of the active ingredient include hydrochloric acid, hydroxymethanesulfonic acid, hydrogen bromide, methanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid, sulfamic acid, Such as glycolic acid, stearic acid, lactic acid, malic acid, pamonic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethonic acid Examples include, but are not limited to, salts formed with various organic and inorganic acids, and various other pharmaceutically acceptable salts (eg, nitrates, phosphates, borates) , Tartrate, citrate, succinate, benzoate, ascorbate, salicylate, etc.) That. A cation such as a quaternary ammonium ion is envisioned as a pharmaceutically acceptable counterion for the anionic moiety. In addition, pharmaceutically acceptable salts of the compounds of the present invention include alkali metals such as sodium, potassium, and lithium; alkaline earth metals such as calcium and magnesium; dicyclohexylamine, tributylamine, and pyridine. Organic bases; and amino acids such as arginine, lysine and the like.

  Pharmaceutically acceptable salts can be synthesized by conventional chemical methods. In general, a salt will form a free base or acid in a suitable solvent or solvent mixture with a stoichiometric amount or excess of an inorganic or organic acid or base that forms the desired salt. It is prepared by reacting.

  In general, the counter ion of the salt can be determined by the reactants used to synthesize the compound. Depending on the reactants, there may be a mixture of salt counterions. For example, if NaI is added to facilitate the reaction, the counterion can be a mixture of Cl and I counteranions. Further, by preparative HPLC, if acetic acid is present in the solvent, the original counter ion may be exchanged by the acetate anion. The counter ion of the salt can be exchanged for various counter ions. The counter ion is preferably exchanged for a pharmaceutically acceptable counter ion so as to form the salt described above. Procedures for exchanging counterions are described in WO2002 / 042265, WO2002 / 042276, and S.I. D. Clas, "Quaternized Collestipol, an improved bill salt absorbent: In Vitro studies." Journal of Pharmaceutical Sciences, 80 (2): 128-131 (1991), the contents of which are incorporated herein by reference. ing. For reasons of clarity, counterions cannot be shown in the chemical structures herein.

(D. Formulation)
The composition further includes one or more other pharmaceutically acceptable ingredient (s) (eg, alum, stabilizer, antimicrobial agent, buffer, colorant, flavoring agent, adjuvant, etc.). May be.

  The composition may be in the form of a tablet or lozenge formulated in a conventional manner. For example, tablets and capsules for oral administration may contain common excipients. Excipients include, but are not limited to, binders, fillers, lubricants, disintegrants, and wetting agents. Binders include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, starch mucus, and polyvinylpyrrolidone. Bulking agents include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, 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 glycolate. Wetting agents include but are not limited to sodium lauryl sulfate. The tablets can be coated according to methods well known in the art.

  The composition may also be a liquid formulation. This includes, but is 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 medium prior to use. Such liquid preparations may contain additives. Additives include, but are not limited to, suspending agents, emulsifiers, non-aqueous media, and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fat. Emulsifiers include, but are not limited to lecithin, sorbitan monooleate, and acacia. Non-aqueous media include, but are not limited to, edible fats and oils, almond oil, coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl p-hydroxybenzoate or propyl p-hydroxybenzoate, and sorbic acid.

  The composition can also be formulated as a suppository. This can include suppository bases including but not limited to cocoa butter or glycerides. The composition may also be formulated for inhalation. This may be in a form including but not limited to solutions, suspensions, or emulsions, and may be aerosolized as a dry powder or using a high pressure gas (eg, dichlorodifluoromethane or trichlorofluoromethane). May be administered in form. The composition also includes a formulated transdermal formulation, including, but not limited to, creams, ointments, lotions, pastes, medicated poultices, patches, or films. It can be a thing.

  The composition may also be formulated for parenteral administration by methods including but not limited to injection or infusion. Injectable formulations may be in the form of suspensions, solutions, or emulsions in oily or aqueous media and include, but are not limited to, suspensions, stabilizers, and dispersants. Agents may be included. The composition may also be provided in powder form for reconstitution with a suitable medium, including but not limited to sterile pyrogen-free water.

  The composition may also be formulated as a depot preparation. This can be administered by implantation or by intramuscular injection. The composition is formulated with a suitable polymeric or hydrophobic material (eg, as an emulsion in an acceptable oil), with an ion exchange resin, or as a poorly soluble derivative (eg, as a poorly soluble salt). Sometimes it is done.

  The composition may also be formulated as a liposomal preparation. Liposome preparations can include liposomes that infiltrate the cells of interest or stratum corneum and fuse with the cell membrane, thereby resulting in delivery of the liposome contents into the cell. For example, as described in US Pat. No. 5,077,211, US Pat. No. 4,621,023, or US Pat. No. 4,508,703, which are incorporated herein by reference. Liposomes such as liposomes can be used. Compositions intended to target skin conditions can be administered before, during or after exposure of mammalian skin to UV or multiple substances that cause oxidative damage. Other suitable formulations can use iosomes. Niosomes are lipid vesicles similar to liposomes, with membranes composed mostly of non-ionic lipids, some of which transport compounds through the stratum corneum It is effective for.

(5. Treatment)
The composition can be used to treat conditions associated with NF-κB activity in vivo by administering aminoacridine to a patient in need thereof. NF-κB activity can be at any level, and its reduction leads to treatment of the condition. NF-κB activity can also be at the basal level. NF-κB activity may also be at a constitutive level. NF-κB activity may also be an induced constitutive level.

  The condition associated with NF-κB activity may be cancer. A variety of cancers can be treated including but not limited to: bladder cancer (accelerated and metastatic bladder cancer), breast cancer, colon cancer (including colorectal cancer), kidney cancer, liver cancer, Lung cancer (including small and non-small cell lung cancer, and lung adenocarcinoma), ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic cancer, rectal cancer, pharyngeal cancer, pancreas Cancer (including exocrine pancreatic cancer), esophageal cancer, stomach cancer, gallbladder cancer, cervical cancer, thyroid cancer, renal cancer, and skin cancer (including squamous cell cancer); leukemia, acute lymphocytic leukemia, acute lymph Construction of lymphatic system including blastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burkitt lymphoma Hematopoietic tumors of the myeloid lineage including acute and chronic myeloid leukemia, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; astrocytoma, neuroblastoma, glioma, and Tumors of the central and peripheral nervous systems, including schwannoma; tumors derived from mesenchyme, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and melanoma, xeroderma pigmentosum, keratinized spine cells Others including tumors, seminoma, follicular thyroid carcinoma, teratocarcinoma, renal cell carcinoma (RCC), pancreatic cancer, myeloma, myeloid leukemia and lymphoblastic leukemia, neuroblastoma, and glioblastoma tumor.

  Transformation induced by tax of HTLV, a causative agent of adult T lymphoblastic leukemia (ATL), may share the same molecular target as that associated with RCC. For example, NF-κB is constitutively active in cells transformed with tax. Similar to RCC, p53 activity is inhibited through activation of NF-κB in cells transformed with tax, and inhibition of p53 does not include p300 sequestration. Based on a common mechanism for inactivation of P53, the composition can also be used to treat HTLV-induced leukemia. Regardless of their p53 status, the majority of human cancers have NF-κB that is constitutively overactivated. The composition can also inhibit NF-κB by reprogramming the trans-activated NF-κB complex into a transcriptional repression complex. It can also be used to treat any tumor regardless of their p53 status. The composition can also be used to treat HIV infection since the HIV LTR is strongly dependent on NF-κB activity.

  The composition may also be used in adjuvant therapy to overcome anticancer drug resistance that can be caused by constitutive NF-κB activation. The anti-cancer agent can be a chemotherapeutic agent described herein.

(A. Administration)
The composition can be administered at regular intervals simultaneously with or together with other anti-cancer treatments such as chemotherapy and radiation therapy. The term “simultaneously” or “simultaneously” as used herein means that other anti-cancer treatments and compositions are 48 hours, 24 hours, 12 hours, 6 hours, 3 hours or less of each other It is meant to be administered in between. The term “at regular intervals”, as used herein, is a composition at a different timing from chemotherapy and at a fixed frequency relative to repeated dosing and / or chemotherapy regimens. Means administration of the product.

  The composition may be administered in any form including, but not limited to, oral, parenteral, sublingual, transdermal, rectal, mucosal, topical, by inhalation, buccal administration, or combinations thereof. it can. 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 that can provide sustained release of the composition and that allows slow controlled intravenous infusion.

(B. Dose)
The therapeutically effective amount of drug required for therapeutic use will vary depending on the nature of the condition being treated, the time for which activity is desired, and the age and condition of the patient, and will ultimately be determined by the attending physician Is done. Desired doses are conveniently administered in a single dose or, for example, multiple doses administered at appropriate intervals as divided doses once, twice, three times, four times or more per day It can be administered as a single dose. Multiple doses are often desired or required.

  When administered in combination with other therapeutic agents, the composition can be administered in relatively small doses. In addition, the required dosage can be relatively reduced by the use of targeting agents. Certain compositions may be administered at relatively high dosages due to factors including, but not limited to, low toxicity, high clearance, and slow cleavage rate of tertiary amines. As a result, the dosage of the composition can be from about 1 ng / kg to about 200 mg / kg, from about 1 μg / kg to about 100 mg / kg, or from about 1 mg / kg to about 50 mg / kg. The dosage of the composition is about 1 μg / kg, 25 μg / kg, 50 μg / kg, 75 μg / kg, 100 μg / kg, 125 μg / kg, 150 μg / kg, 175 μg / kg, 200 μg / kg, 225 μg / kg, 250 μg. / Kg, 275 μg / kg, 300 μg / kg, 325 μg / kg, 350 μg / kg, 375 μg / kg, 400 μg / kg, 425 μg / kg, 450 μg / kg, 475 μg / kg, 500 μg / kg, 525 μg / kg, 550 μg / kg 575 μg / kg, 600 μg / kg, 625 μg / kg, 650 μg / kg, 675 μg / kg, 700 μg / kg, 725 μg / kg, 750 μg / kg, 775 μg / kg, 800 μg / kg, 825 μg / kg, 850 μg / kg, 875 μg / Kg, 900 μg / kg, 925 μg / Kg, 950 μg / kg, 975 μg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg , 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or 100 mg / kg can be any dosage.

(6. Diagnosis method)
The composition can also be used to diagnose whether a patient's tumor can be treated with the composition. Tumor samples can be obtained from patients. Tumor cells can then be transduced with a p53 reporter system (eg, a p53-responsive lacZ reporter). The transduced cells are then incubated with the composition. Production of a signal mediated by p53 over the control indicates that the tumor can be treated with the composition.

(7. Screening method)
The present invention also relates to a method for identifying a substance that modulates NF-κB activity. Agents that modulate NF-κB activity can be identified by methods that include adding candidate modulators of NF-κB activity to a cell-based expression system activated by NF-κB. Here, modulators of NF-κB activity are identified by their ability to change the level of expression activated by NF-κB. Agents that modulate NF-κB activity can also be identified by methods that include adding candidate modulators of NF-κB activity to a cell-based expression system activated by p53. . Here, modulators of NF-κB activity are identified by their ability to alter the level of expression activated by p53. Substances that modulate NF-κB activity can also be added to the expression system activated by NF-κB or p53 by adding candidate aminoacridine and modulators of NF-κB activity, by NF-κB or p53. It can also be identified by a method that includes comparing the level of activated expression to a control. Here, modulators of NF-κB activity are identified by their ability to change the level of the expression system activated by NF-κB or p53 compared to controls.

  The cell may contain functionally silent p53. The cell may also include an NF-κB transactivation complex. The expression system activated by p53 may be a renal cancer cell line. The cell line may be a sarcoma cell line. The cell line may also be a cell line that has amplified mdm2. The cell line may also be a cell line that expresses HPV-E6 or may be a cell line that is capable of expressing HPV-E6.

Candidate substances may be present in a library (ie, a collection of compounds). Such a substance may be encoded, for example, by a DNA molecule in an expression library. Candidate substances are present in conditioned media or in cell extracts. Other such substances are known in the art as “small molecules” having a molecular weight of less than 10 ⁵ daltons, preferably less than 10 ⁴ daltons, and even more preferably less than 10 ³ daltons. Compounds. Such candidate substances may be provided as members of a combinatorial library comprising a plurality of synthetic substances (eg, peptides) prepared according to a plurality of predetermined chemical reactions. Various types of such libraries can be prepared according to established procedures, and members of a library of candidate substances can be screened simultaneously or sequentially as described herein. It will be apparent to those skilled in the art that it can.

  Screening methods can be performed in a variety of formats including in vitro assays, cell-based assays, and in vivo assays. Any cell can be used with the cell-based assay. Preferably, the cells used with the present invention include mammalian cells, more preferably human cells and non-human primate cells. Cell-based screening can be performed using genetically modified tumor cells that express surrogate markers for NF-κB and / or p53 activation. Such markers include, but are not limited to, bacterial β-galactosidase, luciferase, and enhanced green fluorescent protein (EGFP). The expression level of the surrogate marker can be measured using techniques standard in the art including, but not limited to, colorimetric analysis, luminescence analysis, and fluorescence analysis. Representative examples of cells that can be used in cell-based assays include, but are not limited to, renal cell carcinoma cells.

  The conditions under which the expected modulator is added to the cell, for example by mixing, allows the cell to undergo apoptosis or signaling in principle when there are no other regulatory compounds that interfere with apoptosis or signaling. It is a condition. Effective conditions include, but are not limited to, appropriate media, temperature, pH, and oxygen conditions that allow cell growth. Suitable media are usually solid or liquid containing growth factors and sources of carbon, nitrogen, and phosphate that can be absorbed, as well as appropriate salts, inorganic compounds, metals, and other nutrients (eg, vitamins). Medium. This includes an effective medium in which the cells can be cultured so that the cells can exhibit apoptosis or signal transduction. For example, for mammalian cells, the medium can include Dulbecco's modified Eagle medium containing 10% fetal bovine serum.

  The cells can be cultured in a variety of containers including, but not limited to, tissue culture flasks, test tubes, microtiter dishes, and petri dishes. Culturing is performed at a temperature, pH, and carbon dioxide content appropriate for the cells. Such culture conditions are also within the skill of the art.

  Methods for adding the expected regulators to cells include electroporation, microinjection, cellular expression (ie, naked nucleic acid molecules, recombinant viruses, retroviral expression vectors, and adenovirus expression). Use of an expression system including), use of ion pairing agents, and use of detergents for cell permeation, but are not limited thereto.

  The present invention has several aspects that are illustrated by the following non-limiting examples.

(Materials and methods)
(cell)
The 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, HCT116, SK-N-SH, W138 cells were obtained from ATCC. Primary cultures of normal kidney epithelial cells (NKE) are described in J. Org. Provided by Didonato (Cleveland Clinic Foundation, OH). The 041 fibroblast cell line derived from a patient with Lee-Fraumeni cancer syndrome is Provided by Stark. Mel7 and Mel29 cells are described in Kichina et al. , (2003). Oncogene
22, 4911-4917. All cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 10 mM Hepes buffer, 55 nM β-mercaptoethanol, and antibiotics.

  Reporter cell lines carrying p53-responsive β-galactosidase are described in Gurova, et al. , (2004). Cancer Res 64, 1951-1958. A reporter cell line carrying p53-responsive luciferase was generated by transfection of p21-ConALuc plasmid followed by selection with G418. Reporter cell lines carrying NF-κB-dependent luciferase were selected for co-transfection of pNF-κBLuc and pEGFP-mito (Clontech) plasmid followed by G418 (marker provided by pEGFP-mito plasmid) Obtained by. Reporter cell lines carrying myc or Clock / Bmal responsive reporters are C.I. Burkhart and M.M. Courtesy of Antoch (Cleveland Clinic Foundation, OH).

  Cells in which p53 expression was inhibited were generated by retroviral transduction of pBabeH1-sip53 or pBabeH1-siGFP vector for siRNA expression followed by selection for puromycin.

(Plasmid)
p53, Arf expression vector, pBabeH1-siHdm2, p21-ConALuc reporter plasmid are described in Gurova, et al. , (2004). Cancer
Res 64, 1951-1958. The pNF-κBLuc plasmid has Supplied by Neznanov (Cleveland Clinic Foundation, ref. 59). The pCDNA3 vector expressing pss-IκB Supplied by Budunava (Northwestern University). The pBabeH1-sip53 and pBabeH1-siGFP vectors for siRNA expression are described in Gurova, et al. , (2004) Cancer
Similar to the pBabeH1-siHDM2 vector described in Res 64,1951-1958, it was created by inserting the H1 promoter and 64 oligonucleotide loop template for siRNA expression into the left LTR of the pBabeH1-puro vector. . The sequence of siRNA for p53 and GFP is described in Brummelkamp, et al. , (2002). Science 296, 550-553. Lentiviral plasmids for expression of p53 or GFP are described in Gurova, et al. , (2004). Cancer Res 64, 1951-1958.

(Chemical substances)
The DiverSet library of 34,000 compounds is available from Chembridge, Inc. Obtained from An intensive library around 30d9 and 9AA can be found in Chembridge, Inc. Offered by All other compounds were obtained from Sigma.

(Retroviral and lentiviral transduction)
Packaging cells plated on 60 mm plates (A293 by Clontech) were transfected with 2 μg retroviral vector DNA using Lipofectamine Plus (Invitrogen) according to the manufacturer's recommendations. The medium was changed after 8 hours. Medium containing virus supplemented with 8 μg / ml polybrene (Polybrene (Sigma)) was harvested 24 and 48 hours after transfection and used for infection. Cells transduced with the virus were selected for resistance to an appropriate selection agent (G418, hygromycin, or puromycin, depending on the vector) that could completely kill uninfected cells.

Recombinant lentiviral stocks carrying p53 or EGFP (control vector) were transformed into pLV-CMV with a packaging plasmid encoding viral structural proteins and G protein of vesicular stomatitis virus using Lipofectamine reagent (Invitrogen). -Prepared using 293 cell lines transfected with p53 and pLV-CMV-EGFP plasmids. Media containing virus from 293T cells was harvested after 48 hours, transferred to target cells in the presence of 4 μg / ml polybrene, and virus was concentrated 50-100 fold by ultracentrifugation. Viral titer (usually 10 ⁸ IU / ml) was used to infect Rat 1a cells (known to be resistant to ectopic expression of p53), followed by selection against puromycin and colony Determined by count.

(Compound library screening)
2 × 10 ⁴ RCC45ConALacZ cells were plated in wells of a 96 well plate in RPMI medium without 200 μL phenol red with standard additives. After overnight incubation, a library of compounds in DMSO solution with controls was added with the aid of a plastic bacterial replicator (200 +/− 100 nL). The final concentration of the compound was approximately 5 μg / ml. The negative control was DMSO and the positive control was doxorubicin solution (0.2, 0.6, and 2 μM). After 24 hours, lysis buffer containing ONPG was added directly to the medium on ice. After 3 hours incubation at 37 ° C., β-galactosidase activity was estimated from the absorbance readings of a Wallack 1420 plate reader (Perkin Elmer) at λ = 430 mm. All compounds that elicited an ONPG response stronger than the most effective concentration of doxorubicin were considered first hits.

(Reporter assay)
In preparation for co-transfection, plasmids expressing 2 × 10 ⁵ cells in 6-well plates and expressing various concentrations of p53, Arf, Ss-IκB, or siHDM after overnight incubation. 0.5 μg reporter plasmid (p21-ConALuc or pNF-κBLuc) in combination with was transfected using Lipofectamine Plus reagent (Gibco BRL). Corresponding empty vectors were added to all transfections in amounts up to 2 μg total DNA. Normalization of transfection efficiency was performed by adding 0.2 μg of pCMV-LacZ plasmid. Luciferase activity and β-galactosidase activity were measured in lysates prepared 48 hours after transfection in cell lysis buffer (Promega) by luciferase assay system (Promega) or β-galactosidase enzyme system (Promega). Luminescent and colorimetric reactions were read with a Wallack 1420 plate reader (Perkin Elmer). Preparation of an embedded reporter. 2 × 10 ⁴ cells incorporating the reporter were plated into 96 well plates. After overnight incubation, media from cells producing compound or lentivirus was added. Cell lysates were prepared at various times using reporter lysis buffer (Promega). Luciferase or β-galactosidase activity, and protein concentration were measured in aliquots of cell lysates using standard kits (Promega, Luciferase and β-galactosidase assay systems, Biorad Protein Assay Kit).

(Cell viability assay)
5 × 10 ³ cells were plated in 6-well plates and treated with various concentrations of drugs for 24 hours. Thereafter, fresh medium without drug was added. The number of colonies was estimated after 5-6 days of incubation. Cell viability was estimated as a percentage of the intensity of methylene blue staining of treated cells compared to untreated controls (methylene blue was extracted from stained colonies with 0.1% SDS and quantified with a spectrophotometer. did).

(Cell cycle analysis)
10 ⁶ cells were plated on 100 mm plates and after overnight incubation, various concentrations of 9AA or doxorubicin were added. At the end of the incubation period, the cells were harvested, fixed and stained with propidium iodide. DNA content was measured using a FACScalibur (Becton Dickinson) and analyzed using CellQuest software.

(Western blot analysis)
Cells were treated with RIPA buffer (25 mM Tris HCl, pH 7.2, 125 mM NaCl, 1% with 1 mM PMSF (Sigma), 10 μg / ml aprotinin (Sigma), and 10 μg / ml leupeptin (Sigma). NP40, 1% sodium deoxycholate, 1 mM EDTA). Protein concentration was determined using the BioRad Dc protein assay kit. Equal amounts of protein were run on a 4-20% gradient precast gel (Novex) and blotted onto a PVDF membrane (Amersham). The following antibodies were used: anti-p53 monoclonal mouse DO1 (Santa-Cruz), anti-p21 monoclonal mouse F-5 (Santa-Cruz), anti-mdm2 monoclonal mouse SMP14 (Santa-Cruz). The phosphorylation state of p53 was determined according to the manufacturer's recommendations according to Cell signaling phospho-p53 sampler kit, anti-p65 (C20, Santa Cruz), antiphospho-p65 (ser536, Cell Signaling), anti-IκBa (C21, Santa Cruz ), Anti-p50 (NLS, Santa Cruz). HRP-conjugated secondary antibody was purchased from Santa-Cruz. Data quantification was performed using Quantity One (BioRad).

(Immunofluorescence immunostaining)
Cells in the chamber slide were washed with PBS where they were fixed with 10% phosphate buffered formalin at room temperature, 100% methanol at -20 ° C, and acetone at -20 ° C. The slides were then blocked for 1 hour in a solution of 3% BSA, 0.1% Triton X100 in PBS. Anti-p65 antibody (C-20, Santa-Cruz) was added at a concentration of 1 μg / ml in blocking solution. A secondary anti-rabbit Cy2 binding antibody (Sigma) was used. All washes were performed using a blocking solution.

(Assay for DNA-topoisomerase II activity)
HT1080 cells were labeled with 0.02 to 0.04 mCi / mL [ ¹⁴ C] thymidine, specific activity 53 mCi / mmol (Amersham) for 24 hours. Labeled HT1080 cells were treated with various concentrations of etoposide (VP-16), amsacrine (m-AMSA), or 9-aminoacridine for 1 hour. Induction of DNA cleavage mediated by Topo II was determined by measuring the precipitation of the protein DNA complex using an improvement of the SDS-KCl technique.

(Proteasome inhibition assay)
The proteasome assay kit is available from Boston Biochem, Inc. And used as recommended by the manufacturer.

(Electrophoretic mobility assay (EMSA))
Nuclear extracts are described in Chernov, et al. , (1997). Prepared as described in Oncogene 14, 2503-2510. Annealed oligonucleotide corresponding to the NF-κB binding site (Santa-Cruz) was radiolabeled with [α- ³² P] dCTP by Klenow polymerase and then with [γ- ³² P] dATP by T4 polynucleotide kinase. 10 ⁷ cpm labeled oligonucleotides were affinity purified on a Probe Quant column (Amersham). Radiolabeled oligonucleotides were added to 10 μg protein nuclear extract with 1 μg poly-dIdC (Amersham) to prevent non-specific binding and incubated for 30 minutes at room temperature. For supershift, 200 ng of anti-p65 antibody, anti-p50 antibody, or anti-antibody was added to the reaction (all antibodies by Santa Cruz). After a 30 minute incubation, all of the reaction mixture was loaded onto a 4% polyacrylamide gel in 0.5 × TBE buffer and run at 200V for 2 hours. The dried gel was exposed to an X-ray film for 30 minutes to 1 hour.

(Animal experimentation)
5-6 week old male NIH Swiss athymic nude mice were purchased from Harlan. 5 × 10 ⁶ tumor cells were inoculated into the flank of mice in 100 μL of PBS. When the tumor reached a diameter of 5 mm, intraperitoneal injection of the drug was started in a 100 μl solution of 50% DMSO in PBS (except quinacrine dissolved in PBS). As vehicle, 50% DMSO in PBS was used. Tumor size was measured in three dimensions every other day.

Example 1
(Readout for isolation of p53 activators based on RCC cells)
The transactivation function of p53 is inhibited in RCC cells by yet unknown inhibitors, which means that this type of tumor cell, as well as other tumor cells that have similar inhibition to p53, It suggests drug-mediated restoration of p53 function as an approach to selectively kill. To test whether reactivation of p53 is toxic to RCC cells, we expressed p53 ectopically in five RCC-derived cell lines in an attempt to deplete inhibitors. I let you. Cells were supplemented with an integrated p53-responsive reporter (ConALacZ) to monitor p53 reactivation. p53 cDNA was transduced using a lentiviral vector with a CMV promoter. The p53-deficient lung adenocarcinoma cell line H1299 (which is reactive to wild-type p53) and the rat fibroblast-like cell line Rat1 (which is resistant to human p53) were used as controls. .

  As shown in FIG. 1, dormant p53 in RCC could be reactivated, and this reactivation led to tumor cell death. Starting from specific expression levels, p53 became simultaneously cytotoxic and became active in inducing a reporter in RCC45 cells (FIGS. 1a and b). This indicates that cells (eg, RCC cells) can be used in a cell-based reporter system for screening for substances that can reactivate p53. This also indicates that reactivation of p53 in tumor cells (eg, RCC cells) can be cytotoxic.

(Example 2)
Identification of aminoacridine as a potent P53 activator in RCC by screening a library of compounds
In order to isolate small molecules with therapeutic potential that can also be used as tools to elucidate the mechanism of repression of RCC-specific p53, we have identified p53 trans in RCC. Direct cell-based screening of compounds capable of restoring activation was performed. RCC45ConALacZ cells were used to screen various chemical libraries of 34,000 compounds (Chembridge Corporation). β-galactosidase activity was measured in cell lysates after 24 hours of incubation with compounds. Twenty-eight compounds that induced higher β-galactosidase activity than 1 μM doxorubicin were considered first hits (FIG. 2a). The most active compound (compound 30d9) produced a 22-fold induction of the reporter in RCC45 cells, which acted 7-fold stronger than doxorubicin. A library of structural analogs consisting of 40 compounds constructed near compound 30d9 was screened using the same cell-based reporter assay. Two substances of formula 1 were found to be active.

  Thereafter, a library of 59 derivatives of compound 30d9 was screened. This included the anti-cancer drug amsacrine (amsa) and the antimalarial drug quinacrine. Twelve of the compounds tested reactivated p53 in RCC45 cells and their activity ranged from comparable (eg, amsa) to 7-10 fold higher activity than doxorubicin. . Compounds 30d9 and 9AA were the strongest (Figure 2b). Quinacrine showed a moderate level of activity. SAR analysis showed that aminoacridine can reactivate p53.

(Example 3)
(Aminoacridine is a potent activator of p53 in various cell types)
9AA was much stronger than doxorubicin not only in the induction of the p53 reporter gene in RCC45 cells but also in the induction of endogenous p53 responsive genes as determined by analysis of protein levels of p21 / Waf1 and Hdm2 ( Figures 3a and b). Interestingly, in MCF7 cells where the p53 pathway is highly active, the effect of 9AA was weaker than doxorubicin (FIGS. 3a and b).

  We tested the effect of 9AA on p53 transactivation in several other reporter cell lines with wild-type p53. In the majority of cells tested, 9AA stimulates reporter activity that is much stronger than drugs that damage doxorubicin or other DNA used (Figure 3c and data not shown), indicating that a common mechanism It is implicated in the negative control of active p53 in various tumor cell types. The strongest induction of p53 by 9AA was observed in human fibrosarcoma HT1080 cells used in parallel with the RCC cell line in many assays. The effect of 9AA was p53 dependent. This is because stimulation of reporter activity was not detected in p53 null H1299 cells or cells in which p53 expression was inhibited by siRNA (FIGS. 3c and d).

  9AA activated p53 at concentrations as low as 1 μM (FIG. 3e). The rate of activation is abnormally slow compared to stimuli that damage DNA. P53-dependent transactivation induced by 9AA became slightly detectable at 12 hours and reached a maximum after approximately 36 hours of treatment (FIG. 3f). One hour incubation with 9AA was sufficient to initiate detectable p53 activation after several hours (FIG. 3f).

Example 4
(Aminoacridine is toxic to tumors with wild-type p53)
To test whether p53 activation changes into p53-dependent cytotoxicity, we compared the effect of 9AA on the viability of cells with different p53 status. To this end, the inventors have created a series of cell lines that are syngeneic pairs that express either anti-p53 expressing constructs or control siRNA expressing constructs. The extent of p53 down-regulation by siRNA was examined by Western immunoblotting. It was found that 9AA was toxic to all cell variants tested. However, this was less toxic to cells with reduced expression of p53, and a maximal difference was observed in HT1080 cells (FIGS. 4a and b).

  Depending on dose, 9AA caused p53-dependent growth arrest (3 μM) or apoptosis (20 μM, FIG. 4 c). Low doses of 9AA still did not cause apoptosis after longer time incubations (up to 48 hours), whereas high doses of this compound induced apoptosis without prior arrest of growth. . In contrast to doxorubicin, the change in the distribution of cells in the cycle of cells lacking p53 is more intense than in control cells, probably due to a lack of control of the G1 checkpoint, in contrast to doxorubicin, A slight change was observed in p53-deficient cells treated with 9AA (FIG. 4c).

  Interestingly, either chemotherapeutic agents that act through mechanisms that cause DNA damage (eg, camptothecin, doxorubicin) or chemotherapeutic agents that act by affecting the microtubule network (eg, taxol, vinblastine) Toxicity was also revealed to be independent of p53 (FIG. 4d). This suggests that aminoacridine kills tumor cells through a different mechanism than conventional chemotherapeutic agents. Since normal cells also have active p53, we tested the toxicity of 9AA against normal kidney epithelial cells and human diploid fibroblast WI38. Both of these normal cell types were more resistant to 9AA compared to RCC or other tumor cells (FIGS. 4e and f).

(Example 5)
(Aminoacridine-based drugs have antitumor effects in vivo)
The in vivo antitumor effects of aminoacridine were tested in a xenograft tumor model using HT1080 cells that differ in their p53 status and have a p53 luciferase reporter grown subcutaneously in nude mice. The activity of the p53-dependent luciferase reporter in tumor cells was used to test the bioavailability of 9AA in vivo. Both 9AA and quinacrine were able to activate p53 in tumors (FIG. 5a), and both compounds showed similar properties as the above experiments (data not shown).

  To fully elicit p53 dependence on the effect of quinacrine, mice were inoculated with HT1080 sip53 cells (left flank) and HT1080 siGFP cells (right flank). After the tumor reached a diameter of 5 mm, mice were injected intraperitoneally with 50 mg / kg quinacrine daily and 5-fluorouracil (5FU, 35 mg / kg) was used for comparison. As shown in FIG. 5b, quinacrine inhibited the growth of tumors expressing p53 to the same extent as 5FU and had no effect on the growth of p53-deficient tumors. This indicates that aminoacridine inhibits xenograft tumor growth in vivo in a p53-dependent manner (FIG. 5b).

Mice were also inoculated with 2 × 10 ⁶ human prostate cancer cells using PC3 p53 negative or DU145 cells containing mutant p53. 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 approximately 50% in mice treated with quinacrine. Interestingly, this indicates that aminoacridine also inhibits xenograft tumor growth in vivo in a p53-dependent manner.

(Example 6)
(Mechanism of p53 activation mediated by aminoacridine)
We first tested whether aminoacridine activates p53 through DNA damage, a well-known mechanism of action for compounds that induce p53. Since one of the 9AA derivatives, amsa, causes DNA damage through the toxicity of topoisomerase II, we have compared to another inhibitor of topoisomerase II, amsa and etoposide, that 9AA is topoisomerase in vitro. It was tested and found to have no effect on II activity (FIG. 6a). Furthermore, 9AA has no effect on the phosphorylation status of p53, as can be expected from drugs that damage DNA that activates p53 via kinases that are sensitive to DNA cleavage (ATM, ATR, etc.). Did not give. Doxorubicin used in these experiments as a positive control induced phosphorylation of p53, confirming that signaling by upstream p53 is functional in RCC (FIG. 6b). This result shows that aminoacridine activates p53 by a mechanism distinct from DNA damage, along with previous results showing that various types of DNA damage do not activate p53 in RCC. ing.

  Proteasome inhibitors constitute another class of p53 activators that cause the accumulation of unmodified p53 protein. Accumulation of unphosphorylated p53 in response to treatment with 9AA and significant localization of the drug in the cytoplasm (monitored by fluorescence microscopy) suggests that aminoacridine can act as an inhibitor of proteasome degradation. I did. This hypothesis is based on the use of a direct in vitro assay (FIG. 6c) and by monitoring the effect of 9AA on the level of IκB, another target for proteasome degradation [21] Used as an indicator unrelated to proteasome activity). MG132, an inhibitor of proteasome degradation used as a positive control, resulted in the accumulation of a phosphorylated form of IκB, but surprisingly had the opposite effect depending on the treatment with 9AA, which A gradual decrease and subsequent complete disappearance was led (Fig. 6d and e). These results indicate that aminoacridine does not activate p53 by inhibition of the proteasome.

(Example 7)
(Aminoacridine inhibits NF-κB)
As shown in Example 6, the degradation of IκB was unexpectedly induced by 9AA. Based on these results, we focused on the possible effects of aminoacridine on the NF-κβ pathway. We used p53-null H1299 cells incorporating an NF-κB responsive reporter to test the effect of 9AA on the transcriptional activity of NF-κB. We found that both 9AA and quinacrine inhibited the basal activity of the reporter and the activity induced by TNF- (Fig. 7a). They were effective when added before stimulation with TNF (−24 to 0 hours), simultaneously with stimulation with TNF (0 hours), or after stimulation with TNF (0 to 6 hours) (Data not shown). They also inhibited TNF-stimulated induction of IκB, an integral part of the NF-κB feedback regulatory loop (FIG. 7b). Surprisingly, the block of NF-κB-dependent transactivation by 9AA and quinacrine occurred simultaneously with a simultaneous dose-dependent increase in the DNA binding activity of NF-κB (FIG. 7c). Some increase is still observed in the absence of stimulation with TNF, which is mainly related to the p50 / p50 homodimer (FIG. 7d). This increase was particularly strong when the drug was administered in combination with TNF, which involved both p65 / p50 and p50 / p50 NF-κB complexes (FIGS. 7c and d).

  Increased DNA binding activity of NF-κB complex correlates with nuclear accumulation of NF-κB complex containing p65 when stimulated with TNF in the presence of 9AA when observed by immunofluorescence staining Was. We have found that p65 enters the nucleus independently of the presence of 9AA, but the drug significantly extends its presence time in the nucleus (FIG. 7e). This is probably due to a lack of IκB, which plays a role in shuttle transport of NF-κB from the nucleus.

  These results indicate that aminoacridine is a potent inhibitor of NF-κB transactivation acting through an unusual mechanism. In contrast to previously described inhibitors that act by stabilizing IκB, aminoacridine can act by converting the NF-κB complex to a transcriptionally inactive state. This is trapped in the nucleus due to the lack of induction of its shuttle factor IκB.

  Among the mechanisms that can respond to functional inactivation of the NF-κB complex, histone deacetylation is achieved by deficiency of p65 phosphorylation (reported to be essential for NF-κB activity). Enzyme (HDAC) is recruited to the complex, resulting in the conversion of the chromatin at the transcription start site to an inactive form of chromatin. To test this possibility, we analyzed the effect of 9AA on the phosphorylation of p65 in the nucleus of cells treated with TNF. In cells treated with TNF alone, the proportion of phosphorylated p65 increased in parallel with total p65. In cells treated with TNF and 9AA, the proportion of phosphorylated p65 did not increase (FIG. 7f). These results indicate that in the presence of aminoacridine, p65 preferentially enters the nucleus in a phosphorylated state and thus probably in an inactive form.

  Inactive NF-κB exists as a complex with HDAC in the unstimulated nucleus. Inhibition of HDAC activity by trichostatin A (TSA) results in NF-κB-dependent transcriptional activation, which is not accompanied by further stimulation. If inactivation of NF-κB by aminoacridine is also associated with HDAC, it should have no effect on the TSA-stimulated induction of NF-κB. Indeed, treatment of NF-κB reporter cells with TSA resulted in significant activation of NF-κB-dependent transcription that could not be blocked by 9AA (FIG. 7h). These results indicate that aminoacridine can result in the accumulation of inactive NF-κB complexes and that their inactivation is related to HDAC.

  Blocking phosphorylation of p65 may not be the only mechanism for aminoacridine anti-NF-κB activity. We have shown that treatment with 9AA results in an increase in p50 in the cytoplasmic and nuclear pools (FIG. 7g). p50 / p50 homodimer (the proportion increased with 9AA-mediated accumulation of this NF-κB subunit (FIG. 7d)) was also implicated in the formation of an inactive NF-κB complex. There was a possibility.

(Example 8)
(Aminoacridine activates p53 in RCC through inhibition of NF-κB)
Aminoacridine produces two strong effects in RCC cells. They activate p53 in an unusual manner not related to DNA damage or inhibition of proteasome degradation. This also inhibits NF-κB by converting the transcriptionally active NF-κB complex to a transcriptionally inactive form in an abnormal manner. This is probably due to inhibition of p65 / RelA phosphorylation. The effect of aminoacridine on p53 and NF-κB is very specific. This is because they have no effect on transcription regulated by c-Myc, or N-Myc, androgen receptor, or other tested factors such as CLOCK / BMAL1 (data not shown) ). We have determined whether the activation of p53 and inhibition of NF-κB by aminoacridine is related to, or independent of, drug activity. I was interested in knowing if this is an early event. The present inventors could easily exclude the possibility that the activation of p53 may be a factor in suppressing NF-κB. This is because all of the effects of aminoacridine on the NF-κB pathway were seen in p53 deficient cells (H1299) and even p53 wild type cells (HT1080) cells. In addition, inhibition of NF-κB by aminoacridine can be detected within 1 hour after compound administration, but their effects on p53 are usually slow and treatment of 12 to 16 hours is necessary to begin to see effects. It is necessary (FIG. 2d). To study the opposite model (inhibition of NF-κB by aminoacridine drives activation of p53), we (i) p53 activity when NF-κB is inhibited by another mechanism. And (ii) the effect of aminoacridine on p53 activation in NF-κB-deficient cells was analyzed.

  To further confirm the dual effect of 9AA and quinacrine on p53 and NF-κB, we treated with two doses of 9AA (2 μM and 10 μM, causing growth arrest and apoptosis, respectively). Changes in the overall gene expression profiles of the two RCC cell lines, RCC45 and RCC54, were analyzed using microarray hybridization. RNA was isolated 16 hours after treatment, and this treatment was sufficient to induce p53 before signs of cell death occurred. Only 0.6% of the 36847 genes displayed on the array changed their mRNA levels by at least 2-fold with at least one dose in both cell lines. The most up-regulated genes include p21, mdm2, and several other p53 targets (Table 1). On the other hand, IκBα, IL-8, and some chemokines and cytokines (Table 2) (all encoded by NF-κB responsive genes) were strongly suppressed as a result of treatment. These results confirmed the dual effect of aminoacridine as an inducer of p53 and an inhibitor of NF-κB transcription.

  To suppress NF-κB activity in RCC cells, we used the IκB supersuppressor (ss-IκB), a stable IκB mutant lacking both phosphorylation sites. . Transduction of this mutant into RCC ACHN cells resulted in a 3-fold inhibition of NF-κB reporter activity (FIG. 8a), which is constitutive of all the RCC cell lines tested by NF-κB. It was consistent with our observation that it was active. Importantly, the activity of the p53-responsive reporter in the same cells increased 5-fold when NF-κB inhibitory ss-IκB was transduced (FIG. 8b). Similar effects were observed in another RCC strain, RCC54, as well as in non-RCC cells, HT1080 (data not shown). All of these responded to 9AA by p53 activation (FIG. 3a). Of note, ss-IκB activated p53 in RCC much more strongly than transduction of “direct” regulators of the p53 pathway (eg, Arf, siRNA to Hdm2, or p53 itself). (Figure 8b). Thus, the inventors have shown that blocking NF-κB activity can restore p53 transactivation in RCC cells and that aminoacridine probably acts through this mechanism.

  In order to test whether inhibition of NF-κB by aminoacridine is solely responsible for its p53 activation effect, we have NF-κB inhibited by ss-IκB. RCC cells were treated with 9AA. The present inventors could not create an RCC cell line that stably expresses ss-IκB. This is because inhibition of NF-κB interfered with the viability of RCC cells (unpublished observation). Therefore, the effect of aminoacridine on cells in which NF-κB was suppressed was tested in a transient transfection experiment. Here, the introduction of ss-IκB was combined with either NF-κB or a p53-responsive reporter. Transfection of ss-IκB into ACHN inhibited NF-κB reporter activity to a level where no further inhibition occurred with treatment with 9AA. Under these conditions, 9AA can no longer activate the p53-responsive reporter (FIG. 8c), indicating that the p53 activation effect of aminoacridine is mediated by inhibition of NF-κB function. And NF-κB activity is responsive to p53 inhibition in RCC.

Example 9
(Sensitization of resistant tumor cells)
We next tested the ability of aminoacridine to sensitize tumor cells that are resistant to death ligands. We have treated tumor cells derived from prostate, kidney, lung, and breast that were originally resistant to treatment with death ligand in the presence of non-toxic or low-toxic concentrations of aminoacridine. When found, it was found to be dramatically sensitized to TNF, Fas, and TRAIL (FIG. 9).

(Example 10)
(Anti-tumor application of aminoacridine)
We tested the efficacy of aminoacridine against conventional chemotherapy. For this purpose, the inventors have identified several sets of tumor cells derived from RCC and tumor cells derived from other than RCC (6 types for each type), as well as a set of normal kidney cells (NKE). The IC50% of these chemotherapeutic agents (doxorubicin, taxol, and 5-fluorouracil), 9AA, and quinacrine (QC) were compared. Non-RCC cell lines included MCF7 (wt p53), HT1080 (wt p53), H1299 (p53 null), U2OS (wt p53), LNCaP (wt p53), and HCT116 (wt p53). The average IC50% of RCC was higher than the non-RCC cell line for all chemotherapeutic agents used and was close to that of normal cells. This was consistent with the clinical report (Figure 10). 9AA and quinacrine efficiently restored p53-mediated transactivation in nine of the ten RCC cell lines tested (data not shown). These compounds were equally effective against both RCC and non-RCC cells, regardless of the latter p53 status. See also Table 3.

  The inventors next confirmed that aminoacridine produces similar effects in ex vivo organ cultures. A normal kidney fragment from the same patient as the new RCC specimen was placed in a short organ culture in medium containing a lentiviral vector with a p53-responsive lacZ reporter (FIG. 10B) for 24 hours. . Tissue samples were then treated with quinacrine or doxorubicin for an additional 24 hours, then fixed with glutaraldehyde and stained using X-gal (blue staining) for β-galactosidase activity. As shown in FIG. 10B, quinacrine induced expression of a p53-responsive reporter in tumor samples, but not doxorubicin. With any drug, no reporter induction could be detected in normal kidney samples (a normal kidney structure expressing endogenous β-galactosidase activity was seen). X-gal positive staining was not observed in all tumor samples that were not transduced with the reporter. Therefore, p53 function was restored by aminoacridine. This assay can also be used as a prognostic criterion that can predict tumor responsiveness to aminoacridine treatment.

  The combination of two important properties (inhibition of NF-κB activity and activation of p53) in one compound provides a possible use of aminoacridine. Tumor cell resistance to TRAIL, one of the most promising naturally occurring anticancer cytokines, is usually associated with constitutively active NF-κB and inhibition of p53. This controls the expression of DR5, one of the TRAIL receptors. 9AA was able to induce the expression of TRAIL reporter (DR5) in the two RCC cell lines tested (Table 1). Therefore, compounds capable of simultaneously inhibiting NF-κB and activating p53 are expected to mask tumor cells that are resistant to TRAIL. We tested this by treating RCC54 and RCC45, TRAIL resistant RCC cell lines, in combination with quinacrine. Normal kidney epithelial cells were used for comparison. As shown in FIG. 10C, quinacrine reversed tumor TRAIL resistance, but not normal kidney cells, indicating another form of application of aminoacridine to cancer.

  Finally, we tested the effect of quinacrine on the growth of tumor xenografts formed by ACHN cells subcutaneously injected into nude mice and was observed when 5-fluorouracil (5FU) was used. Similar to the effect, we found about 50% inhibition of tumor growth. However, there was no significant weight loss (up to 20%) with 5FU administration (FIG. 10D).

Claims (1)

Invention described in this specification.

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