WO2017091952A1 - Akt2在诊断和治疗肿瘤中的用途 - Google Patents

Akt2在诊断和治疗肿瘤中的用途 Download PDF

Info

Publication number
WO2017091952A1
WO2017091952A1 PCT/CN2015/096011 CN2015096011W WO2017091952A1 WO 2017091952 A1 WO2017091952 A1 WO 2017091952A1 CN 2015096011 W CN2015096011 W CN 2015096011W WO 2017091952 A1 WO2017091952 A1 WO 2017091952A1
Authority
WO
WIPO (PCT)
Prior art keywords
akt2
tumor
group
inhibitor
dexamethasone
Prior art date
Application number
PCT/CN2015/096011
Other languages
English (en)
French (fr)
Inventor
谢彦晖
谢咪雪
Original Assignee
谢彦晖
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 谢彦晖 filed Critical 谢彦晖
Priority to EP15909469.7A priority Critical patent/EP3375445A4/en
Priority to CN201580084316.6A priority patent/CN108472296A/zh
Priority to PCT/CN2015/096011 priority patent/WO2017091952A1/zh
Priority to US15/778,479 priority patent/US10849906B2/en
Priority to CN202210336057.3A priority patent/CN115109850A/zh
Priority to JP2018546727A priority patent/JP6975720B2/ja
Publication of WO2017091952A1 publication Critical patent/WO2017091952A1/zh
Priority to JP2021181430A priority patent/JP7303407B2/ja

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo

Definitions

  • the invention belongs to the field of biological and medical applications, relates to tumor diagnosis and treatment, in particular to Akt kinase subtype Akt2 detecting agent for diagnosing glucocorticoid-resistant tumors and Akt2 inhibitors combined with glucocorticoids for treating tumors, especially lymphocytic leukemia As well as myeloma and lymphoma.
  • Akt1 and Akt2 act as Akt subtypes, which play a role in regulating cell growth and regulating blood glucose. It has been suggested that Aktl is an important target for glucocorticoid resistance in cells. However, there has been no report on the correlation between Akt2 in the development of lymphocyte-derived tumors and the occurrence of drug resistance and prognosis.
  • Glucocorticoid is one of the clinically effective drugs that can initiate lymphocyte apoptosis and is the first line of chemotherapy for various hematological lymphomas.
  • Glucocorticoid resistance is a common problem in the treatment of clinical lymphoid tumors and is an important cause of treatment failure. At present, although the research on glucocorticoid resistance has made some progress, the specific molecular resistance mechanism is still unclear.
  • glucocorticoid resistance is not associated with up-regulation of glucocorticoid receptor (GR), GR structural function, chaperone expression and gene mutation, and GR mutations are extremely rare; prednisone resistance and multidrug resistance genes (eg ABCB1, ABCB4, ABCC1, ABCG2, and MVP) have nothing to do; another study found many target genes that may cause glucocorticoid resistance, and initially confirmed that glucocorticoid sensitivity and drug resistance are mediated by different cellular signaling pathways. . Therefore, there is a need in the art for new methods for treating glucocorticoid-resistant tumors.
  • GR glucocorticoid receptor
  • the invention provides a method of diagnosing a glucocorticoid-resistant tumor comprising measuring the expression of Akt2 in said tumor cell, wherein Akt2 expression is increased in said measured tumor cell relative to a glucocorticoid-sensitive tumor cell High indicates that the tumor is glucocorticoid resistant.
  • the invention provides the use of a detection agent for detecting Akt2, such as an antibody to Akt2 protein, or a probe or primer for detecting Akt2 mRNA, for use in the preparation of a composition or kit for diagnosing a glucocorticoid-resistant tumor .
  • a detection agent for detecting Akt2 such as an antibody to Akt2 protein, or a probe or primer for detecting Akt2 mRNA
  • the invention provides a detection agent for detecting Akt2, such as an antibody to the Akt2 protein, or a probe or primer for detecting Akt2 mRNA for use in diagnosing a glucocorticoid-resistant tumor.
  • a detection agent for detecting Akt2 such as an antibody to the Akt2 protein, or a probe or primer for detecting Akt2 mRNA for use in diagnosing a glucocorticoid-resistant tumor.
  • the invention provides a method of treating a tumor, such as promoting tumor sensitivity to glucocorticoid therapy or treating a glucocorticoid-resistant tumor, comprising administering to a tumor patient a therapeutically effective amount of an Akt2 inhibitor and a glucocorticoid.
  • the present invention provides a pharmaceutical composition for treating a tumor, for example, promoting or enhancing the sensitivity of a tumor to glucocorticoid therapy or treating a glucocorticoid-resistant tumor, including an Akt2 inhibitor and a glucocorticoid, and
  • the pharmaceutically acceptable carrier, excipient and/or diluent present is selected.
  • the invention provides the use of an Akt2 inhibitor for the preparation of a pharmaceutical composition for treating a tumor, for example, promoting tumor sensitivity to glucocorticoid therapy or treating a glucocorticoid resistant tumor.
  • the pharmaceutical composition comprises a glucocorticoid and optionally a pharmaceutically acceptable carrier, excipient and/or diluent.
  • the invention provides an Akt2 inhibitor for use in treating a tumor, for example, to promote or enhance the sensitivity of a tumor to glucocorticoid therapy or to treat a glucocorticoid resistant tumor.
  • the tumor is a lymphocyte-derived tumor, such as lymphocytic leukemia, lymphoma such as B-cell lymphoma or T-cell lymphoma or myeloma.
  • the tumor is a T cell derived tumor such as a T lymphocyte leukemia, a T cell lymphoma, and a myeloma.
  • the lymphocytic leukemia is selected from the group consisting of acute lymphocytic leukemia and chronic lymphocytic leukemia.
  • the tumor is selected from the group consisting of acute T lymphocytic leukemia.
  • the glucocorticosteroid is selected from the group consisting of dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, beclomethasone, prednisolone, prednisone, methylprednisone.
  • Dragon hydrocortisone, cortisone acetate, budesonide, beclomethasone dipropionate, ciclesonide, cortisone, methylprednisolone, clobetasol butyrate, flupromone, propionate Clozamodium, mometasone furoate, fluoxetine, clobetasone propionate, fluhexoxacin, halotasone, diflufenone and its derivatives.
  • Figure 1 Correlation between dexamethasone and Akt/FoxO3a pathway protein expression.
  • Figure 1A CCRF-CEM flow cytometry: Apoptosis rate of CCRF-CEM cells induced by different concentrations of dexamethasone.
  • Figure 1B Akt/FoxO3a/Bim signaling in CCRF-CEM cells after dexamethasone treatment Western blot of pathway protein expression.
  • p-Akt phosphorylated Akt
  • p-FoxO3a phosphorylated FoxO3a.
  • Figure 1C Apoptosis rate induced by different concentrations of dexamethasone in CEM-DR cells.
  • Figure 1D Akt/FoxO3a/Bim signaling pathway protein expression in CCRF-CEM and CEM-DR cells.
  • Figure 2 Effect of cell pathway inhibitors in combination with glucocorticoids on apoptosis.
  • Figure 2A Apoptosis rate of CCRF-CEM groups under the action of Akt inhibitor and PI3K inhibitor LY294002.
  • Figure 2B Western blot of Akt/FoxO3a/Bim signaling pathway protein expression in CCRF-CEM cells of Akt inhibitor and PI3K inhibitor LY294002.
  • Figure 2C Apoptosis rate of CCRF-CEM cells after combination of various concentrations of Akt inhibitor and dexamethasone.
  • Figure 2D Apoptosis rate of CCRF-CEM cells under the action of Akt inhibitors.
  • Figure 2E Apoptosis rate of Molt-4 cells in the presence of Akt inhibitor and PI3K inhibitor LY294002.
  • Figure 2F Apoptosis rate of Jurkat group under the action of Akt inhibitor and PI3K inhibitor LY294002.
  • Figure 2G Apoptosis rate of CCRF-CEM cells in the presence of 2-DG.
  • Figure 2H Akt/FoxO3a/Bim signaling pathway protein expression in CCRF-CEM cells under 2-DG.
  • Figure 2I Western blot of FoxO3a protein expression in CCRF-CEM cells of the Notch1 pathway inhibitor dapt.
  • Figure 2J Apoptosis rate of CCRF-CEM groups under the action of Notch1 pathway inhibitor dapt.
  • Figure 2K Apoptosis rate of tumor lymphocytes under the action of SGK inhibitor GSK.
  • Figure 2L Apoptosis rate of CCRF-CEM cells compared to other cell proliferation pathway inhibitors by Akt inhibitors.
  • Figure 2M Apoptosis rate of SP2/0 cells under the action of Akt inhibitors.
  • Figure 2N Apoptosis rate of Raji cells under the action of Akt inhibitors.
  • Figure 3 Sensitization of glucocorticoids by Akt inhibitors in nude mice.
  • Figure 3A Tumor formation in each group of CCRF-CEM tumor-bearing mice.
  • Figure 3B Subcutaneous tumor mass size of each group of CCRF-CEM tumor-bearing mice.
  • Figure 3C HE staining of tumors of each group of CCRF-CEM tumor-bearing mice.
  • Figure 3D Comparison of overall survival of tumor-bearing mice in each group.
  • Figure 3E Apoptosis rate of spleen cells in tumor-bearing mice of each group.
  • Figure 3F Liver transaminase ALT and AST indicators after administration of tumor-bearing mice in each group.
  • Figure 4 Akt inhibitor in combination with glucocorticoids.
  • Figure 4A Apoptosis rate of CCRF-CEM cells induced by 0.1 ⁇ M dexamethasone concentration combined with different concentrations of Aktl, Akt2, and Aktl/2 inhibitors.
  • Figure 4B CCRF-CEM cell activity under different concentrations of dexamethasone in combination with Akt subtype inhibitors.
  • Figure 4C Apoptosis rate of CCRF-CEM groups under the action of Akt subtype inhibitors.
  • Figure 4D Apoptosis rate of CCRF-CEM groups in the presence of Akt subtype inhibitors.
  • Figure 4E Cell viability of each group of CCRF-CEM in a fixed concentration ratio of Akt subtype inhibitor combined with dexamethasone.
  • Figure 4F IC50 values of dexamethasone in CCRF-CEM groups under the action of Akt subtype inhibitors.
  • Figure 4G CEM-DR cell activity under different concentrations of dexamethasone combined with Akt isoform inhibitor.
  • Figure 4H Apoptosis rate of CEM-DR groups under the action of Akt subtype inhibitors.
  • Figure 4I Cell viability of CEM-DR groups in a fixed concentration ratio of Akt subtype inhibitor combined with dexamethasone.
  • Figure 4J Apoptosis rate of Jurkat group under the action of Akt subtype inhibitors.
  • Figure 4K Cell viability of Jurkat group in combination with Akt subtype inhibitor and dexamethasone at a fixed concentration ratio.
  • Figure 4L Apoptosis rate of Daudi groups under the action of Akt subtype inhibitors.
  • Figure 4M Apoptosis rate of Daudi group in combination with Akt subtype inhibitor and dexamethasone at a fixed concentration ratio.
  • Figure 4N IC50 values of dexamethasone in CEM-DR, Jurkat, and Daudi cells under the action of Akt subtype inhibitors.
  • Figure 5 Effect of Akt subtype inhibitor in combination with glucocorticoids on cell viability.
  • Figure 5A Total and phosphorylated Aktl and Akt2 protein expression in cells of each group of CCRF-CEM cells.
  • Figure 5B Total FoxO3a protein expression in cells of each group of CCRF-CEM cells.
  • Figure 5C Phosphorylation of FoxO3a protein expression in cells of each group of CCRF-CEM cells.
  • Figure 5D Bim protein expression in cells of each group of CCRF-CEM cells.
  • Figure 5E Expression levels of Aktl and Akt2 in various lymphoma cells and hepatocytes.
  • Figure 5F Fluorescence of Jurkat cells transfected with small interfering RNA.
  • Figure 5G Small interfering RNA interferes with Aktl and Akt2 protein expression in Jurkat cells.
  • Figure 5H Apoptosis rate of each group after transfection of Jurkat cells with small interfering RNA.
  • Figure 5I Expression of Akt1 mRNA and Akt2 mRNA in lymphocytes of patients with newly diagnosed and relapsed refractory acute lymphoblastic leukemia.
  • Figure 5J ROC analysis of Akt2 mRNA in lymphocytes of patients with newly diagnosed and relapsed refractory acute lymphoblastic leukemia.
  • Figure 6 Effect of Aktl, Akt2 inhibitors combined with dexamethasone on normal liver cells.
  • Figure 6A Inhibition of cellular activity of normal liver cells L-02 by combination of Aktl, Akt2 inhibitor and dexamethasone.
  • Figure 6B Expression of total and phosphorylated Akt isoforms in L-02 cells under the action of Aktl and Akt2 inhibitors.
  • Figure 6C Total FoxO3a protein expression in L-02 cells after 24 hours of Akt isoform inhibition.
  • Figure 6D Phosphorylation of FoxO3a protein in L-02 cells after 24 hours of Akt isoform inhibition.
  • Figure 6E Bim protein expression in L-02 cells after 24 hours of Akt subtype inhibitor action.
  • Figure 6F Comparison of L-02 activity of hepatocytes after 24 hours of Akt subtype inhibitor action.
  • Figure 6G Peripheral blood alanine aminotransferase (ALT) index in each group of nude mice.
  • Figure 6H Peripheral blood aspartate aminotransferase (AST) index of each group of nude mice.
  • Figure 6I Peripheral blood total bilirubin (TBIL) index of each group of nude mice.
  • Figure 6J Peripheral blood white blood cell counts of nude mice in each group.
  • Figure 6K Peripheral blood red blood cell counts of each group of nude mice.
  • Figure 6L Peripheral blood hemoglobin in each group of nude mice.
  • Figure 6M Peripheral blood platelet counts of nude mice in each group.
  • Figure 6N Blood glucose levels of nude mice in each group.
  • Figure 6O Peripheral serum creatinine index of each group of nude mice.
  • Figure 7 Effect of Akt inhibitor in combination with dexamethasone in nude mice.
  • Figure 7A Subcutaneous tumor size after administration of each group of CCRF-CEM tumor-bearing mice.
  • Figure 7B Each group of CCRF-CEM tumor-bearing mice after administration Subcutaneous tumor size comparison.
  • Figure 7C Spleen size after administration of each group of CCRF-CEM tumor-bearing mice.
  • Figure 7D Overall survival of each group of CCRF-CEM tumor-bearing mice after administration.
  • Figure 7E HE staining of tumors in each group of nude mice: the arrow indicates the area of necrosis in the tumor.
  • Figure 7F Ki-67 staining of tumors in each group of nude mice.
  • Figure 7G HE staining of spleen in each group of nude mice.
  • Figure 7H Spleen CD3 staining of each group of nude mice.
  • Figure 7I Spleen TDT staining of each group of nude mice.
  • Figure 8 Pathological section of organs in nude mice.
  • Figure 8A Liver pathology sections of each group of nude mice (HE: 10*20).
  • Figure 8B Liver pathological sections of each group of nude mice (HE: 10*40).
  • Figure 8C Cardiac pathological sections of each group of nude mice (HE: 10*20).
  • Figure 8D Pathological sections of lungs of each group of nude mice (HE: 10*20).
  • Figure 8E Renal pathological sections of each group of nude mice (HE: 10*20).
  • Akt2 expression is increased as compared with sensitive cells.
  • Akt2 inhibitor By inhibiting the Akt2 protein with an Akt2 inhibitor, glucocorticoid-resistant tumor cells are more sensitive to glucocorticoids.
  • Akt2 subtype inhibitors significantly increase the sensitivity of glucocorticoid-induced lymphocyte apoptosis; in drug-resistant cell lines, Akt2 subtype inhibitors have a good synergistic effect with glucocorticoids. Reverse glucocorticoid resistance.
  • Akt2 subtype inhibitors can significantly down-regulate the intracellular p-FoxO3a/total FoxO3a ratio, up-regulate the expression of the pro-apoptotic protein Bim, and increase the intracellular FoxO3a/Bim signaling pathway to increase glucocorticoid-induced lymphocyte apoptosis. Increase the sensitivity of lymphocyte glucocorticoids.
  • Akt2 protein expression is associated with glucocorticoid sensitivity in cells, and overexpression of Akt2 protein in cells may be an important mechanism for lymphocyte resistance to glucocorticoids.
  • Akt2 can be used as a more precise therapeutic target for reversing the glucocorticoid resistance of lymphoma, and can also be used as a target to enhance the sensitivity of lymphoma to glucocorticoid therapy, and can also predict whether lymphocytes are glucocorticoid resistant. index.
  • Akt2 subtype inhibitors had the least toxicity and had no effect on blood system, liver function, kidney function and blood glucose in mice.
  • Akt2 subtype inhibitors can synergize with glucocorticoids such as dexamethasone to effectively reduce tumor and spleen size of tumor-bearing mice, cause liquefaction and necrosis of the tumor, and prolong overall survival;
  • Akt2 subtype inhibitor is glucocorticoid sensitization The drug with the best effect and minimal side effects.
  • the invention provides a method of diagnosing a glucocorticoid-resistant tumor comprising measuring Akt2 levels in said tumor cells, wherein said measured tumor is relative to a glucocorticoid-sensitive tumor cell An increase in Akt2 levels in the cells indicates that the tumor is glucocorticoid resistant.
  • an elevated level of Akt2 means an increase in the expression level of the Akt2 protein and/or an increase in the activity of the Akt2 protein.
  • Measuring the expression level of Akt2 can be carried out by various methods known in the art for measuring expression levels, for example, at the nucleic acid level by measuring the level of mRNA or measuring the level of protein at the protein level.
  • Measurement of the activity of the Akt2 protein can also be carried out by various methods known in the art for measuring Akt2 activity, such as phosphorylation activity.
  • the glucocorticoid-sensitive tumor refers to a tumor cell semi-inhibitory concentration (IC50) value ⁇ 10 ⁇ M
  • the clinical application includes a tumor in which the glucocorticoid treatment protocol can be effectively alleviated.
  • IC50 tumor cell semi-inhibitory concentration
  • the tumor cell semi-inhibitory concentration (IC50) value of dexamethasone is ⁇ 10 ⁇ M
  • the tumor is a dexamethasone-sensitive tumor.
  • a glucocorticoid-resistant tumor refers to a tumor cell semi-inhibitory concentration (IC50) value of ⁇ 10 ⁇ M, and these tumors are resistant to glucocorticoid action, resulting in a decrease in the therapeutic effect of glucocorticoid; sensitivity to glucocorticoids Compared to tumors, glucocorticoid-resistant tumors require more glucocorticoids to achieve the same therapeutic effect or even effective treatment with glucocorticoids. For example, if the tumor cell semi-inhibitory concentration (IC50) value of dexamethasone is ⁇ 10 ⁇ M, the tumor is a dexamethasone-resistant tumor.
  • IC50 tumor cell semi-inhibitory concentration
  • glucocorticoids are any glucocorticoid drugs that can be used by those skilled in the art in the treatment of tumors.
  • the glucocorticosteroid is selected from the group consisting of dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, beclomethasone, prednisolone, prednisone, methylprednisone.
  • the glucocorticoid is Dexamethasone or a derivative thereof.
  • the "level increase" in the present invention can be determined, for example, by using glucocorticoid-sensitive tumor cells as a control group to determine the range of Akt2 protein levels in the control cells, and then if the corresponding level of Akt2 protein in the tumor cells to be measured is to be measured. Above the range of Akt2 protein levels in the control group, the level of Akt2 protein in the tumor cells to be tested is considered to be elevated.
  • level rise means that the obtained test value is increased compared to a reference value such as an intermediate value or an average value observed in glucocorticoid-sensitive tumor cells, for example, at least about 5%, at least about 10 %, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90 %, at least about 95% or at least about 100% or more.
  • the level of Akt2 can be measured using an Akt2 detecting agent.
  • Akt2 detection agent refers to a molecule or compound capable of detecting Akt2 at the protein and/or nucleic acid level, especially mRNA level, and may be a polypeptide, a nucleic acid, a carbohydrate, a lipid, a small molecular weight compound, an oligonucleotide, an oligopeptide, an RNA interference. (RNAi), antisense RNA, recombinant protein, antibody, or a conjugate or fusion protein thereof.
  • RNAi see Milhavet O, Gary DS, Mattson MP. (Pharmacol Rev. 2003 Dec; 55(4): 629-48.
  • the method of the invention can determine the level (concentration or absolute amount) of Akt2 protein in a tumor sample from a patient using a ligand that binds to the Akt2 protein.
  • An elevated level of Akt2 protein in the sample indicates the presence of a glucocorticoid-resistant tumor in the patient.
  • the ligands described herein can be receptor targeting substances, cytokines, hormones, growth factors, receptor-specific antibodies, and pattern recognition receptor (PRR) ligands.
  • the ligand is an antibody (or antigen binding fragment thereof).
  • the Akt2 protein in the sample can be revealed or analyzed using any technique known to those skilled in the art, especially, for example, using specific ligands such as antibodies or fragments or antibody derivatives.
  • the ligand is a specific antibody to the Akt2 protein or a fragment of such an antibody (such as Fab, Fab', CDR, etc.) or a derivative of such an antibody (such as a single chain antibody, ScFv).
  • the presence or amount of a target protein in a sample can be detected by detecting a complex of the target and the ligand, such as with a labeled ligand, with a second labeled detection ligand, and the like.
  • a complex of the target and the ligand such as with a labeled ligand, with a second labeled detection ligand, and the like.
  • Well-known immunological techniques including ELISA, RIA, and the like can be used.
  • an “antigen-binding fragment” of an antibody refers to production by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies, including Fab, Fab', F(ab') 2 , Fv, and single chain antibodies (svFc).
  • the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, and an antibody that may be a marker, and a fragment, variant or derivative of the antibody.
  • the antibody label can be a radioactive label, a fluorescent label, an enzyme label, a chemiluminescent label, or a biotin group label.
  • the preparation and use of antibodies or fragments thereof are well known.
  • the specific antibody of the target protein can be produced by conventional techniques, in particular by immunizing a non-human animal with an immunogen comprising the protein (or an immunogenic fragment thereof) and recovering the antibody (polyclonal) or producing a cell (to produce a monoclonal antibody) .
  • Techniques for the preparation of polyclonal or monoclonal antibodies, ScFv fragments, and human or humanized antibodies are described, for example, in Harlow et al., Antibodies: A Laboratory Manual, CSH Press, 1988; Ward et al., Nature 341 (1989) 544; Bird et al., Science 242 (1988) 423; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999; /02602; US 5,223,409; US 5,877,293; WO 93/01288.
  • Proteins can also be detected using techniques known to those skilled in the art of mass spectrometry, and such techniques are generally assigned to proteome analysis to detect specific signature sequences in the sample.
  • the method of the present invention may determine the level of Akt2 mRNA in the tumor using any detection method for detecting the amount of Akt2 mRNA, wherein an increase in the level of Akt2 mRNA in the tumor indicates that the tumor is glucocorticoid resistant Drug tumor.
  • the above detection methods include various techniques capable of detecting nucleic acids in a sample, such as Northern blotting, selective hybridization, use of substrates coated with oligonucleotide probes such as nucleic acid molecule arrays, DNA chips, etc., by, for example, RT-PCR, quantification PCR or nucleic acid amplification of ligation PCR, and the like.
  • nucleic acid probes or primers that are capable of selectively or specifically detecting a nucleic acid target in a sample.
  • hybridization is carried out by those skilled in the art and can be carried out with reference to standard conditions (Sambrook, Fritsch, Maniatis (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press), for example hybridization can be carried out under high, medium or low stringency conditions.
  • one skilled in the art can perform amplification using various known methods such as PCR, LCR, transcription-mediated amplification (TMA), strand displacement amplification (SDA), NASBA, allele-specific oligonucleotides.
  • TMA transcription-mediated amplification
  • SDA strand displacement amplification
  • NASBA allele-specific oligonucleotides.
  • ASO adenosine monophosphate
  • SSCA single-strand conformational analysis
  • FISH in situ hybridization
  • the Akt2 detecting agent is a primer for detecting Akt2 mRNA or a cDNA thereof, for example, the sequence shown in SEQ ID NO: 3 or 4.
  • the invention provides the use of an Akt2 detecting agent described herein in the preparation of a composition or kit for the diagnosis of a glucocorticoid resistant tumor.
  • kit refers to a combination of an Akt2 detecting agent described herein with another item for purposes of, but not limited to, administering, diagnosing, and evaluating the activity of the Akt2 detecting agent. Or nature.
  • the kit optionally includes instructions for use.
  • the invention provides a method of treating a patient having a tumor, particularly a glucocorticoid-resistant tumor characterized by elevated Akt2 expression, comprising administering to the patient a therapeutically effective amount of a glucocorticoid and Akt2 inhibition
  • Agents for example, pharmaceutical compositions comprising an Akt2 inhibitor and a glucocorticoid.
  • the administration can be by any suitable route including, but not limited to, parenteral, such as subcutaneous, as well as oral or oral or nasal mucosa.
  • the method of the invention comprises administering to the patient any suitable dose ratio of a glucocorticoid such as a glucocorticoid to an Akt2 inhibitor such as CCT128930, for example, the molar concentration ratio thereof can be, for example, 1:10 to 10: 1, for example 1:10 to 5:1, 1:10 to 4:1, 1:10 to 3:1, 1:10 to 2:1, 1:10 to 1:1, 1:10 to 1:2 , 1:10 to 1:3, 1:10 to 1:4 or 1:10 to 1:5.
  • the method of the invention comprises administering to the patient a glucocorticoid such as dexamethasone and an Akt2 inhibitor such as CCT128930 in a molar concentration ratio of 1:8.
  • treating means that the tumor of the subject is partially or completely eliminated, or remains stable after treatment and no longer progresses.
  • Treatment includes prevention, treatment, and/or cure.
  • Prevention refers to preventing the occurrence of potential tumors and/or preventing the progression of tumors or the progression of tumors.
  • Prevention of tumorigenesis includes alleviating or eliminating one or more risk factors that cause tumors; since it is usually not possible to determine whether a tumor has never occurred, so prevention also includes Reduce the risk of developing or having a tumor.
  • terapéuticaally effective amount refers to an amount of an agent, compound, material in a dosage formulation that is at least sufficient to produce a therapeutic effect in a subject.
  • a particular dose that is therapeutically effective can be initially estimated using a variety of techniques known in the art. For example, in a cell culture assay, an agent can be formulated in an animal model to achieve a circulating concentration range that includes the IC50 determined in cell culture. Dosage ranges suitable for human subjects can be determined, for example, using data from cell culture experiments and other animal studies.
  • Dosage levels and protocols can be determined based on known dosages and protocols, and if desired, can be extrapolated based on the nature of the Akt2 inhibitor and/or can be based on multiple The factors are determined by experience. The factors include the subject's weight, overall health, age, activity of the particular compound used, sex, diet, time of administration, rate of secretion, combination of drugs, severity and duration of the disease, as well as the patient's disease susceptibility and physician's judgment. After the patient's condition has improved, a maintenance dose of the compound or composition can be administered, if desired, the dosage, dosage form, and frequency of administration, or a combination thereof, can vary. The exact dose and schedule should be based on the judgment of the physician and the specific patient.
  • an Akt2 inhibitor refers to a molecule capable of inhibiting the expression and/or activity of Akt2 at the nucleic acid level and/or protein level.
  • Akt2 inhibitors available in the art can be used in the present invention.
  • the Akt2 inhibitor may be a small molecule compound such as a compound represented by the following formula (I), (II), (III), (IV), (V) or (VI).
  • the Akt2 inhibitor may be an interfering RNA molecule of mRNA; or may be an antagonist molecule of an Akt2 protein, such as a ligand, aptamer or antibody.
  • the Akt2 inhibitor is an antibody to the Akt2 protein.
  • the Akt2 inhibitor is a double stranded RNA (dsRNA), such as a short interfering RNA (siRNA) or a short hairpin RNA (shRNA).
  • dsRNA double stranded RNA
  • siRNA short interfering RNA
  • shRNA short hairpin RNA
  • the double stranded RNA can be any type of RNA including, but not limited to, mRNA, snRNA, microRNA and tRNA.
  • RNA interference is particularly useful for specifically inhibiting the production of specific RNAs and/or proteins.
  • dsRNA molecules suitable for the present invention are within the abilities of those skilled in the art, with particular reference to Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029 and WO 01/34815.
  • Preferred siRNA molecules include the same nucleotide sequence as about 19 to 23 contiguous nucleotides of the target mRNA.
  • siRNA Represents an siRNA molecule in which less than about 50 nucleotides are base paired with a complementary sequence located on the same RNA molecule, the sequence and the complementary sequence being at least about 4 to 15 nucleotides unpaired region (in The two regions complementary to each other produce a single-stranded loop formed on the stem structure.
  • siRNA design criteria see, for example, Elbashire et al., 2001; Amarzguioui et al., 2004; Reynolds et al., 2004). Details can be found in suppliers such as Ambion, Dharmacon, GenScript and OligoEngine.
  • the dsRNA used in the methods of the invention can be produced by any method known in the art, such as by in vitro transcription, recombination, or by synthetic means.
  • the siRNA can be produced in vitro by using a recombinase (such as T7 RNA polymerase) and a DNA oligonucleotide template, or can be prepared in vivo, for example, in cultured cells.
  • the nucleic acid molecule is produced synthetically.
  • the Akt2 inhibitor is an Akt2 selective or specific inhibitor.
  • selective and specificity are used interchangeably when used in the context of an inhibitor, meaning that the inhibitor has an inhibitory effect only on the target, or the inhibitory effect on the target is relatively It has a higher inhibitory effect on other compounds or molecules, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000. 10,000 times, etc.
  • CCT128930 (Selleckchem), shown in formula (II) below, is a potent ATP-competitive selective Akt2 inhibitor with an IC50 of 6 nM in a cell-free assay, acting on Akt2 over a closely related PKA kinase selection. The sex is 28 times higher.
  • the Akt2 inhibitor is a compound of (II)
  • the Akt2 inhibitor is an interfering RNA molecule, such as set forth in SEQ ID NO: 7 or 8.
  • the Akt2 inhibitor can be administered in combination with other therapeutic agents or methods, prior to other therapeutic agents or methods, intermittently with other therapeutic agents or methods, or after other therapeutic agents or methods, including but not limited to others Biological small molecule compounds and surgery.
  • the present invention provides a pharmaceutical composition for treating a tumor, particularly a glucocorticoid-resistant tumor characterized by elevated Akt2 expression, comprising the Akt2 of the present invention Inhibitors and glucocorticoids, and optionally pharmaceutically acceptable carriers, excipients and/or diluents.
  • the Akt2 inhibitor and/or glucocorticoid may be formulated in a pharmaceutical composition.
  • the pharmaceutical compositions may be formulated for any suitable route of administration, such as for oral, nasal, parenteral, intravenous, intramuscular, intradermal, subcutaneous, buccal, inhalation, intramucosal or topical administration.
  • a pharmaceutical composition of the invention may be in any pharmaceutical dosage form as defined in the art, such as a capsule, a pill, a tablet, a powder, a granule (eg, beads, granules or crystals), an aerosol, a spray, a foam, a solution , dispersing agents, tinctures, syrups, elixirs, suspensions, ointments and emulsions.
  • the pharmaceutical composition can be formulated as a solid, liquid, gel or other form.
  • the composition When the composition is formulated for oral administration, it can be formulated as a tablet or capsule, such as enteric coated tablets or capsules which are enteric coated.
  • the Akt2 inhibitor and glucocorticosteroid may be formulated in a pharmaceutical composition of the invention in any suitable ratio, for example, the molar ratio of glucocorticoid to Akt2 inhibitor may be from 10:1 to 1:10. For example, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5. In one embodiment, the molar concentration ratio of a glucocorticoid such as dexamethasone to an Akt2 inhibitor such as CCT128930 in the pharmaceutical composition of the invention is 1:0.8.
  • compositions of the present invention may comprise pharmaceutically acceptable carriers, excipients and/or diluents including, for example, but not limited to, lactose, sucrose, starch, talc, magnesium stearate, magnesium oxide, crystalline cellulose. , methyl cellulose, carboxymethyl cellulose, gelatin, glycerin, sodium alginate, brine and water, may also contain additives such as fillers, binders, moisturizers, glidants, stabilizers, preservatives, Emulsifiers and additional solvents or solubilizers or substances that effect storage effects.
  • pharmaceutically acceptable carriers including, for example, but not limited to, lactose, sucrose, starch, talc, magnesium stearate, magnesium oxide, crystalline cellulose.
  • methyl cellulose, carboxymethyl cellulose, gelatin, glycerin, sodium alginate, brine and water may also contain additives such as fillers, binders, moisturizers, glidants, stabilizers,
  • the invention provides the use of an Akt2 inhibitor of the invention in the manufacture of a pharmaceutical composition for the treatment of a tumor, particularly a glucocorticoid-resistant tumor characterized by elevated Akt2 expression.
  • the pharmaceutical composition comprises an Akt2 inhibitor and a glucocorticoid, and optionally a pharmaceutically acceptable carrier, excipient, and/or diluent.
  • the tumor of the invention is a lymphocyte-derived tumor, such as lymphocytic leukemia, lymphoma such as B-cell lymphoma or T-cell lymphoma or myeloma.
  • the tumor is a T cell derived tumor such as a T lymphocyte leukemia, a T cell lymphoma, and a myeloma.
  • the lymphocytic leukemia is selected from the group consisting of acute lymphocytic leukemia and chronic lymphocytic leukemia.
  • the lymphoma is selected from the group consisting of a B cell lymphoma and a T cell lymphoma.
  • the tumor is a myeloma.
  • the tumor is selected from the group consisting of Burkitt's lymphoma, T lymphocytic leukemia such as acute T lymphocytic leukemia and myeloma.
  • the B cell lymphoma is selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma, such as mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma/chronic lymphocytes Leukemia, mantle cell lymphoma (MCL), diffuse large B-cell lymphoma, and follicular lymphoma.
  • MALT mucosa-associated lymphoid tissue lymphoma
  • MCL mantle cell lymphoma
  • follicular lymphoma follicular lymphoma
  • the T cell lymphoma is selected from the group consisting of adult T cell leukemia/lymphoma (ATL), peripheral T cell lymphoma, and unshaped (PTCL-U) angioimmunoblastic T cell lymphoma ( AITL), vascular immunological mother T-cell lymphoma (AITL), subcutaneous panniculitis-like T-cell lymphoma (SCPTCL), skin y ⁇ T-cell lymphoma (CGD-TCL), hepatosplenic T-cell lymphoma (HSTCL), enteropathy Intestinal T-cell lymphoma (EITCL).
  • ATL adult T cell leukemia/lymphoma
  • PTCL-U unshaped angioimmunoblastic T cell lymphoma
  • AITL vascular immunological mother T-cell lymphoma
  • SCPTCL subcutaneous panniculitis-like T-cell lymphoma
  • CCD-TCL skin y ⁇ T-cell lymphoma
  • HTCL he
  • the tumor is acute T lymphocytic leukemia.
  • the glucocorticoid is any glucocorticoid drug that can be used by those skilled in the art in treating tumors.
  • the glucocorticosteroid is selected from the group consisting of dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, beclomethasone, prednisolone, prednisone, methylprednisone.
  • the glucocorticosteroid is dexamethasone or a derivative thereof.
  • Range and amount may be expressed as “about” a particular value or range. It also includes the exact amount. Thus “about 5%” means “about 5%” and “5%”.
  • a pharmaceutically acceptable carrier is meant to include or not include the pharmaceutically acceptable carrier.
  • Example 1 Correlation between dexamethasone activity and Akt/FoxO3a pathway protein expression
  • CCRF-CEM cells human acute T lymphocyte leukemia cell line purchased from the Shanghai Institute of Biochemistry and Cell Research, Chinese Academy of Sciences
  • RPMI1640 complete medium Gibco
  • FBS fetal bovine serum
  • Dexamethasone DEX, Shanghai Shenggong Biological Co., Ltd.
  • Apoptosis was detected by Annexin V-FITC PI double staining (flow cytometry cassette (FITC, PI double staining), Shanghai Shenggong Biological Co., Ltd.): The collected cells were treated with phosphate buffered saline (PBS) (135 mM).
  • PBS phosphate buffered saline
  • Western Blot assay protein Akt antibody (rabbit source), phosphorylated-Akt antibody (rabbit source), FoxO3a antibody (rabbit source), phosphorylated-FoxO3a antibody (rabbit source) and GAPDH antibody (rabbit source) used in the examples of the present invention ) and the secondary antibodies were purchased from CST.
  • a suitable amount of color developing solution (DAB 4 mg, 30% hydrogen peroxide 15 ⁇ L, 0.01 M Tris-cl (pH 7.5) 5 mL) was applied to the PVDF membrane, and the image was detected by a bioluminescence image colorimeter and a gel imaging system (Thermo). Strip analysis was performed using Image Lab software.
  • Akt is a major regulator of phosphorylation of inactivated FoxO3a in lymphocytes
  • FoxO3a is an indispensable participant in the process of dexamethasone promoting lymphocyte apoptosis.
  • Example 2 Abnormal activation of the Akt pathway is a mechanism by which lymphoma cells produce glucocorticoid resistance
  • CCRF-CEM cells were cultured in RPMI1640 complete medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1 ⁇ M dexamethasone (DEX, Shanghai Biotech Co., Ltd.) at 5% CO2, 37 °C.
  • CEM-DR cells were cultured to logarithmic growth phase and different concentrations of dexamethasone were added, and apoptosis was detected by Annexin V-FITC PI double staining and protein was detected by Western Blot.
  • Bim antibody (rabbit source) was purchased from CST.
  • RESULTS As shown in Fig. 1C, the apoptosis-inducing effect of the cells was not significantly increased under the action of 25 ⁇ M, 50 ⁇ M and 100 ⁇ M dexamethasone. When the concentration of dexamethasone reached 200 ⁇ M, the apoptosis effect was significantly increased. As shown in Fig. 1D, compared with CCRF-CEM cells, CEM-DR cells had abnormally increased total Akt expression, phosphorylated FoxO3a (Ser253) expression was abnormally increased, and apoptosis protein Bim expression was down-regulated. CEM: CCRF-CEM cells.
  • Example 3 Akt inhibitors can significantly enhance glucocorticoid sensitivity
  • Akt inhibitor Akt IV we compared the sensitization effect of Akt inhibitor Akt IV with other cell proliferation pathway inhibitors on glucocorticoid-induced apoptosis of tumor lymphocytes.
  • Other cell proliferation pathway inhibitors include the PI3K inhibitor LY294002, the Notch1 pathway inhibitor dapt (abnormal Notch pathway causes thymocytes to resist hormone-induced apoptosis), and the glycolysis inhibitor 2-DG (research confirmed that 2-DG can increase sugar) Corticosteroid sensitivity) and the SGKs pathway (glucocorticoid-inducing kinase) inhibitor GSK.
  • Cellular CCRF-CEM human acute T lymphocyte leukemia cell line
  • Jurkat human chronic lymphocyte leukemia cell line
  • Molt4 human acute T lymphocyte leukemia cell line
  • Daudi human Burkitt's lymphoma cell line
  • Raji human Burkitt's lymphoma cell line
  • L1210 rat source
  • lymphoblastic leukemia cells and SP2/0 murine myeloma cells
  • the cells were cultured in RPMI1640 complete medium (Gibco) containing 10% fetal calf serum (FBS, Gibco) in a 5% CO 2 , 37 ° C incubator (Thermo) to logarithmic growth phase, and then The culture medium was added with different concentrations of inhibitor and/or dexamethasone/ethanol/DMSO, and the culture was continued (only cells added with dexamethasone/ethanol/DMSO were further cultured for 48 hours, and the cells to which the inhibitor was added were further cultured for 24 hours.
  • the apoptosis was detected by Annexin V-FITC PI double staining as described in Example 1, and the protein was detected by Western Blot.
  • Akt inhibitors Akt IV (Calbiochem) and PI3K inhibitor LY294002 (Promega) significantly increased the apoptosis effect of dexamethasone-induced CCRF-CEM cells compared with dexamethasone alone. (p ⁇ 0.01) (Akt inhibitor: 1 ⁇ M; dexamethasone: 1 ⁇ M; LY294002: 30 ⁇ M; ethanol: 0.1%).
  • the glycolysis inhibitor 2-DG significantly increased the apoptosis effect of dexamethasone-induced CCRF-CEM cells compared with dexamethasone alone (p ⁇ 0.01).
  • the expression of phosphorylated FoxO3a was decreased in the 2-DG-conjugated dexamethasone group, the total FoxO3a expression was increased, and the pro-apoptotic factor Bim was significantly up-regulated (p ⁇ 0.01).
  • the Notch1 pathway inhibitor dapt significantly increased the apoptosis effect of dexamethasone-induced CCRF-CEM cells compared with dexamethasone alone (p ⁇ 0.01).
  • the phosphorylated FoxO3a expression in the dapt-conjugated dexamethasone group was significantly reduced compared to the dexamethasone alone group.
  • Akt inhibitors are superior to other cell proliferation pathway inhibitors, and combined with dexamethasone to cause apoptosis. Strongest (p ⁇ 0.01).
  • DMSO murine myeloma cell line SP2/0 and the human Burkitt's lymphoma cell line Raji, the Akt inhibitor was combined with the dexamethasone group. Rice pine can significantly increase the apoptosis effect of cells (P ⁇ 0.01), thereby increasing the sensitivity of glucocorticoids.
  • the nude mouse Bal b/c purchased from the Animal Research Center of Fudan University School of Pharmacy was raised in the Specefic pathogen Free (SPF) animal room of the Animal Experimental Center of Fudan University.
  • SPF Specefic pathogen Free
  • the logarithmic growth of CCRF-CEM cells was collected and resuspended in serum-free RPMI1640 medium at a cell density of approximately 1 x 10 8 /ml.
  • Four-week-old immunodeficient female nude mice were selected, and a cell suspension of 0.1 ml/cell (about 1 ⁇ 10 7 cells/cell) was injected subcutaneously into the armpit. After growing for four weeks, the tumors were 300-500 mm 3 , randomly grouped and tested.
  • mice were injected intraperitoneally with dexamethasone 0.1 mg/A and Akt inhibitor 1.25 ⁇ g/day for 7 consecutive days.
  • the overall survival time, tumor volume and spleen cell apoptosis rate of tumor-bearing mice were measured, and pathological HE staining was performed in parallel.
  • Akt inhibitors were able to significantly reduce subcutaneous tumor size (p ⁇ 0.01) in combination with dexamethasone alone.
  • Akt inhibitor combined with dexamethasone significantly prolonged the overall survival of tumor-bearing mice compared to the dexamethasone alone group (p ⁇ 0.05).
  • liver enzymes ALT and AST were tested in peripheral blood of mice.
  • mice prepared as described in Example 4 were tested for peripheral blood liver enzymes ALT and AST after intraperitoneal injection of dexamethasone 0.1 mg/a and Akt inhibitor 1.25 ⁇ g/day for 7 consecutive days. Planning Materials for Higher Medical Colleges: Experimental Zoology (2nd Edition)).
  • liver enzymes ALT and AST were significantly increased in mice treated with Akt inhibitor (p ⁇ 0.05, 0.01) compared with saline group (NC). Therefore, Akt inhibitors have significant hepatotoxicity, which affects the prospects of clinical application.
  • Example 6 Comparison of the synergistic effect of Akt subtype inhibitor combined with glucocorticoid on apoptosis of lymphoid tumor cells
  • the cells were cultured in RPMI1640 complete medium containing 10% fetal calf serum at 5% CO 2 at 37 ° C to logarithmic growth phase as described in Example 3, and then dexamethasone and various concentrations were added to the medium.
  • Akt inhibitor continued culture (in which only dexamethasone/DMSO cells were added for 48 hours, while inhibitor-added cells were further cultured for 24 hours), and Annexin V-FITC PI double staining was performed as described in Example 1. Apoptosis and Western Blot assay for protein, and CCK-8 for cell viability.
  • CCK-8 cell viability (Cell-Counting Kit (CCK-8) kit, Dongren Chemical Co., Ltd.): (1) cells were seeded in 96-well plates at no more than 1 ⁇ 10 4 cells per well. 5 duplicate wells, 200 ⁇ l culture system per well, each well was added with the corresponding concentration of drugs according to the experiment requirements, and cultured in a 5% CO 2 , 37 ° C incubator for 48 hours; (2) 10 ⁇ l of CCK- was added to each well.
  • Akt1 inhibitor A-674563 Selleckchem
  • Akt2 inhibitor CCT128930 Selleckchem
  • Akt1/2 co-inhibitor Akti-1/2 Santa Cruz
  • Akt subtype inhibitors significantly inhibited CCRF-CEM cell activity.
  • DMSO concentration: 0.1%) dexamethasone combined with Akt subtype inhibitors significantly increased the apoptosis effect of CCRF-CEM cells compared with the dexamethasone alone group (p ⁇ 0.01). ).
  • Aktl, Akt2, and Aktl1/2 inhibitors As shown in Figures 4E and 4F, by using Aktl, Akt2, and Aktl1/2 inhibitors, the concentration of dexamethasone (IC50 value) in which CCRF-CEM cell activity was inhibited by 50% was reduced from the original 0.3 ⁇ M to 0.18 ⁇ M, 0.13, respectively. ⁇ M, 0.03 ⁇ M.
  • Akt1, Akt2 and Akt1/2 inhibitor concentrations are 0.8 ⁇ M
  • Akt subtypes Inhibitors can significantly inhibit cell viability.
  • Figure 4H DMSO concentration: 0.1%), dexamethasone combined with Akt subtype inhibitors were significantly increased compared with the dexamethasone alone group. Apoptotic effect of CEM-DR cells (p ⁇ 0.01).
  • the concentration of dexamethasone (IC50 value) in which Jurkat cell activity was inhibited by 50% was decreased from 224 ⁇ M to 208 ⁇ M, 74 ⁇ M, and 63 ⁇ M, respectively, by using Aktl, Akt2, and Aktl/2 inhibitors.
  • Akt subtype inhibitors reduced the IC50 value of dexamethasone compared to the dexamethasone alone.
  • Akt subtype inhibitors can significantly reduce the IC50 value of dexamethasone compared with the dexamethasone alone group, which is significant.
  • the glucocorticoids act synergistically, and the synergistic effects of Akt2 and Aktl/2 inhibitors with glucocorticoids are significantly better than Aktl inhibitors.
  • Example 7 Comparison of the combined index of inhibition of lymphocyte activity by Akt subtype inhibitors in combination with glucocorticoids
  • the CCK-8 method (CCK-8 kit) was used to observe the inhibition rate of two drugs (dexamethasone and inhibitor) on various lymphocyte strains alone and in combination, and then calculate the respective semi-inhibitory concentrations using the medium effect equation.
  • the CompuSyn software was used to calculate the Combination Index (CI) when the two drugs were combined.
  • cell proliferation inhibition rate (1 - experimental group OD value mean / control OD value mean) ⁇ 100%, and analyze the two drugs by Chou-Talalay joint index method The effect of the interaction.
  • Akt subtype inhibitors combined with dexamethasone can exert a high synergistic effect and significantly inhibit lymphoid tumor cell activity, and Akt2 and Akt1/2 inhibitors are significantly superior to Aktl inhibitors; in B lymphoid tumor cells, Aktl1/2 inhibitors are synergistically synergistic with dexamethasone, and Akt2 inhibitors are additive with dexamethasone.
  • the combination of Akt1 inhibitor and dexamethasone has a low synergistic effect.
  • Table 1 Joint index of each Akt isoform inhibitor and dexamethasone in multiple lymphoid tumor cells
  • Example 8 Mechanism of sensitization of glucocorticoid-induced lymphocyte apoptosis by Akt2 inhibitors: up-regulation of intracellular FoxO3a/Bim signaling pathway
  • CCRF-CEM cells were cultured in logarithmic RPMI1640 complete medium (Gibco) containing 10% fetal calf serum (FBS, Gibco) in a 5% CO 2 , 37 ° C incubator (Thermo).
  • DMSO, dexamethasone and different inhibitors DMSO: 0.1%, dexamethasone: 0.1 ⁇ M, inhibitor: 0.8 ⁇ M
  • Thermo were then added to the medium, and the culture was continued (only DMSO or dexamethasone was added).
  • the cells were further cultured for 48 hours, while the cells to which the inhibitor was added were further cultured for 24 hours), and the protein was detected by Western Blot as described in Example 1.
  • CCRF-CEM cells were able to effectively inhibit p-Akt1 by Akt1 inhibitor, but caused a compensatory increase in p-Akt2.
  • Akt2 inhibitor do not inhibit Akt2 phosphorylation, but inhibit phosphorylation activation of Akt downstream targets, leading to a compensatory increase in p-Akt1.
  • Akt1/2 inhibitor inhibited the phosphorylation activation of Akt itself, and the expression of p-Akt1 and p-Akt2 was decreased compared with the DMSO group, but the expression of p-Akt1 and p-Akt2 in the DMSO group was very small, so the Akt1/2 group The p-Akt1 and p-Akt2 protein bands in the DEX+Akt1/2 group were not shown.
  • the total FoxO3a protein in the DEX+Akt1 group, the DEX+Akt2 group and the DEX+Akt1/2 group was significantly up-regulated (P ⁇ 0.05, 0.01, 0.05);
  • the expression of total FoxO3a protein in the DEX+Akt1 group, DEX+Akt2 group and DEX+Akt1/2 group was not significantly up-regulated (p>0.05).
  • Figure 5C compared with the DEX group, the expression of p-FoxO3a protein in the DEX+Akt2 group and the DEX+Akt1/2 group was significantly down-regulated (p ⁇ 0.01); the expression of p-FoxO3a protein in the DEX+Akt1 group was observed.
  • Akt2 inhibitor and Akt1/2 inhibitor significantly down-regulate the ratio of p-FoxO3a/total FoxO3a in cells by up-regulating the intracellular FoxO3a/Bim signaling pathway, up-regulate the expression of pro-apoptotic protein Bim, and increase the apoptosis effect of glucocorticoid-induced lymphocytes.
  • Akt1 inhibitors did not significantly affect the intracellular FoxO3a/Bim signaling pathway, a result that explains the relatively weaker sensitization of lymphocyte glucocorticoids by Akt1 inhibitors.
  • Akt1 and Akt2 primer sequences are as follows:
  • Akt1 sense strand primer 5'-GCTGGACGATAGCTTGGA-3' (SEQ ID NO: 1)
  • Antisense strand primer 5'-GATGACAGATAGCTGGTG-3' (SEQ ID NO: 2)
  • Akt2 sense strand primer 5'-GGCCCCTGATCAGACTCTA-3' (SEQ ID NO: 3)
  • Antisense strand primer 5'-TCCTCAGTCGTGGAGGAGT-3' (SEQ ID NO: 4)
  • the cDNA reaction product was obtained.
  • the above reverse-transcribed cDNA was diluted 1:10 as a template, and real time PCR reaction was performed using ABI's Power SYBR Green PCR Master Mix.
  • the reaction system is as follows
  • the 18srRNA was then used as a homologous internal reference and the results were analyzed using the 2 - ⁇ ct method.
  • Akt1, Akt2 RNA small interference method cell transfection
  • the transfected cells were passaged for 3-5 passages after resuscitation, and the transfection reagent was selected with INTERFERinTM.
  • the transfection procedure was carried out in strict accordance with the instructions of the transfection reagent. Briefly, logarithmic growth phase cells were plated at 2x10 5 /well. When transfected, cells were replaced with fresh complete medium (no antibiotics added), siRNA was diluted with Opti-MEM, and appropriate amount of INTERFERin TM was added and mixed. After incubating for 10 min at room temperature, it was added dropwise to the cells, and the cells were collected as appropriate.
  • the cells were transfected with green fluorescein-labeled siRNA (FAM-siRNA) (the final concentration of siRNA was 20 ⁇ M, and 1.25 ⁇ l/well was added), and the transfection efficiency was observed with a fluorescence microscope after 24 hours.
  • FAM-siRNA green fluorescein-labeled siRNA
  • Akt1 siRNA GGCCCAACACCUUCAUCAUTT (SEQ ID NO: 5)
  • Akt2 siRNA GGUUCUUCCUCAGCAUCAATT (SEQ ID NO: 7)
  • Akt2 in glucocorticoid-sensitive cells CCRF-CEM was minimal, and the Akt2 protein band could not be displayed; the highly resistant cell CEM-DR cultured by CCRF-CEM, intracellular Akt2 The protein expression was significantly higher than that of CCRF-CEM, and the protein bands were clear.
  • the expression of Akt2 protein in the other two glucocorticoid-resistant cells Jurkat, Daudi and normal hepatocytes was significantly higher than that of the sensitive cell CCRF-CEM.
  • Akt1 mRNA and Akt2 mRNA levels in bone marrow lymphocytes from 10 patients with acute lymphoblastic leukemia who were initially treated and 11 patients with refractory relapse (glucose-based chemotherapy regimen, 7.2 patients with refractory recurrence)
  • Fig. 5I compared with the newly diagnosed group, the expression level of Akt2 mRNA was significantly increased in the relapsed and refractory group (p ⁇ 0.01), and the expression level of Akt1 mRNA was not significantly different (p>0.05).
  • Akt2 was used as an indicator to detect the degree of glucocorticoid resistance in patients.
  • the area under the ROC curve was 0.9818, the optimal judgment threshold was 16.39, the diagnostic sensitivity was 90%, and the specificity was 100%.
  • Akt2 overexpression of Akt2 in cells may be an important mechanism for lymphocyte resistance to glucocorticoids: up-regulated Akt2 inactivates FoxO3a by phosphorylation, up-regulates p-FoxO3a/total FoxO3a ratio, and down-regulates pro-apoptotic factors
  • up-regulated Akt2 inactivates FoxO3a by phosphorylation, up-regulates p-FoxO3a/total FoxO3a ratio, and down-regulates pro-apoptotic factors
  • Bim protein inhibits the intracellular FoxO3a/Bim signaling pathway, leading to the production of glucocorticoid resistance.
  • L-02 human normal liver cell line was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Research, Chinese Academy of Sciences. Nude mice were purchased from the Animal Research Center of the School of Pharmacy of Fudan University and were raised in the Animal Research Center of the Animal Research Center of Fudan University School of Medicine in the Specefic Pathogen Free (SPF) animal house.
  • SPF Specefic Pathogen Free
  • CCK-8 detection was performed as described in Examples 6 and 7, and Western Blot detection was performed as described in Examples 1 and 2.
  • CCRF-CEM cells The logarithmic growth of CCRF-CEM cells was collected and resuspended in serum-free RPMI1640 medium at a cell density of approximately 1 x 10 8 /ml.
  • Four-week-old immunodeficient female nude mice were selected, and a cell suspension of 0.1 ml/cell (about 1 ⁇ 10 7 cells/cell) was injected subcutaneously into the armpit. After growing for four weeks, the tumors were 300-500 mm 3 , randomly grouped and tested. Mice were injected intraperitoneally with dexamethasone 0.1 mg/A, Akt1, Akt2 or Aktl/2 inhibitor once daily for 7 days. On the 8th day, blood was taken from the eyelids, and blood samples were sent to the Shanghai Animal Testing Center for peripheral blood testing.
  • Figures 6A-6F show experimental results of L02 hepatocyte cell line, wherein DMSO: 0.1%; DEX: 0.1 ⁇ M; Aktl inhibitor, Akt2 inhibitor and Aktl/2 inhibitor: 0.8 ⁇ M.
  • dexamethasone had no damage to hepatocytes; Akt1 inhibitor had the most serious damage to hepatocytes, and hepatocyte activity did not recover within 48 hours after administration; Akt1/2 inhibitor also had some damage to hepatocytes.
  • the hepatocyte activity was reduced to 50.5% at 12 hours after administration, and the hepatocyte activity gradually recovered after 24 hours after administration; Akt2 inhibitor had the least damage to hepatocytes, and the hepatocyte activity was reduced to 56.1% at 6 hours after administration. Liver cell activity gradually recovered after 6 hours of administration, and the hepatocyte activity of this group was always higher than that of the Aktl inhibitor and the Aktl1/2 inhibitor group 24 hours after administration.
  • Akt subtype inhibitors Akt subtype inhibitors
  • ALT elevation was significantly increased in mice treated with Aktl1/2 inhibitors compared with NC (p ⁇ 0.01).
  • AST elevation was significantly increased in mice treated with Aktl and Aktl/2 inhibitors compared to the NC group (p ⁇ 0.05).
  • Fig. 6I the TBIL of the mice treated with Aktl and Aktl1/2 inhibitors was significantly increased compared with the NC group (p ⁇ 0.01, 0.05).
  • Fig. 6G ALT elevation was significantly increased in mice treated with Aktl1/2 inhibitors compared with NC (p ⁇ 0.01).
  • AST elevation was significantly increased in mice treated with Aktl and Aktl/2 inhibitors compared to the NC group (p ⁇ 0.05).
  • Fig. 6I the TBIL of the mice treated with Aktl and Aktl1/2 inhibitors was significantly increased compared with the NC group (p ⁇ 0.01, 0.05).
  • Fig. 8A and Fig. 8B the morphology of the hepatocytes of the tumor-bearing mice in each group was normal, no degeneration and necrosis, no hyperplasia of fibrous tissue, and no infiltration of inflammatory cells in the hepatic stroma.
  • Fig. 8C the myocardial cells of each group of tumor-bearing mice were normal, and no inflammatory cell infiltration was observed in the myocardial interstitial.
  • Fig. 8C the myocardial cells of each group of tumor-bearing mice were normal, and no inflammatory cell infiltration was observed in the myocardial interstitial.
  • the alveolar filling of the tumor-bearing mice in each group was good, the morphology of the alveolar cells was normal, and there was no obvious bleeding and exudation in the alveolar cavity.
  • Fig. 8E the morphological structure of the glomerular tubules of the tumor-bearing mice in each group was normal, no proliferation of mesangial cells, and no infiltration of inflammatory cells in the renal interstitial.
  • the log phase-grown CCRF-CEM cells were harvested and resuspended in serum-free RPMI1640 medium at a cell density of approximately 1 x 10 8 /ml.
  • Four-week-old immunodeficient female nude mice were selected, and the cell suspension was injected subcutaneously with 0.1 ml/cell (about 1 ⁇ 10 7 cells/cell), and grown for four weeks.
  • the tumors were 300-500 mm 3 , randomly grouped and tested. .
  • the mice were intraperitoneally injected with dexamethasone 0.1 mg/A, Aktl, Akt2, and Aktl/2 inhibitors once a day for 7 consecutive days.
  • the overall survival time, tumor volume and spleen volume of the tumor-bearing mice were measured.
  • the spleens of the DEX group, the DEX+Akt1 group, the DEX+Akt2 group, and the DEX+Akt1/2 group were significantly reduced (p ⁇ 0.01, 0.01, 0.01, 0.01);
  • the spleen volume was significantly reduced (p ⁇ 0.05), and the spleen volume of the DEX+Akt1 group and the DEX+Akt1/2 group was not significantly reduced (p>0.05, 0.05).
  • the total survival time of the tumor-bearing mice in the DEX+Akt2 group and the DEX+Akt1/2 group was longer than that in the DEX group (p ⁇ 0.05, 0.01).
  • Akt subtype inhibitors can synergistically promote glucocorticoids in vivo to promote lymphocyte apoptosis, and Akt subtype inhibitors have a significant sensitizing effect on glucocorticoids, among which Akt2 and Aktl1/2 inhibitors are superior in sensitization.
  • Akt1 inhibitor Akt1 inhibitor.
  • Akt2 In glucocorticoid-resistant tumor lymphocyte strains, the expression of Akt2 in cells increased significantly compared with sensitive cell lines. The difference in Akt2 expression in lymphocytes may affect and reflect the sensitivity of glucocorticoids to some extent. Overexpression of Akt2 protein may be an important mechanism for lymphocyte resistance to glucocorticoids. Akt2 may be used as a reversal lymphoma. More precise therapeutic targets for glucocorticoid resistance. It can also predict whether lymphocytes are a diagnostic indicator of glucocorticoid resistance.
  • Akt2 subtype inhibitors significantly increased the sensitivity of glucocorticoid-induced lymphocyte apoptosis; in drug-resistant cell lines, Akt2 subtype inhibitors have a good synergistic effect with glucocorticoids. , can reverse glucocorticoid resistance.
  • Akt2 subtype inhibitors can significantly down-regulate the ratio of p-FoxO3a/total FoxO3a in cells, up-regulate the expression of pro-apoptotic protein Bim, and enhance the intracellular FoxO3a/Bim signaling pathway. Glucocorticoid-induced lymphocyte apoptosis effects, thereby increasing lymphocyte glucocorticoid sensitivity.
  • Akt2 subtype inhibitors have the least toxicity, no effect on the blood system, liver function, kidney function, blood sugar of mice.
  • Akt2 subtype inhibitor can synergistically dexamethasone to effectively reduce the tumor and spleen size of tumor-bearing mice, causing liquefaction and necrosis of the tumor, and prolonging the overall survival time; Akt2 subtype inhibitor is the most sensitizing effect of glucocorticoids Good drug with the least side effects.

Abstract

治疗特征为Akt2表达升高的糖皮质激素耐药肿瘤的药物组合物，其包含Akt2抑制剂和糖皮质激素以及任选存在的药物可接受的载体、赋形剂和/或稀释剂；Akt2检测剂在检测糖皮质激素耐药肿瘤中的用途；Akt2抑制剂在制备用于治疗肿瘤的药物组合物中的用途。

Description

AKT2在诊断和治疗肿瘤中的用途 技术领域
本发明属于生物学和医学应用领域，涉及肿瘤诊断和治疗，特别涉及Akt激酶亚型Akt2检测剂诊断糖皮质激素耐药肿瘤以及Akt2抑制剂与糖皮质激素组合来治疗肿瘤，特别是淋巴细胞白血病以及骨髓瘤和淋巴瘤。
发明背景
PI3K/Akt通路广泛存在于细胞中，是参与细胞生长、增殖和分化调节的信号传导通路，与人类多种肿瘤的发生发展密切相关。Akt1、Akt2作为Akt亚型，分别发挥了调控细胞生长增殖和调节血糖的作用。已有研究认为Akt1是细胞产生糖皮质激素耐药的重要靶点。但是，尚未有报道Akt2在淋巴细胞来源肿瘤发生发展与治疗耐药发生、预后判断等的相关性。
糖皮质激素(GC)是临床有效的药物之一，能够启动淋巴细胞凋亡，是多种血液淋巴肿瘤化疗的一线用药。糖皮质激素耐药是临床淋巴肿瘤治疗中的常见的难题，是导致治疗失败的重要原因。目前，对糖皮质激素耐药的研究虽取得了一定进展，但其具体的分子耐药机制尚不清楚。新近研究发现糖皮质激素耐药与糖皮质激素受体(GR)数量上调、GR结构功能、分子伴侣表达和基因突变无关，且GR突变极为罕见；泼尼松的耐药与多药耐药基因(如ABCB1、ABCB4、ABCC1、ABCG2和MVP)无关；另有研究发现很多可能导致糖皮质激素耐药的靶基因，并初步肯定糖皮质激素敏感和耐药是由不同的细胞信号通路介导的。因此本领域需要新的方法来治疗糖皮质激素耐药肿瘤。
发明内容
一方面，本发明提供了一种诊断糖皮质激素耐药肿瘤的方法，包括测量所述肿瘤细胞中Akt2的表达，其中相对于糖皮质激素敏感肿瘤细胞，所述被测量肿瘤细胞中Akt2表达升高指示该肿瘤是糖皮质激素耐药的。
另一方面，本发明提供了检测Akt2的检测剂，例如Akt2蛋白的抗体、或检测Akt2 mRNA的探针或引物，在制备用于诊断糖皮质激素耐药肿瘤的组合物或试剂盒中的用途。
另一方面，本发明提供了检测Akt2的检测剂，例如Akt2蛋白的抗体、或检测Akt2 mRNA的探针或引物，用于诊断糖皮质激素耐药肿瘤。
另一方面，本发明提供了一种治疗肿瘤例如促进肿瘤对糖皮质激素治疗的敏感性或治疗糖皮质激素耐药肿瘤的方法，包括给予肿瘤患者治疗有效量的Akt2抑制剂以及糖皮质激素。
另一方面，本发明提供了一种用于治疗肿瘤例如促进或提高肿瘤对糖皮质激素治疗的敏感性或治疗糖皮质激素耐药肿瘤的药物组合物，包括Akt2抑制剂和糖皮质激素以及任选存在的药物可接受的载体、赋形剂和/或稀释剂。
另一方面，本发明提供了Akt2抑制剂在制备用于治疗肿瘤例如促进肿瘤对糖皮质激素治疗的敏感性或治疗糖皮质激素耐药肿瘤的药物组合物中的用途。在一个实施方案中，所述药物组合物包括糖皮质激素以及任选存在的药物可接受的载体、赋形剂和/或稀释剂。
另一方面，本发明提供了Akt2抑制剂用于治疗肿瘤例如促进或提高肿瘤对糖皮质激素治疗的敏感性或治疗糖皮质激素耐药肿瘤。
在本发明的一个实施方案中，所述肿瘤是淋巴细胞来源肿瘤，例如淋巴细胞白血病、淋巴瘤如B细胞淋巴瘤或T细胞淋巴瘤或骨髓瘤。在本发明的一个实施方案中，所述肿瘤是T细胞来源肿瘤如T淋巴细胞白血病、T细胞淋巴瘤和骨髓瘤。在本发明的一个实施方案中，所述淋巴细胞白血病选自急性淋巴细胞白血病和慢性淋巴细胞白血病。在一个实施方案中，所述肿瘤选自急性T淋巴细胞白血病。
在本发明的一个实施方案中，所述糖皮质激素选自地塞米松、倍他米松、曲安西龙、曲安奈德、倍氯米松、泼尼松龙、泼尼松、甲基强的松龙、氢化可的松、醋酸可的松、布地奈德、二丙酸倍氯米松、环索奈德、可的松、甲泼尼龙、丁酸氯倍他松、氟氢松、丙酸倍氯米松、糠酸莫米松、氯氟舒松、丙酸氯倍他松、氯氟舒松、卤美他松、双醋二氟松及其衍生物。
附图说明
图1：地塞米松与Akt/FoxO3a通路蛋白表达的相关性。图1A：CCRF-CEM流式细胞术：CCRF-CEM细胞在不同浓度地塞米松诱导下的凋亡率。图1B：地塞米松作用后CCRF-CEM细胞内Akt/FoxO3a/Bim信号通 路蛋白表达的Western印迹。p-Akt：磷酸化Akt，p-FoxO3a：磷酸化FoxO3a。图1C：CEM-DR细胞不同浓度地塞米松诱导下的凋亡率。图1D：CCRF-CEM与CEM-DR细胞内Akt/FoxO3a/Bim信号通路蛋白表达。
图2：细胞通路抑制剂与糖皮质激素联用对于细胞凋亡的作用。图2A：Akt抑制剂与PI3K抑制剂LY294002作用下CCRF-CEM各组细胞凋亡率。图2B：Akt抑制剂与PI3K抑制剂LY294002作用下CCRF-CEM各组细胞内Akt/FoxO3a/Bim信号通路蛋白表达的Western印迹。图2C：各浓度Akt抑制剂与地塞米松联合后CCRF-CEM细胞凋亡率。图2D：Akt抑制剂作用下CCRF-CEM细胞凋亡率。图2E：Akt抑制剂与PI3K抑制剂LY294002作用下Molt-4各组细胞凋亡率。图2F：Akt抑制剂与PI3K抑制剂LY294002作用下Jurkat各组细胞凋亡率。图2G：2-DG作用下CCRF-CEM各组细胞凋亡率。图2H：2-DG作用下CCRF-CEM各组细胞内Akt/FoxO3a/Bim信号通路蛋白表达。图2I：Notch1通路抑制剂dapt作用下CCRF-CEM各组细胞内FoxO3a蛋白表达的Western印迹。图2J：Notch1通路抑制剂dapt作用下CCRF-CEM各组细胞凋亡率。图2K：SGK抑制剂GSK作用下肿瘤淋巴细胞凋亡率。图2L：Akt抑制剂与其他细胞增殖通路抑制剂比较的CCRF-CEM细胞凋亡率。图2M：Akt抑制剂作用下SP2/0细胞凋亡率。图2N：Akt抑制剂作用下Raji细胞凋亡率。
图3：Akt抑制剂在裸鼠中对于糖皮质激素的增敏作用。图3A：各组CCRF-CEM荷瘤小鼠成瘤情况。图3B：各组CCRF-CEM荷瘤小鼠皮下瘤块大小。图3C：各组CCRF-CEM荷瘤小鼠瘤块HE染色。图3D：各组荷瘤小鼠总体生存期比较。图3E：各组荷瘤小鼠脾脏细胞凋亡率。图3F：各组荷瘤小鼠用药后肝转氨酶ALT、AST指标。
图4：Akt抑制剂与糖皮质激素联合作用。图4A：0.1μM地塞米松浓度联合不同浓度Akt1、Akt2、Akt1/2抑制剂诱导下CCRF-CEM细胞的凋亡率。图4B：不同浓度地塞米松联合Akt亚型抑制剂作用下CCRF-CEM细胞活性。图4C：Akt亚型抑制剂作用下CCRF-CEM各组细胞凋亡率。图4D：Akt亚型抑制剂作用下CCRF-CEM各组细胞凋亡率。图4E：Akt亚型抑制剂联合地塞米松固定浓度配比下CCRF-CEM各组细胞活性。图4F：Akt亚型抑制剂作用下CCRF-CEM各组细胞地塞米松IC50值。图4G：不同浓度地塞米松联合Akt亚型抑制剂作用下CEM-DR细胞活性。图4H： Akt亚型抑制剂作用下CEM-DR各组细胞凋亡率。图4I：Akt亚型抑制剂联合地塞米松固定浓度配比下CEM-DR各组细胞活性。图4J：Akt亚型抑制剂作用下Jurkat各组细胞凋亡率。图4K：Akt亚型抑制剂联合地塞米松固定浓度配比下Jurkat各组细胞活性。图4L：Akt亚型抑制剂作用下Daudi各组细胞凋亡率。图4M：Akt亚型抑制剂联合地塞米松固定浓度配比下Daudi各组细胞凋亡率。图4N：Akt亚型抑制剂作用下CEM-DR、Jurkat、Daudi各组细胞地塞米松IC50值。
图5：Akt亚型抑制剂联合糖皮质激素对于细胞活性的作用。图5A：CCRF-CEM细胞各组细胞内总及磷酸化Akt1、Akt2蛋白表达。图5B：CCRF-CEM细胞各组细胞内总FoxO3a蛋白表达。图5C：CCRF-CEM细胞各组细胞内磷酸化FoxO3a蛋白表达。图5D：CCRF-CEM细胞各组细胞内Bim蛋白表达。图5E：多种淋巴肿瘤细胞及肝细胞内Akt1、Akt2表达水平。图5F：小干扰RNA转染Jurkat细胞荧光图。图5G：小干扰RNA干扰Jurkat细胞内Akt1、Akt2蛋白表达。图5H：小干扰RNA转染Jurkat细胞后各组细胞凋亡率。图5I：初治与复发难治急性淋巴细胞白血病患者淋巴细胞内Akt1mRNA、Akt2 mRNA表达水平。图5J：初治与复发难治急性淋巴细胞白血病患者淋巴细胞内Akt2 mRNA的ROC分析。
图6：Akt1、Akt2抑制剂联合地塞米松对正常肝脏细胞的作用。图6A：Akt1、Akt2抑制剂与地塞米松联合用药对正常肝脏细胞L-02的细胞活性抑制。图6B：Akt1、Akt2抑制剂作用下L-02细胞内总及磷酸化Akt亚型蛋白表达。图6C：Akt亚型抑制剂作用24小时后L-02细胞内总FoxO3a蛋白表达。图6D：Akt亚型抑制剂作用24小时后L-02细胞内磷酸化FoxO3a蛋白表达。图6E：Akt亚型抑制剂作用24小时后L-02细胞内Bim蛋白表达。图6F：Akt亚型抑制剂作用24小时后肝细胞L-02活性比较。图6G：各组裸鼠外周血丙氨酸氨基转移酶(ALT)指标。图6H：各组裸鼠外周血天冬氨酸氨基转移酶(AST)指标。图6I：各组裸鼠外周血总胆红素(TBIL)指标。图6J：各组裸鼠外周血白细胞计数。图6K：各组裸鼠外周血红细胞计数。图6L：各组裸鼠外周血血红蛋白。图6M：各组裸鼠外周血血小板计数。图6N：各组裸鼠血糖指标。图6O：各组裸鼠外周血肌酐指标。
图7：Akt抑制剂联合地塞米松在裸鼠中的作用。图7A：各组CCRF-CEM荷瘤小鼠用药后皮下肿瘤大小。图7B：各组CCRF-CEM荷瘤小鼠用药后 皮下肿瘤大小比较。图7C：各组CCRF-CEM荷瘤小鼠用药后脾脏大小。图7D：各组CCRF-CEM荷瘤小鼠用药后总体生存期。图7E：裸鼠各组瘤体HE染色：箭头所指为瘤体内坏死区域。图7F：裸鼠各组瘤体Ki-67染色。图7G：裸鼠各组脾脏HE染色。图7H：裸鼠各组脾脏CD3染色。图7I：裸鼠各组脾脏TDT染色。
图8：裸鼠器官病理切片。图8A：各组裸鼠肝脏病理切片(HE：10*20)。图8B：各组裸鼠肝脏病理切片(HE：10*40)。图8C：各组裸鼠心脏病理切片(HE：10*20)。图8D：各组裸鼠肺脏病理切片(HE：10*20)。图8E：各组裸鼠肾脏病理切片(HE：10*20)。
发明详述
除非另有说明，所有技术和科学术语都具有本领域技术人员常见的含义。所有专利，专利申请，公开出版物，GenBank序列，网站以及其他公开材料除非另有说明包含在此作为参考。如果本发明的术语有多种定义，以本部分中为准。描述URL或其他鉴定子或地址时，应理解所述鉴定子可改变并且因特网上的具体信息随时更新，相关信息可通过搜索因特网找到。其公开内容可作为公众所得的依据。
本发明人发现在糖皮质激素耐药肿瘤细胞中，与敏感细胞相比，Akt2表达升高。通过使用Akt2抑制剂抑制Akt2蛋白，糖皮质激素耐药肿瘤细胞对于糖皮质激素的敏感性增加。在T淋巴肿瘤细胞中，Akt2亚型抑制剂显著增加糖皮质激素诱导淋巴细胞凋亡的敏感性；在耐药细胞株中，Akt2亚型抑制剂与糖皮质激素联合具有良好的协同作用，能够逆转糖皮质激素耐药性。Akt2亚型抑制剂能够显著下调细胞内p-FoxO3a/总FoxO3a比值，上调促凋亡蛋白Bim的表达，通过增强细胞内FoxO3a/Bim信号通路，增加糖皮质激素诱导的淋巴细胞凋亡效应，从而增加淋巴细胞糖皮质激素的敏感性。
不希望受任何理论局限，本发明人认为Akt2蛋白表达与细胞对糖皮质激素的敏感性相关，Akt2蛋白在细胞内的过度表达可能是引起淋巴细胞对糖皮质激素耐药的重要机制。Akt2可作为逆转淋巴肿瘤糖皮质激素耐药性更精确的治疗靶点，也可作为增强淋巴肿瘤对糖皮质激素治疗敏感性的靶点，也可预示淋巴细胞是否是糖皮质激素耐药的诊断指标。
体内外试验均证实Akt2亚型抑制剂毒性最小，对小鼠血液系统、肝脏功能、肾脏功能、血糖均无影响。Akt2亚型抑制剂能够协同糖皮质激素例如地塞米松有效缩小荷瘤小鼠的瘤体和脾脏大小，引起瘤体液化坏死，并延长总生存时间；Akt2亚型抑制剂是糖皮质激素增敏效果最佳且毒副作用最小的药物。
因此，在本发明的一个方面，本发明提供了一种诊断糖皮质激素耐药肿瘤的方法，包括测量所述肿瘤细胞中Akt2水平，其中相对于糖皮质激素敏感肿瘤细胞，所述被测量肿瘤细胞中Akt2水平升高指示该肿瘤是糖皮质激素耐药的。
在本发明中，Akt2水平升高是指Akt2蛋白的表达水平升高和/或Akt2蛋白的活性升高。测量Akt2的表达水平可以通过本领域已知的各种测量表达水平的方法进行，例如在核酸水平通过测量mRNA的水平或在蛋白水平测量蛋白的水平进行。测量Akt2蛋白的活性也可以通过本领域已知的各种测量Akt2活性例如磷酸化活性的方法进行。
在本发明中，所述糖皮质激素敏感肿瘤是指肿瘤细胞半抑制浓度(IC50)值＜10μM，临床应用包含糖皮质激素治疗方案能有效被缓解的肿瘤。例如如果地塞米松的肿瘤细胞半抑制浓度(IC50)值＜10μM，则该肿瘤为地塞米松敏感肿瘤。
在本发明中，糖皮质激素耐药肿瘤是指肿瘤细胞半抑制浓度(IC50)值≥10μM，这些肿瘤对于糖皮质激素作用出现耐受，导致糖皮质激素的治疗作用下降；与糖皮质激素敏感肿瘤相比，糖皮质激素耐药肿瘤需要更多的糖皮质激素以达到相同治疗效果或者甚至不能使用糖皮质激素有效治疗。例如如果地塞米松的肿瘤细胞半抑制浓度(IC50)值≥10μM，则该肿瘤为地塞米松耐药肿瘤。
在本发明中，糖皮质激素是本领域技术人员在治疗肿瘤时可以使用的任何糖皮质激素类药物。在本发明的一个实施方案中，所述糖皮质激素选自地塞米松、倍他米松、曲安西龙、曲安奈德、倍氯米松、泼尼松龙、泼尼松、甲基强的松龙、氢化可的松、醋酸可的松、布地奈德、二丙酸倍氯米松、环索奈德、可的松、甲泼尼龙、丁酸氯倍他松、氟氢松、丙酸倍氯米松、糠酸莫米松、氯氟舒松、丙酸氯倍他松、氯氟舒松、卤美他松、双醋二氟松及其衍生物。在本发明的一个实施方案中，所述糖皮质激素是地 塞米松或其衍生物。
本发明中的“水平升高”可以例如如下确定，即以糖皮质激素敏感肿瘤细胞作为对照组，确定对照组细胞中的Akt2蛋白水平的范围，然后如果待测量肿瘤细胞中Akt2蛋白的相应水平高于所述对照组细胞Akt2蛋白水平的范围，则认为该待测肿瘤细胞中Akt2蛋白的水平升高。
本文所述“水平升高”是指获得的检测值与参照值例如在糖皮质激素敏感肿瘤细胞中观察到的中间值或者平均值相比升高，例如升高至少大约5％、至少大约10％、至少大约15％、至少大约20％、至少大约25％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％、至少大约95％或者至少大约100％或者更多。
在本发明中，可以使用Akt2检测剂测量Akt2的水平。Akt2检测剂是指能够在蛋白和/或核酸水平特别是mRNA水平检测Akt2的分子或化合物，可以是多肽、核酸、碳水化合物、脂质、小分子量化合物、寡核苷酸、寡肽、RNA干扰(RNAi)、反义RNA、重组蛋白质、抗体、或者其缀合物或融合蛋白。关于RNAi，可见Milhavet O，Gary DS，Mattson MP.(Pharmacol Rev.2003 Dec；55(4)：629-48.Review.)，关于反义RNA，可见Opalinska JB，Gewirtz AM.(Sci STKE.2003 Oct 28；2003(206)：pe47)。例如，为了确定疑似患有糖皮质激素耐药肿瘤的患者中Akt2蛋白的水平，本发明的方法可以使用结合Akt2蛋白的配体测定来自患者的肿瘤样品中Akt2蛋白的水平(浓度或绝对量)，在所述样品中Akt2蛋白水平升高指示所述患者中存在糖皮质激素耐药肿瘤。
本文所述配体可以是受体靶向物质、细胞因子、激素、生长因子、受体特异性抗体以及模式识别受体(PRR)配体。在一个实施方案中，所述配体是抗体(或其抗原结合片段)。可以利用本领域技术人员已知的任意技术揭示或者分析样品中的Akt2蛋白，尤其是例如利用特异性配体，如抗体或者片段或者抗体衍生物。优选所述配体是所述Akt2蛋白的特异性抗体或者这类抗体的片段(如Fab，Fab′，CDR等等)或者这类抗体的衍生物(诸如单链抗体，ScFv)。通过检测靶和配体的复合物如利用标记的配体、利用第二种标记的检测配体等等可以检测样品中靶蛋白的存在或者量。可以使用公知的免疫技术包括ELISA，RIA等等。
本文所述“抗体”、“抗原结合片段”或“免疫原性部分”均具有本领域技术 人员通常已知的含义。例如抗体的“抗原结合片段”是指通过重组DNA技术或者通过酶或化学切割完整的抗体而产生的，包括Fab、Fab′、F(ab′)2、Fv和单链抗体(svFc)。抗体可以是多克隆抗体、单克隆抗体、嵌合抗体、人源化抗体、人抗体及可以是标记的抗体以及所述抗体的片段、变体或衍生物。抗体标记可以是放射性标记、荧光标记、酶标记、化学发光标记或者生物素基团标记。
抗体或其片段其制备及使用是熟知的。可以通过常规技术尤其是利用包括所述蛋白(或者其免疫原性片段)的免疫原免疫非人动物并回收抗体(多克隆)或者生成细胞(以产生单克隆抗体)产生靶蛋白的特异性抗体。用于制备多克隆或者单克隆抗体、ScFv片段以及人的或者人源化抗体的技术例如在下列文献中描述：Harlow et al.，Antibodies：A Laboratory Manual，CSH Press，1988；Ward et al.，Nature 341(1989)544；Bird et al.，Science 242(1988)423；Harlow，E.and Lane，D.，Antibodies：A Laboratory Manual，Cold SpringHarbor Laboratory Press，Cold Spring Harbor，New York，1999；WO94/02602；US5,223,409；US5,877,293；WO93/01288。
还可以利用质谱分析相关领域的技术人员已知的技术检测蛋白质，这类技术一般来说被分到蛋白质组分析名下，以便检测样品中的特异性特征序列。
或者，本发明的方法可以使用检测Akt2的mRNA量的任何检测方法来测定所述肿瘤中Akt2的mRNA的水平，其中所述肿瘤中Akt2的mRNA的水平升高指示所述肿瘤是糖皮质激素耐药肿瘤。上述检测方法包括能够检测样品中核酸的各种技术，如Northern印迹，选择性杂交，使用包被寡核苷酸探针的基材例如核酸分子阵列、DNA芯片等，通过例如RT-PCR、定量PCR或者连接PCR的核酸扩增等等。这些方法可以包括使用能够选择性或者特异性检测样品中核酸靶的核酸探针或者引物。例如，本领域技术人员已知并且可以参考标准条件(Sambrook，Fritsch，Maniatis(1989)Molecular Cloning，Cold Spring Harbor Laboratory Press)进行杂交，例如可以在高、中或者低严格性条件下进行杂交。或者，本领域技术人员可以利用已知的各种方法进行扩增，如PCR、LCR、转录介导的扩增(TMA)、链置换扩增(SDA)、NASBA、等位基因特异性寡核苷酸(ASO)的使用、等位基因特异性扩增、Southern印迹、单链构像分析(SSCA)、原位杂交(例如FISH)、凝胶迁移、 异源双链分析等等。
在本发明的一个实施方案中，所述Akt2检测剂是用于检测Akt2 mRNA或其cDNA的引物，例如SEQ ID NO：3或4所示的序列。
另一方面，本发明还提供了本文所述的Akt2检测剂在制备用于诊断糖皮质激素耐药肿瘤的组合物或试剂盒中的用途。
如本文所用，“试剂盒”是指本文描述的Akt2检测剂与另一项目的组合，所述项目用于如下目的，所述目的包括但不限于施用、诊断和评价所述Akt2检测剂的活性或性质。试剂盒任选地包括使用说明。
另一方面，本发明提供了一种治疗患有肿瘤、特别是特征在于Akt2表达升高的糖皮质激素耐药性肿瘤的患者的方法，包括给予患者施用治疗有效量的糖皮质激素以及Akt2抑制剂，例如包含Akt2抑制剂以及糖皮质激素的药物组合物。所述施用可以通过任何合适途径，包括但不限于胃肠外，如皮下，以及口服或经口或鼻的粘膜。在一个实施方案中，本发明所述方法包括给所述患者施用任何合适剂量比例的糖皮质激素例如糖皮质激素与Akt2抑制剂例如CCT128930，例如其摩尔浓度比例可以是例如1∶10至10∶1，例如1∶10至5∶1、1∶10至4∶1、1∶10至3∶1、1∶10至2∶1、1∶10至1∶1、1∶10至1∶2、1∶10至1∶3、1∶10至1∶4或1∶10至1∶5。在一个实施方案中，本发明所述方法包括给所述患者施用摩尔浓度比例是1∶8的糖皮质激素例如地塞米松与Akt2抑制剂例如CCT128930。
如本文所用，“治疗”患有肿瘤的对象是指对象的肿瘤部分或完全消除，或者在治疗后保持稳定不再进展。治疗包括预防、治疗和/或治愈。预防是指防止潜在肿瘤发生和/或防止肿瘤恶化或肿瘤的进展，防止肿瘤发生包括减轻或消除导致肿瘤发生的一或多种风险因子；因为通常不能确定肿瘤否从未发生，因此防止还包括降低发生或患有肿瘤的风险。
如本文所用，“治疗有效量”或“治疗有效剂量”是指至少足以在对象中产生治疗作用的剂量配制品中的药剂、化合物、材料的量。对于本发明的Akt2抑制剂，治疗有效的具体剂量可利用本领域已知的多种技术初步估计。例如，细胞培养测定法中，可在动物模型中配制药剂以实现包括在细胞培养中测定的IC50的循环浓度范围。适合人对象的剂量范围可例如利用得自细胞培养实验和其他动物研究的数据确定。剂量水平和方案可基于已知的剂量和方案确定，如果需要可基于Akt2抑制剂性质外推和/或可基于多 种因素经验确定。所述因素包括对象的体重，总体健康，年龄，所用特定化合物的活性，性别，饮食，施用时间，分泌速度，药物组合，疾病严重度和病程，以及患者的疾病易感性和医师的判断。患者病情改善后，可施用维持剂量的化合物或组合物，如果需要，剂量，剂型以及施用频率或其组合可以改变。精确的剂量和方案应根据医师的判断以及具体患者的情况。
在本发明中，Akt2抑制剂是指能够在核酸水平和/或蛋白水平抑制Akt2的表达和/或活性的分子。本领域可获得的Akt2抑制剂均可用于本发明。例如，Akt2抑制剂可以是小分子化合物，例如下式(I)、(II)、(III)、(IV)、(V)或(VI)所示的化合物。
Figure PCTCN2015096011-appb-000001
或者，Akt2抑制剂可以是mRNA的干扰RNA分子；或者可以是Akt2蛋白的拮抗剂分子，例如配体、适配体或者抗体。在一个实施方案中，所述Akt2抑制剂是Akt2蛋白的抗体。在另一个实施方案中，所述Akt2抑制剂是双链RNA(dsRNA)，例如短干扰RNA(siRNA)或短发夹RNA(shRNA)。所述双链RNA可以是任意类型的RNA，包括但不限于mRNA，snRNA，微小RNA和tRNA。RNA干扰(RNAi)尤其可用于特异性抑制特定RNA和/或蛋白的产生。适于本发明的dsRNA分子的设计和产生在本领域技术人员的能力范围内，尤其参考Waterhouse等(1998)，Smith等(2000)，WO 99/32619，WO 99/53050，WO 99/49029和WO 01/34815。优选的siRNA分子包括与靶mRNA的约19到23个连续核苷酸相同的核苷酸序列。“shRNA” 表示一种siRNA分子，其中少于约50个核苷酸与位于相同RNA分子上的互补序列碱基配对，所述序列与互补序列被至少约4到15个核苷酸的不配对区域(在碱基互补的两个区域产生的茎结构上形成单链环)分开。存在公认的siRNA设计标准(见例如Elbashire et al.，2001；Amarzguioui et al.，2004；Reynolds et al.，2004)。详细内容可以参见供应商如Ambion，Dharmacon，GenScript和OligoEngine。一旦设计好，用于本发明方法的dsRNA可以通过本领域已知的任意方法产生，例如通过体外转录，重组，或通过合成方式。siRNA可以在体外产生，通过使用重组酶(如T7 RNA聚合酶)和DNA寡核苷酸模板，或可以在体内制备，例如在培养的细胞中。在优选的实施方案中，核酸分子是合成产生的。
在本发明的一个实施方案中，所述Akt2抑制剂是Akt2选择性或特异性抑制剂。在本发明中，用于抑制剂时，术语“选择性”和“特异性”可互换使用，意指所述抑制剂仅对于所述靶具有抑制作用，或者对于所述靶的抑制作用相对于对其它化合物或分子具有更高的抑制作用，例如高至少大约1、2、3、4、5、6、7、8、9、10、20、30、40、50、100、500、1000、10000倍等。例如，如下式(II)所示的CCT128930(Selleckchem公司)是一种有效的ATP竞争性的选择性Akt2抑制剂，无细胞试验中IC50为6nM，作用于Akt2比作用于紧密相关的PKA激酶选择性高28倍。在本发明的一个实施方案中，所述Akt2抑制剂是(II)所示的化合物
Figure PCTCN2015096011-appb-000002
在本发明的另一个实施方案中，所述Akt2抑制剂是干扰RNA分子，例如SEQ ID NO：7或8所示。
Akt2抑制剂可与其他治疗剂或方法组合、在其他治疗剂或方法之前、与其他治疗剂或方法间歇、或在其他治疗剂或方法之后施用，所述其他治疗剂或方法包括但不限于其他生物小分子化合物和手术。
另一方面，本发明还提供了一种用于治疗肿瘤、特别是特征在于Akt2表达升高的糖皮质激素耐药性肿瘤的药物组合物，包括本发明所述的Akt2 抑制剂和糖皮质激素以及任选存在的药物可接受的载体、赋形剂和/或稀释剂。
在本发明的一个实施方案中，所述Akt2抑制剂和/或糖皮质激素可以被配制在药物组合物中。所述药物组合物可以配制为用于任何合适的施用途径，如用于口服、鼻、胃肠外、静脉内、肌内、皮内、皮下、口腔、吸入、粘膜内或局部施用。例如，本发明的药物组合物可以是本领域确定的任何药物剂量形式，例如包囊、丸剂、片剂、粉末、颗粒(例如珠、颗粒或晶体)、气雾剂、喷雾剂、泡沫、溶液、分散剂、酊剂、糖浆、酏剂、悬浮液、软膏和乳剂。所述药物组合物可以配制为固体、液体、凝胶或其他形式。当组合物配制为用于口服施用时，其可以配制为片剂或包囊，如肠溶包衣的片剂或包囊是肠溶包衣的。
在一个实施方案中，所述Akt2抑制剂与糖皮质激素可以任何合适比例配制在本发明的药物组合物中，例如糖皮质激素与Akt2抑制剂的摩尔浓度比例可以是10∶1至1∶10，例如5∶1、4∶1、3∶1、2∶1、1∶1、1∶2、1∶3、、1∶4或1∶5。在一个实施方案中，本发明所述药物组合物中糖皮质激素例如地塞米松与Akt2抑制剂例如CCT128930的摩尔浓度比例是1∶0.8。
本发明的所述药物组合物可以包含药物可接受的载体、赋形剂和/或稀释剂，例如包括但不限于乳糖，蔗糖，淀粉，滑石粉，硬脂酸镁，氧化镁，结晶纤维素，甲基纤维素，羧甲基纤维素，明胶，甘油，海藻酸钠，盐水和水，也可以包含添加剂，如充填剂、结合剂、增湿剂、助流剂、稳定剂、防腐剂、乳化剂及另外的溶剂或者增溶剂或者实现储存效应的物质。
另一方面，本发明还提供了本发明所述Akt2抑制剂在制备用于治疗肿瘤、特别是特征在于Akt2表达升高的糖皮质激素耐药性肿瘤的药物组合物中的用途。在一个实施方案中，所述药物组合物包括Akt2抑制剂和糖皮质激素以及任选存在的药物可接受的载体、赋形剂和/或稀释剂。
在本发明的一个实施方案中，本发明所述肿瘤是淋巴细胞来源肿瘤，例如淋巴细胞白血病、淋巴瘤如B细胞淋巴瘤或T细胞淋巴瘤或骨髓瘤。在本发明的一个实施方案中，所述肿瘤是T细胞来源肿瘤如T淋巴细胞白血病、T细胞淋巴瘤和骨髓瘤。在本发明的一个实施方案中，所述淋巴细胞白血病选自急性淋巴细胞白血病和慢性淋巴细胞白血病。在本发明的一个实施方案中，所述淋巴瘤选自B细胞淋巴瘤和T细胞淋巴瘤。在本发明的 一个实施方案中，所述肿瘤是骨髓瘤。在本发明的一个实施方案中，所述肿瘤选自Burkitt′s淋巴瘤、T淋巴细胞白血病例如急性T淋巴细胞白血病和骨髓瘤。
在本发明的一个实施方案中，所述B细胞淋巴瘤选自霍奇金淋巴瘤以及非霍奇金淋巴瘤，例如黏膜相关淋巴组织淋巴瘤(MALT)、小淋巴细胞淋巴瘤/慢性淋巴细胞白血病、套细胞淋巴瘤(MCL)、弥漫性大B细胞淋巴瘤和滤泡型淋巴瘤。
在本发明的一个实施方案中，所述T细胞淋巴瘤选自成人T细胞性白血病/淋巴瘤(ATL)、外周T细胞淋巴瘤、未定型(PTCL-U)血管免疫母T细胞淋巴瘤(AITL)、血管免疫母T细胞淋巴瘤(AITL)、皮下脂膜炎样T细胞淋巴瘤(SCPTCL)、皮肤yδT细胞淋巴瘤(CGD-TCL)、肝脾T细胞淋巴瘤(HSTCL)、肠病型肠道T细胞淋巴瘤(EITCL)。
在本发明的一个实施方案中，所述肿瘤是急性T淋巴细胞白血病。
在本发明中，所述糖皮质激素是本领域技术人员在治疗肿瘤时可以使用的任何糖皮质激素类药物。在本发明的一个实施方案中，所述糖皮质激素选自地塞米松、倍他米松、曲安西龙、曲安奈德、倍氯米松、泼尼松龙、泼尼松、甲基强的松龙、氢化可的松、醋酸可的松、布地奈德、二丙酸倍氯米松、环索奈德、可的松、甲泼尼龙、丁酸氯倍他松、氟氢松、丙酸倍氯米松、糠酸莫米松、氯氟舒松、丙酸氯倍他松、氯氟舒松、卤美他松、双醋二氟松及其衍生物。在本发明的一个实施方案中，所述糖皮质激素是地塞米松或其衍生物。
如本文所用，范围和量可以表示为“大约”特定数值或范围。大约也包括精确量。因此“大约5％”意味着“大约5％”以及“5％”。
如本文所用，“任选存在的”或“任选地”意味着随后描述的事件或情况发生或不发生，以及所述描述包括所述事件或情况发生的情况和其不发生的情况。例如，任选存在的药物可接受的载体意味着包括或不包括所述药物可接受的载体。
实施例
本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。本发明的范围由所附权利要求书 限定，结合本说明书和本领域一般常识，本领域普通技术人员可以清楚地明白权利要求书所限定的范围。在不偏离本发明的精神和范围的前提下，本领域技术人员可以对本发明的技术方案进行任何修改或改变，这种修改和改变也包含在本发明的范围内。
实施例1：地塞米松活性与Akt/FoxO3a通路蛋白表达水平的相关性
将CCRF-CEM细胞(人源急性T淋巴细胞白血病细胞株，购于中国科学院上海生物化学与细胞研究所细胞库)以1×105细胞/ml密度接种在细胞培养皿(CORNING公司)中补加了10％胎牛血清(FBS，Gibco公司)的RPMI1640完全培养基(Gibco公司)中，在5％CO2、37℃恒温培养箱(Thermo公司)中培养至对数生长期；然后向培养皿中加入终浓度为0.1μM或1μM的地塞米松(DEX，上海生工生物有限公司)，在5％CO2、37℃继续培养48小时，收集细胞。对照组不添加任何试剂。所有实验一式三份进行。
Annexin V-FITC PI双染法检测细胞凋亡(流式细胞凋亡盒(FITC、PI双染法)，上海生工生物有限公司)：将收集的细胞用磷酸盐缓冲液(PBS)(135mM NaCl，2.7mM KCl，1.5mM KH2PO4和8mM K2HPO4，pH 7.2)清洗1次；用去离子水将4×结合缓冲液稀释成1×结合缓冲液，然后用195μl的1×结合缓冲液重悬细胞，使细胞密度为2-5×105/ml；加5μl的Annexin V-FITC至195μl细胞重悬液，混匀后，避光，室温孵育10分钟；用200μl的1×结合缓冲液洗涤细胞一次，然后将细胞重悬于190μl的1×结合缓冲液；加入10μl的碘化丙啶(20μg/ml)。采用流式细胞仪(Beckman公司)检测，并用Summit软件分析细胞凋亡率。
Western Blot法检测蛋白：本发明实施例所用Akt抗体(兔源)、磷酸化-Akt抗体(兔源)、FoxO3a抗体(兔源)、磷酸化-FoxO3a抗体(兔源)和GAPDH抗体(兔源)以及二抗都购自CST公司。收集细胞于离心管中，以1000转/分钟速度离心，弃去上清液，加4℃预冷的PBS清洗2次；加细胞裂解液(150mM NaCL、1％NP-40、0.1％SDS、2μg/ml Aprotinin、2μg/ml Leupeptin、1mM PMSF、1.5mM EDTA、1mM钒酸钠)，冰上裂解细胞40分钟，以20000转/分钟、4℃离心细胞裂解液15分钟，收集上清液；加入十二烷基硫酸钠聚丙烯酰胺(SDS-PAGE)凝胶电泳蛋白上样缓冲液(1.25mL 1MTris-HCL(pH6.8)，0.5g SDS，25mg BPB，2.5mL甘油，去离 子水5mL)，煮沸5分钟，使蛋白质变性；将含有变性蛋白的上样缓冲液在10％浓度的SDS-PAGE上以120V电压电泳，然后以130V、1.5小时转移至PVDF膜(上海生工生物有限公司)上；转膜完毕后，立即把PVDF膜用Western洗涤液(150mmol/L NaCl，50mmol/L Tris-HCl(PH 7.5))漂洗1-2分钟；加入5％脱脂奶粉(上海生工生物有限公司)，缓慢摇动，室温封闭60分钟；加入按照适当比例用5％脱脂奶粉稀释的一抗，在室温缓慢摇动孵育过夜；回收一抗，用洗涤液洗涤5-10分钟，共洗涤3次；加入按照适当比例用Western洗涤液稀释的辣根过氧化物酶标记的二抗，室温缓慢摇动孵育1小时；回收二抗，加入Western洗涤液，缓慢摇动洗涤5-10分钟，共洗涤3次。在PVDF膜上滴适量显色液(DAB 4mg，30％的双氧水15μL，0.01M Tris-cl(pH7.5)5mL)，用生物发光影像显色仪和凝胶成像系统(Thermo公司)检测图像，使用Image Lab软件进行条带定量分析。
数据处理用Excel及Stata软件，数据比较用t检验，P＜0.05认为有统计学意义。
结果：如图1A所示，对地塞米松敏感的CCRF-CEM，随着地塞米松浓度增加，细胞凋亡效应逐渐增加。如图1B所示，与空白组相比，应用0.1μM地塞米松处理后的细胞内磷酸化Akt表达明显下调，磷酸化Akt(Ser473)/总Akt水平明显下降(p＜0.05)；磷酸化FoxO3a(Ser 253)表达相应减少，总FoxO3a表达相应增加，磷酸化FoxO3a(Ser 253)/总FoxO3a蛋白水平显著下调(p＜0.05)；且上述变化随地塞米松浓度增加更趋明显。
因此，在淋巴细胞中Akt是磷酸化失活FoxO3a的主要调控激酶，且在地塞米松发挥促淋巴细胞凋亡过程中，FoxO3a是不可或缺的参与者。
实施例2：Akt路径的异常激活是淋巴肿瘤细胞产生糖皮质激素耐药的机制
将CCRF-CEM细胞在含有10％胎牛血清(FBS，Gibco公司)和1μM地塞米松(DEX，上海生工生物有限公司)的RPMI1640完全培养基(Gibco公司)中在5％CO2、37℃恒温培养箱(Thermo公司)中培养，长到85％～90％传代，吸出旧液1/2-2/3，加入新鲜培养基进行传代，传至第20代的子代细胞，用1μM，25μM，50μM，100μM浓度的地塞米松作用48小时后，流式细胞法检测细胞凋亡率未见明显升高，提示获得了糖皮质激素耐药细胞株 CEM-DR。然后如实施例1所述，将CEM-DR细胞培养至对数生长期并加入不同浓度的地塞米松后进行Annexin V-FITC PI双染法检测细胞凋亡以及Western Blot法检测蛋白。Bim抗体(兔源)购自CST公司。
结果：如图1C所示，该细胞在25μM、50μM、100μM地塞米松作用下，细胞的诱导凋亡效应未见显著升高；当地塞米松浓度到达200μM时，细胞凋亡效应显著升高。如图1D所示，与CCRF-CEM细胞相比，CEM-DR细胞存在总Akt表达异常增高，磷酸化FoxO3a(Ser253)表达异常增高，凋亡蛋白Bim表达下调。CEM：CCRF-CEM细胞。
通过与敏感细胞株CCRF-CEM比较，我们发现CEM-DR细胞内存在总Akt异常增高。
实施例3：Akt抑制剂能够显著增强糖皮质激素的敏感性
我们比较了Akt抑制剂Akt IV与其他细胞增殖通路抑制剂对糖皮质激素诱导肿瘤淋巴细胞凋亡的增敏效果。其他细胞增殖通路抑制剂包括PI3K抑制剂LY294002，Notch1通路抑制剂dapt(异常Notch通路可导致胸腺细胞抵抗激素诱导的凋亡)，糖酵解抑制剂2-DG(研究证实2-DG可增加糖皮质激素敏感性)以及SGKs途径(糖皮质激素诱导激酶)抑制剂GSK。
方法：细胞CCRF-CEM(人源急性T淋巴细胞白血病细胞株)、Jurkat、Molt4(人源急性T淋巴细胞白血病细胞株)、Daudi、Raji(人源Burkitt’s淋巴瘤细胞株)、L1210(鼠源淋巴母细胞白血病细胞)和SP2/0(鼠源骨髓瘤细胞)均购于中国科学院上海生物化学与细胞研究所细胞库。
将细胞在含有10％胎牛血清(FBS，Gibco公司)的RPMI1640完全培养基(Gibco公司)中在5％CO2、37℃恒温培养箱(Thermo公司)中培养至对数生长期，然后向培养基中加入不同浓度的抑制剂和/或地塞米松/乙醇/DMSO，继续培养(其中只加入地塞米松/乙醇/DMSO的细胞继续培养48小时，而加入抑制剂的细胞继续培养24小时)，如实施例1所述进行Annexin V-FITC PI双染法检测细胞凋亡以及Western Blot法检测蛋白。
结果：如图2A所示，与单用地塞米松相比，Akt抑制剂Akt IV(Calbiochem公司)、PI3K抑制剂LY294002(Promega公司)均能显著增加地塞米松诱导CCRF-CEM细胞的凋亡效应(p＜0.01)(Akt抑制剂：1μM；地塞米松：1μM；LY294002：30μM；乙醇：0.1％)。如图2B所示(乙醇： 浓度0.1％)，与单用地塞米松组相比，Akt抑制剂联合地塞米松组以及PI3K抑制剂LY294002联合地塞米松组显著下调CCRF-CEM细胞内磷酸化FoxO3a(Ser 253)表达，上调促凋亡因子Bim的表达。如图2C所示，在CCRF-CEM细胞株中，各浓度的Akt抑制剂均能增加地塞米松诱导的凋亡效应，且随着Akt抑制剂浓度的增加，细胞凋亡率随之升高。如图2E、2F所示(DMSO：浓度0.1％)，在高度糖皮质激素耐药的人源急性T淋巴细胞白血病细胞株Molt4和Jurkat中，与单用地塞米松组相比，Akt抑制剂或PI3K抑制剂LY294002联合地塞米松能显著增加细胞的凋亡率(p＜0.01)，且Akt抑制剂与地塞米松的协同效果优于PI3K抑制剂LY294002(p＜0.01)。
如图2G所示，与单用地塞米松相比，糖酵解抑制剂2-DG(Sigma公司)能显著增加地塞米松诱导CCRF-CEM细胞的凋亡效应(p＜0.01)。如图2H所示，与单用地塞米松组相比，2-DG联合地塞米松组细胞内磷酸化FoxO3a表达减少，总FoxO3a表达升高，促凋亡因子Bim显著上调(p＜0.01)。
如图2J所示，与单用地塞米松相比，Notch1通路抑制剂dapt(Sigma公司)能显著增加地塞米松诱导CCRF-CEM细胞的凋亡效应(p＜0.01)。如图2I所示，与单用地塞米松组相比，dapt联合地塞米松组细胞内磷酸化FoxO3a表达显著减少。
如图2K所示(乙醇：0.1％)，在人源急性T淋巴白血病细胞株CCRF-CEM、鼠源T淋巴母细胞白血病细胞株L1210、人源Burkitt’s淋巴瘤细胞株Raji和Daudi中，地塞米松组与GSK(Bioscience公司)联合地塞米松组细胞凋亡率未见显著差异(p＞0.05)，说明SGKs途径抑制剂未能协同地塞米松增加肿瘤淋巴细胞的凋亡效应。
我们将上述各细胞增殖通路抑制剂对糖皮质激素的增敏效果进行比较，如图2L所示，Akt抑制剂明显优于其他细胞增殖通路抑制剂，与地塞米松联合后引起细胞凋亡效应最强(p＜0.01)。如图2M、2N所示(DMSO：0.1％)，在鼠源骨髓瘤细胞株SP2/0与人源Burkitt’s淋巴瘤细胞株Raji中，与单用地塞米松组相比，Akt抑制剂联合地塞米松能显著升高细胞的凋亡效应(P＜0.01)，从而增加糖皮质激素的敏感性。
实施例4：Akt抑制剂在裸鼠体内的糖皮质激素增敏效果
将购于复旦大学药学院动物研究中心的裸鼠Bal b/c饲养于复旦大学药 学院动物实验中心无特定病原体(Specefic pathogen Free，SPF)级动物房。收集对数期生长的CCRF-CEM细胞，用无血清的RPMI1640培养基重悬细胞，细胞密度约为1×108/ml。选择4周龄的免疫缺陷雌性裸鼠，腋窝处皮下注射细胞悬液0.1ml/只(约为1×107细胞/只)。生长四周，待瘤体达300-500mm3，随机分组并实验。将小鼠用地塞米松0.1mg/只和Akt抑制剂为1.25μg/只腹腔注射，每天一次，共连续7天。测量荷瘤小鼠的总体生存时间，瘤体体积及脾脏细胞凋亡率，并行瘤体病理HE染色。
结果：如图3A、3B所示，与单用地塞米松组相比，Akt抑制剂能够协同地塞米松显著缩小皮下肿瘤大小(p＜0.01)。如图3D所示，与单用地塞米松组相比，Akt抑制剂联合地塞米松能够显著延长荷瘤小鼠的总生存期(p＜0.05)。同时，我们分离了小鼠脾脏的淋巴细胞进行流式凋亡检测，结果如图3E所示，与单用地塞米松组相比，荷瘤小鼠脾脏的淋巴细胞凋亡率显著升高(p＜0.01)。
荷瘤小鼠体内试验再次证实Akt抑制剂显著的糖皮质激素增敏效果。
实施例5：Akt抑制剂具有严重的肝脏毒性
为了进一步探究Akt抑制剂IV对肝脏是否存在损害，我们检测小鼠外周血中肝酶指标ALT、AST。
方法：测量如实施例4所述制备的荷瘤小鼠在每天一次腹腔注射地塞米松0.1mg/只和Akt抑制剂1.25μg/只共连续7天后的外周血肝酶ALT、AST指标(全国高等医药院校规划教材：实验动物学(第2版))。
结果：如图3F所示，与生理盐水组(NC)相比，Akt抑制剂处理后小鼠体内肝酶ALT、AST显著升高(p＜0.05、0.01)。因此，Akt抑制剂存在明显肝脏毒性，影响了临床应用的前景。
实施例6：比较Akt亚型抑制剂与糖皮质激素联合诱导淋巴肿瘤细胞凋亡的协同作用
如实施例3所述，将细胞在含有10％胎牛血清的RPMI1640完全培养基中在5％CO2、37℃培养至对数生长期，然后向培养基中加入地塞米松和不同浓度的Akt抑制剂，继续培养(其中只加入地塞米松/DMSO的细胞继续培养48小时，而加入抑制剂的细胞继续培养24小时)，如实施例1所述 进行Annexin V-FITC PI双染法检测细胞凋亡以及Western Blot法检测蛋白，以及CCK-8检测细胞活力。
CCK-8检测细胞活力(Cell-Counting Kit(CCK-8)试剂盒，东仁化学有限公司)：(1)细胞按每孔不多于1×104细胞接种于96孔板中，并设5个复孔，每孔200μl培养体系，每孔按实验要求加入相应浓度的药物，并在5％CO2、37℃恒温培养箱中培养48小时；(2)向每孔加入10μl的CCK-8溶液，避免在孔中生成气泡，影响光密度(OD)值的读数；(3)将培养板在5％CO2、37℃恒温培养箱内孵育4小时；(4)用酶标仪测定在450nm处的吸光度；(5)绘制细胞活力曲线：按照以下公式计算细胞活力，然后绘制成图表，不同药物浓度或处理时间设为横坐标，细胞存活率为纵坐标：
细胞存活率为(％)＝[(As-Ab)/(Ac-Ab)]×100％，
As：实验孔(含有细胞的培养基、CCK-8、毒性物质)
Ac：对照孔(含有细胞的培养基、CCK-8、没有毒性物质)
Ab：空白孔(不含细胞和毒性物质的培养基、CCK-8)。
结果：我们以Akt1抑制剂A-674563(Selleckchem公司)(抑制Akt1下游靶点磷酸化激活)、Akt2抑制剂CCT128930(Selleckchem公司)(抑制Akt2下游靶点磷酸化激活)以及Akt1/2共同抑制剂Akti-1/2(Santa Cruz公司)(同时抑制Akt1、Akt2自身磷酸化)为研究对象，0.1μM地塞米松浓度联合0.3μM、0.5μM、0.8μM、1μM浓度的Akt各亚型抑制剂处理CCRF-CEM细胞，结果如图4A所示，在0.1μM地塞米松联合0.8μMAkt亚型抑制剂浓度下，细胞凋亡效应最强。如图4B所示，在不同浓度的地塞米松条件下(Akt1、Akt2以及Akt1/2抑制剂浓度：均为0.8μM)，Akt各亚型抑制剂均能显著抑制CCRF-CEM细胞的活性。如图4C、4D所示(DMSO浓度：0.1％)，与单用地塞米松组相比，地塞米松联合Akt各亚型抑制剂均能显著增加CCRF-CEM细胞的凋亡效应(p＜0.01)。如图4E、4F所示，通过应用Akt1、Akt2、Akt1/2抑制剂，CCRF-CEM细胞活性被抑制50％的地塞米松浓度(IC50值)可由原先的0.3μM分别下降至0.18μM、0.13μM、0.03μM。
在培育的高度耐药细胞CEM-DR细胞株中，如图4G所示(Akt1、Akt2以及Akt1/2抑制剂浓度均为0.8μM)，在不同浓度的地塞米松条件下，Akt各亚型抑制剂均能显著抑制细胞的活性。如图4H所示(DMSO浓度：0.1％)，与单用地塞米松组相比，地塞米松联合Akt各亚型抑制剂均能显著增加 CEM-DR细胞的凋亡效应(p＜0.01)。如图4I、4N所示，通过应用Akt1、Akt2、Akt1/2抑制剂，CEM-DR细胞活性被抑制50％的地塞米松浓度(IC50值)可由原先的138μM分别下降至55μM、25μM、11μM。
在耐药人源急性T淋巴细胞白血病细胞株Jurkat，如图4J所示(DMSO浓度：0.1％)，与单用地塞米松组相比，地塞米松联合Akt1、Akt2、Akt1/2均能显著增加细胞的凋亡效应(p＜0.05、0.01、0.01)；且DEX+Akt1组细胞凋亡率显著低于DEX+Akt2组(p＜0.01)。如图4K、4N所示，通过应用Akt1、Akt2、Akt1/2抑制剂，Jurkat细胞活性被抑制50％的地塞米松浓度(IC50值)可由原先的224μM分别下降至208μM、74μM、63μM。
在耐药人源Burkitt’s细胞株Daudi，如图4L所示(DMSO浓度：0.1％)，与单用地塞米松组相比，地塞米松联合Akt1、Akt2、Akt1/2抑制剂均能显著增加细胞的凋亡效应(p＜0.01、0.05、0.01)。如图4M、4N所示，通过应用Akt1、Akt2、Akt1/2抑制剂，Daudi细胞活性被抑制50％的地塞米松浓度(IC50值)可由原先的225μM分别下降至183μM、213μM、118μM。
因此，与单用地塞米松组相比，Akt各亚型抑制剂联用地塞米松降低了地塞米松的IC50值。特别在T淋巴肿瘤细胞株(CCRF-CEM、CEM-DR、Jurkat细胞)中，与单用地塞米松组相比，Akt各亚型抑制剂均能显著降低地塞米松的IC50值，起到显著的糖皮质激素协同作用，且Akt2及Akt1/2抑制剂与糖皮质激素的协同作用显著优于Akt1抑制剂。
实施例7：比较Akt亚型抑制剂与糖皮质激素联合后对淋巴细胞活性抑制的联合指数
采用CCK-8法(CCK-8试剂盒)观察两种药物(地塞米松及抑制剂)单用及合用对多种淋巴细胞株的抑制率，然后用中效方程计算各自的半抑制浓度，并应用CompuSyn软件计算两种药物合用时的联合指数(Combination Index，CI)。
(1)取处于对数生长期的淋巴细胞，将细胞制成5×104/mL的悬液，实验设空白对照组(5孔)、地塞米松组(每种药物浓度3孔，重复3次)、单纯抑制剂组(每种药物浓度3孔，重复3次)、地塞米松联合抑制剂组(每种联合的药物浓度3孔，重复3次)，每孔180μL，接种于96孔培养板内，置于5％CO2、37℃恒温培养箱中培养24小时。
(2)将两种单药的五个不同浓度分别加在96孔板内，地塞米松浓度根据细胞的耐药程度为基础依次分为五种浓度，Akt1抑制剂A-674563(Selleckchem公司)、Akt2抑制剂CCT128930(Selleckchem公司)、Akt1/2抑制剂Akti-1/2(Santa Cruz公司)浓度依次分为五种浓度。根据细胞的耐药情况，两药合用比例固定。地塞米松组培养48小时，抑制剂组培养24小时。
(3)每孔加入10μl CCK-8继续培养2小时，采用自动酶标读数仪比色波长450nm测出光密度(OD)值并计算药物对细胞的增殖抑制率。
(4)用下列公式计算细胞增殖抑制率：细胞增殖抑制率＝(1-实验组OD值均数/对照组OD值均数)×100％，并通过Chou-Talalay联合指数法分析两药物之间相互作用的效果。
结果：我们应用CompuSyn软件计算地塞米松与Akt各亚型抑制剂合用时的CI，CI＜1表明两药协同，CI＝1表明两药相加，CI＞1表明两药拮抗。如表1所示，在T淋巴肿瘤细胞(CCRF-CEM、CEM-DR、Jurkat细胞)中，Akt各亚型抑制剂与地塞米松联合能发挥高度协同作用，明显抑制淋巴肿瘤细胞活性，且Akt2、Akt1/2抑制剂显著优于Akt1抑制剂；在B淋巴肿瘤细胞中，Akt1/2抑制剂与地塞米松联用呈中度协同作用，Akt2抑制剂与地塞米松联用呈叠加作用，Akt1抑制剂与地塞米松联用呈低度协同作用。
表1：多株淋巴肿瘤细胞各Akt亚型抑制剂与地塞米松的联合指数
Figure PCTCN2015096011-appb-000003
注：DEX＝地塞米松
联合指数CI＜1表明两药协同，CI＝1表明两药相加，CI＞1表明两药拈抗；0.9≤CI≤1.1为叠加作用(-)，0.8≤CI＜0.9为低度协同作用(+-)，0.6≤CI＜0.8为中度协同作用(+)，0.4≤CI＜0.6为高度协同作用(++)，0.2≤CI＜0.4为强协同作用(+++)，CI＜0.2为超强协同作用(++++)。
实施例8：Akt2抑制剂发挥糖皮质激素诱导淋巴细胞凋亡的增敏作用的机制：上调细胞内FoxO3a/Bim信号通路
方法：将CCRF-CEM细胞在含有10％胎牛血清(FBS，Gibco公司)的RPMI1640完全培养基(Gibco公司)中在5％CO2、37℃恒温培养箱(Thermo公司)中培养至对数生长期，然后向培养基中加入DMSO、地塞米松和不同的抑制剂(DMSO：0.1％，地塞米松：0.1μM，抑制剂：0.8μM)，继续培养(其中只加入DMSO或地塞米松的细胞继续培养48小时，而加入抑制剂的细胞继续培养24小时)，如实施例1所述进行Western Blot法检测蛋白。
结果：如图5A所示，CCRF-CEM细胞经Akt1抑制剂后能有效抑制p-Akt1，但引起p-Akt2补偿性增高。Akt2抑制剂预处理后，细胞内p-Akt1、p-Akt2表达相应增加。Akt2抑制剂不抑制Akt2磷酸化，却抑制了Akt下游靶点磷酸化活化，同时导致p-Akt1的补偿性增高。Akt1/2抑制剂抑制了Akt自身的磷酸化活化，p-Akt1、p-Akt2表达量较DMSO组减少，但由于DMSO组本身p-Akt1、p-Akt2表达量极少，因此Akt1/2组及DEX+Akt1/2组p-Akt1、p-Akt2蛋白条带未显示。
如图5B所示，较DMSO组相比，DEX+Akt1组、DEX+Akt2组和DEX+Akt1/2组细胞内总FoxO3a蛋白均显著上调(P＜0.05，0.01，0.05)；较DEX组相比，DEX+Akt1组、DEX+Akt2组和DEX+Akt1/2组细胞内总FoxO3a蛋白表达未见明显上调(p＞0.05)。如图5C所示，较DEX组相比，DEX+Akt2组和DEX+Akt1/2组细胞内p-FoxO3a蛋白表达明显下调(p＜0.01)；DEX+Akt1组细胞内p-FoxO3a蛋白表达与DEX组无显著差异(p＞0.05)。如图5D所示，较DMSO组相比，DEX+Akt2组和DEX+Akt1/2组细胞内促凋亡蛋白Bim表达明显上调(p＜0.01)，DEX+Akt1组细胞内Bim蛋白表达与DMSO组无显著差异(p＞0.05)。
Akt2抑制剂、Akt1/2抑制剂通过影响细胞内FoxO3a/Bim信号通路，显著下调细胞内p-FoxO3a/总FoxO3a比值，上调促凋亡蛋白Bim表达，增加糖皮质激素诱导的淋巴细胞凋亡效应，从而发挥对糖皮质激素的增敏作用。该Western Blot结果同样证实了，Akt1抑制剂未能显著影响细胞内FoxO3a/Bim信号通路，这一结果解释了Akt1抑制剂对淋巴细胞糖皮质激素的增敏作用相对较弱的原因。
实施例9：Akt2表达量与淋巴细胞糖皮质激素耐药的关系
实时定量PCR测定：
1.mRNA引物序列设计和合成
Akt1及Akt2引物序列如下：
Akt1正义链引物：5’-GCTGGACGATAGCTTGGA-3’(SEQ ID NO：1)
    反义链引物：5’-GATGACAGATAGCTGGTG-3’(SEQ ID NO：2)
Akt2正义链引物：5’-GGCCCCTGATCAGACTCTA-3’(SEQ ID NO：3)
    反义链引物：5’-TCCTCAGTCGTGGAGGAGT-3’(SEQ ID NO：4)
2.RNA抽提
按RNArose Reagent试剂说明书提取细胞总RNA：
1)将5x106个细胞加入1mlTRlZOL Reagent，室温放置5分钟；
2)加200μl氯仿(与RNAroseReagent之比为1∶5)，振荡15秒，15-30℃放置2-3min，
3)4℃、12000rpm离心15min；
4)将400μl上层水相移入新的RNase-free的离心管中，加入40μl 3M pH值为5.5的醋酸钠溶液和1ml的无水乙醇，-20℃沉淀4小时以上，沉淀出总RNA；
5)4℃、12,000rpm离心10min，弃上清；
6)用75％乙醇1000μl(1∶1)清洗沉淀，-20℃放过夜或继续下步；
7)4℃、12,000rpm离心5min，弃上清，真空抽干或晾干；
8)用RNase-free水30μl溶解RNA，振荡，轻微离心数秒，于55℃-60℃孵育10min，放入-70℃冰箱备用；
9)取少量上述溶液经紫外分光光度计行核酸定量，测量OD260、OD280以确定总RNA的浓度及纯度，调整RNA浓度，1％琼脂糖凝胶电泳观察提取质量。RT-PCR法检测mRNA的表达。
3.mRNA反转录成cDNA：
Figure PCTCN2015096011-appb-000004
Figure PCTCN2015096011-appb-000005
得cDNA反应产物。
4.定量PCR法检测mRNA的表达丰度
将以上反转录的cDNA按1∶10稀释后为模板，使用ABI公司的Power SYBRGreen PCR Master Mix进行realtimePCR反应。反应体系如下
Figure PCTCN2015096011-appb-000006
加水补至总体积20μl，使用ABI公司的RealtimePCR仪7500fast进行反应，反应条件如下：
1)94℃5min
2)94℃30s
3)55℃30s
4)72℃30s
5)读板
6)至2)进行45个循环
7)72℃2min
8)熔解曲线：60℃至95℃，每0.5℃停留90s，读板。
然后使用18srRNA为均一化内参，结果使用2-ΔΔct方法分析。
Akt1、Akt2 RNA小干扰方法：细胞转染
转染的细胞选用复苏后传代3-5代，转染试剂选用INTERFERinTM，转染程序严格按照转染试剂的说明进行。简言之，取对数生长期细胞按2x105/ 孔铺板，转染时给细胞更换新鲜的完全培养基(无添加抗生素)，用Opti-MEM稀释siRNA，加入适量的INTERFERinTM，混匀，室温下孵育10min后滴加到细胞中，适时收集细胞。用绿色荧光素标记的siRNA(FAM-siRNA)转染细胞(siRNA终浓度为20μM，加入1.25μl/孔)，24h后用荧光显微镜观察转染效率。地塞米松组在转染24小时后加入，继续孵育24小时后收细胞检测；对照组在同样时间加入等量的无水乙醇。
Akt1 siRNA：GGCCCAACACCUUCAUCAUTT(SEQ ID NO：5)
            AUGAUGAAGGUGUUGGGCCTT(SEQ ID NO：6)
Akt2 siRNA：GGUUCUUCCUCAGCAUCAATT(SEQ ID NO：7)
            UUGAUGCUGAGGAAGAACCTT(SEQ ID NO：8)
结果：如图5E所示，糖皮质激素敏感细胞CCRF-CEM细胞内Akt2表达量极少，Akt2蛋白条带无法显示；由CCRF-CEM培育而来的高度耐药细胞CEM-DR，细胞内Akt2蛋白表达较CCRF-CEM升高明显，蛋白条带显示清晰；另两种糖皮质激素耐药细胞Jurkat、Daudi以及正常肝细胞L-02细胞内Akt2蛋白表达明显高于敏感株细胞CCRF-CEM。
如图5F、5G所示，我们采用小干扰RNA分别干扰Jurkat细胞内Akt1、Akt2表达。如图5H所示，与地塞米松组相比，被抑制Akt2表达的Jurkat细胞在地塞米松诱导下凋亡效应显著升高(p＜0.01)，被抑制Akt1表达的Jurkat细胞组细胞凋亡率未见升高(p＞0.05)。NC：对照组，转染无siRNA空质粒。
我们对10例初治与11例难治复发(含有糖皮质激素为主的化疗方案，平均7.2疗程后成为难治复发患者)的急性淋巴细胞白血病患者的骨髓淋巴细胞进行Akt1 mRNA、Akt2 mRNA水平的检测，结果如图5I所示，与初治组相比，复发难治组患者体内Akt2 mRNA表达水平显著升高(p＜0.01)，Akt1 mRNA表达水平没有显著差异(p＞0.05)。根据ROC曲线分析，如图5J所示，Akt2作为指标检测患者糖皮质激素耐药程度，ROC曲线下面积为0.9818，最佳判断阈值为16.39，诊断敏感性为90％，特异性为100％。
因此，我们发现Akt2在细胞内的过度表达可能是引起淋巴细胞对糖皮质激素耐药的重要机制：上调的Akt2通过磷酸化失活FoxO3a，上调p-FoxO3a/总FoxO3a比值，下调促凋亡因子Bim蛋白的表达，抑制了细胞内FoxO3a/Bim信号通路，导致糖皮质激素耐药的产生。
实施例10：比较Akt1、Akt2抑制剂对肝细胞的毒性反应
L-02人源正常肝细胞株购于中国科学院上海生物化学与细胞研究所细胞库。裸鼠购于复旦大学药学院动物研究中心并饲养于复旦大学药学院动物实验中心无特定病原体(Specefic pathogen Free，SPF)级动物房。
如实施例6和7所述进行CCK-8检测，以及如实施例1和2所述进行Western Blot检测。
收集对数期生长的CCRF-CEM细胞，用无血清的RPMI1640培养基重悬细胞，细胞密度约为1×108/ml。选择4周龄的免疫缺陷雌性裸鼠，腋窝处皮下注射细胞悬液0.1ml/只(约为1×107细胞/只)。生长四周，待瘤体达300-500mm3，随机分组并实验。将小鼠用地塞米松0.1mg/只，Akt1、Akt2或Akt1/2抑制剂为1.25μg/只腹腔注射，每天一次，共连续7天。第8天眼眶取血，血液标本送至上海动物检测中心行外周血检测。
结果：图6A-6F示出L02肝细胞株的实验结果，其中DMSO：0.1％；DEX：0.1μM；Akt1抑制剂、Akt2抑制剂和Akt1/2抑制剂：0.8μM。
如图6A所示，地塞米松对肝细胞无损害；Akt1抑制剂对肝细胞损伤最为严重，在用药48小时内肝细胞活性均没有恢复；Akt1/2抑制剂对肝细胞也有一定损害，在用药后12小时肝细胞活性降至最低为50.5％，在用药后24小时后肝细胞活性逐渐恢复；Akt2抑制剂对肝细胞损害最小，用药后6小时肝细胞活性降至最低为56.1％，在用药6小时后肝细胞活性逐渐恢复，且在用药后24小时，该组肝细胞活性始终高于Akt1抑制剂和Akt1/2抑制剂组。
如图6B所示，L-02细胞经Akt1抑制剂、Akt2抑制剂预处理后，细胞内p-Akt1、p-Akt2表达相应增加。如图6C所示，较DMSO组相比，DEX+Akt1组、DEX+Akt2组和DEX+Akt1/2组细胞内总FoxO3a蛋白均显著上调(p＜0.05，0.01，0.05)。如图6D所示，较DMSO组相比，DEX+Akt1、DEX+Akt2组和DEX+Akt1/2组细胞内p-FoxO3a蛋白表达明显下调(p＜0.05，0.05，0.05)。如图6E所示，较DMSO组相比，DEX+Akt2组和DEX+Akt1/2组细胞内促凋亡蛋白Bim表达明显上调(p＜0.05)，DEX+Akt1组细胞内Bim蛋白表达与DMSO组无显著差异(p＞0.05)。Akt亚型抑制剂处理肝细胞L-0224小时后，如图6F所示，DEX+Akt2组细胞活性显著高于DEX+Akt1组和DEX+Akt1/2组(p＜0.01，0.05)。上述结果显示，用药24小时后，虽然肝细 胞在Akt2信号被抑制下地塞米松仍然可通过FoxO3a诱导Bim表达产生部分凋亡作用，但Akt1抑制剂、Akt1/2抑制剂较Akt2抑制剂对肝细胞活性抑制作用更显著，我们推测是由于Akt1靶点抑制后导致细胞生长增殖相关的mTOR通路被抑制，从而对肝细胞活性的影响更大；而Akt2靶点为主的FoxO3a/Bim通路的抑制也可调控糖皮质激素诱导肝细胞凋亡，而对肝细胞活性产生一定的影响，但该过程对肝细胞活性的影响与Akt1/mTOR通路被抑制后相比更轻微，且因补偿性p-Akt1增高增强了Akt1/mTOR信号通路使肝细胞活性易于恢复。
我们观察了Akt各亚型抑制剂在裸鼠体内的毒性，如图6G所示，与NC组相比，Akt1/2抑制剂处理后的小鼠ALT升高明显(p＜0.01)。如图6H所示，与NC组相比，Akt1及Akt1/2抑制剂处理后的小鼠AST升高明显(p＜0.05)。如图6I所示，与NC组相比，Akt1及Akt1/2抑制剂处理后的小鼠TBIL升高明显(p＜0.01，0.05)。如图6J、6M-6O所示，与NC组相比，DEX组、DEX+Akt1组、DEX+Akt2组以及DEX+Akt1/2组外周血白细胞、血小板、肌酐、血糖无显著差异(p＞0.05)。如图6K-6L所示，与NC组相比，DEX组、DEX+Akt1组、DEX+Akt2组以及DEX+Akt1/2组外周血RBC、HGB水平显著升高，该结果是由于糖皮质激素刺激后，红细胞由储备池向循环池转移，导致外周血红细胞计数增多。NC：对照组，注射生理盐水。
同时我们解剖各组荷瘤小鼠重要脏器病理标本，包括心脏、肺脏、肾脏、肝脏，行病理切片及HE染色。如图8A、8B所示，各组荷瘤小鼠肝细胞形态结构正常，无变性坏死，纤维组织未见增生，肝细胞间质未见炎症细胞浸润。如图8C所示，各组荷瘤小鼠心肌细胞形态正常，心肌间质未见炎症细胞浸润。如图8D所示，各组荷瘤小鼠肺泡充盈良好，肺泡细胞形态结构正常，肺泡腔内无明显出血及渗出。如图8E所示，各组荷瘤小鼠肾小球肾小管形态结构正常，系膜细胞未见增生，肾间质未见炎症细胞浸润。上述病理结果说明，虽然Akt1、Akt1/2亚型抑制剂对肝脏细胞造成一定损伤，导致小鼠外周血肝酶及总胆红素升高，但用药7天时间里，上述抑制剂未造成肝脏组织形态学的改变。
实施例11：荷瘤小鼠体内验证Akt亚型抑制剂的糖皮质激素增敏效果
收集对数期生长的CCRF-CEM细胞并用无血清的RPMI1640培养基重 悬细胞，细胞密度约为1×108/ml。选择4周龄的免疫缺陷雌性裸鼠，腋窝处皮下注射细胞悬液0.1ml/只(约为1×107细胞/只)，生长四周，待瘤体达300-500mm3，随机分组并实验。将小鼠用地塞米松0.1mg/只，Akt1、Akt2、Akt1/2抑制剂为1.25μg/只腹腔注射，每天一次，共连续7天。测量荷瘤小鼠的总体生存时间，瘤体及脾脏体积，并行瘤体病理切片HE及Ki-67染色，脾脏病理切片HE、CD3及TdT染色。
结果：如图7A、7B所示，与NC对照组(注射生理盐水)相比，DEX+Akt1组、DEX+Akt2组以及DEX+Akt1/2组瘤体体积均明显缩小(p＜0.01、0.01、0.01)；与DEX组相比，DEX+Akt2组和DEX+Akt1/2组瘤体体积显著缩小(p＜0.01、0.05)，DEX+Akt1组瘤体体积未见明显缩小(p＞0.05)。
如图7C所示，与NC组相比，DEX组、DEX+Akt1组、DEX+Akt2组以及DEX+Akt1/2组脾脏均明显缩小(p＜0.01、0.01、0.01、0.01)；与DEX组相比，DEX+Akt2组脾脏显著缩小(p＜0.05)，DEX+Akt1组及DEX+Akt1/2组脾脏体积未见明显缩小(p＞0.05、0.05)。
如图7D所示，DEX+Akt2组及DEX+Akt1/2组荷瘤小鼠总生存时间较DEX组延长(p＜0.05，0.01)。
因此，Akt各亚型抑制剂在体内能协同糖皮质激素有效促进淋巴细胞凋亡，Akt亚型抑制剂对糖皮质激素的增敏效果显著，其中Akt2、Akt1/2抑制剂增敏效果优于Akt1抑制剂。
结论：
1、糖皮质激素耐药的肿瘤淋巴细胞株中，细胞内Akt2表达量较敏感细胞株增加明显。淋巴细胞内Akt2表达量差异可能一定程度上影响并反映糖皮质激素的敏感程度，Akt2蛋白在细胞内的过度表达可能是引起淋巴细胞对糖皮质激素耐药的重要机制，Akt2可作为逆转淋巴肿瘤糖皮质激素耐药更精确的治疗靶点。也可预示淋巴细胞是否糖皮质激素耐药的诊断指标。
2、在T淋巴肿瘤细胞中，Akt2亚型抑制剂显著增加糖皮质激素诱导淋巴细胞凋亡的敏感性；在耐药细胞株中，Akt2亚型抑制剂与糖皮质激素联合具有良好的协同作用，能够逆转糖皮质激素耐药。
3、Akt2亚型抑制剂能够显著下调细胞内p-FoxO3a/总FoxO3a比值，上调促凋亡蛋白Bim的表达，通过增强细胞内FoxO3a/Bim信号通路，增 加糖皮质激素诱导的淋巴细胞凋亡效应，从而增加淋巴细胞糖皮质激素的敏感性。
4、体内外试验均证实Akt2亚型抑制剂毒性最小，对小鼠血液系统、肝脏功能、肾脏功能、血糖均无影响。
5、Akt2亚型抑制剂能够协同地塞米松有效缩小荷瘤小鼠的瘤体和脾脏大小，引起瘤体液化坏死，并延长总生存时间；Akt2亚型抑制剂是糖皮质激素增敏效果最佳且毒副作用最小的药物。

Claims (15)

  1. 一种用于治疗特征为Akt2表达升高的糖皮质激素耐药肿瘤的药物组合物，其包含Akt2抑制剂和糖皮质激素以及任选存在的药物可接受的载体、赋形剂和/或稀释剂。
  2. 权利要求1的药物组合物，其中所述Akt2抑制剂选自小分子化合物，或者Akt2蛋白的拮抗剂例如配体、适配体或抗体，或者抑制Akt2 mRNA的双链RNA分子。
  3. 权利要求1或2的药物组合物，其中所述Akt2抑制剂是选自下式(I)、(II)、(III)、(IV)、(V)和(VI)所示的化合物
    Figure PCTCN2015096011-appb-100001
  4. 权利要求1-3任一项的药物组合物，其中所述Akt2抑制剂是Akt2特异性抑制剂，例如式(II)所示的化合物CCT128930，Akt2蛋白的抗体或者Akt2 mRNA的干扰RNA分子，例如SEQ ID NO：7或8所示的核苷酸分子。
    Figure PCTCN2015096011-appb-100002
  5. 权利要求1-4任一项的药物组合物，其中所述糖皮质激素选自地塞米松、倍他米松、曲安西龙、曲安奈德、倍氯米松、泼尼松龙、泼尼松、甲基强的松龙、氢化可的松、醋酸可的松、布地奈德、二丙酸倍氯米松、环索奈德、可的松、甲泼尼龙、丁酸氯倍他松、氟氢松、丙酸倍氯米松、糠酸莫米松、氯氟舒松、丙酸氯倍他松、氯氟舒松、卤美他松、双醋二氟松及其衍生物，特别是地塞米松。
  6. 权利要求1-5任一项的药物组合物，其中所述肿瘤选自淋巴细胞来源肿瘤，例如选自淋巴细胞白血病如急性淋巴细胞白血病或慢性淋巴细胞白血病、淋巴瘤如B细胞淋巴瘤或T细胞淋巴瘤、和骨髓瘤，优选T细胞来源肿瘤如T淋巴细胞白血病、T细胞淋巴瘤和骨髓瘤。
  7. 权利要求1-6任一项的药物组合物，其中所述肿瘤选自B细胞淋巴瘤如Burkitt’s淋巴瘤、T淋巴细胞白血病例如急性T淋巴细胞白血病、和骨髓瘤。
  8. Akt2检测剂在制备用于检测糖皮质激素耐药肿瘤的组合物或试剂盒中的用途，所述Akt2检测剂例如是Akt2蛋白的配体、适配体或抗体，或者是检测Akt2 mRNA或其cDNA的探针或引物。
  9. 权利要求8的用途，其中所述Akt2检测剂选自SEQ ID NO：3和4所示的核苷酸分子。
  10. Akt2抑制剂在制备用于治疗特征为Akt2表达升高的肿瘤例如促进或提高肿瘤对糖皮质激素治疗的敏感性或治疗糖皮质激素耐药肿瘤的药物组合物中的用途，其中所述药物组合物任选地包含糖皮质激素和/或药物可接受的载体、赋形剂和/或稀释剂。
  11. 权利要求10的用途，其中所述Akt2抑制剂选自小分子化合物例如选自下式(I)、(II)、(III)、(IV)、(V)或(VI)所示的化合物，Akt2蛋白的拮抗剂例如配体、适配体或抗体，或者抑制Akt2 mRNA的双链RNA
    Figure PCTCN2015096011-appb-100003
    Figure PCTCN2015096011-appb-100004
  12. 权利要求10或11的用途，其中所述Akt2抑制剂是Akt2特异性抑制剂，例如式(II)所示的化合物，Akt2蛋白的抗体或者Akt2 mRNA的干扰RNA分子，例如SEQ ID NO：7或8所示的核苷酸分子
    Figure PCTCN2015096011-appb-100005
  13. 权利要求8-12任一项的用途，其中所述糖皮质激素选自地塞米松、倍他米松、曲安西龙、曲安奈德、倍氯米松、泼尼松龙、泼尼松、甲基强的松龙、氢化可的松、醋酸可的松、布地奈德、二丙酸倍氯米松、环索奈德、可的松、甲泼尼龙、丁酸氯倍他松、氟氢松、丙酸倍氯米松、糠酸莫米松、氯氟舒松、丙酸氯倍他松、氯氟舒松、卤美他松、双醋二氟松及其衍生物，特别是地塞米松。
  14. 权利要求8-13任一项的用途，其中所述肿瘤选自淋巴细胞来源肿瘤，例如选自淋巴细胞白血病如急性淋巴细胞白血病或慢性淋巴细胞白血病、淋巴瘤如B细胞淋巴瘤或T细胞淋巴瘤、和骨髓瘤，优选选自T细胞来源肿瘤如T淋巴细胞白血病、T细胞淋巴瘤和骨髓瘤。
  15. 权利要求8-14任一项的用途，其中所述肿瘤选自B细胞淋巴瘤如Burkitt’s淋巴瘤、T淋巴细胞白血病例如急性T淋巴细胞白血病、和骨髓瘤。
PCT/CN2015/096011 2015-11-30 2015-11-30 Akt2在诊断和治疗肿瘤中的用途 WO2017091952A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP15909469.7A EP3375445A4 (en) 2015-11-30 2015-11-30 USE OF AKT2 IN THE DIAGNOSIS AND TREATMENT OF A TUMOR
CN201580084316.6A CN108472296A (zh) 2015-11-30 2015-11-30 Akt2在诊断和治疗肿瘤中的用途
PCT/CN2015/096011 WO2017091952A1 (zh) 2015-11-30 2015-11-30 Akt2在诊断和治疗肿瘤中的用途
US15/778,479 US10849906B2 (en) 2015-11-30 2015-11-30 Use of Akt2 in diagnosis and treatment of tumor
CN202210336057.3A CN115109850A (zh) 2015-11-30 2015-11-30 Akt2在诊断和治疗肿瘤中的用途
JP2018546727A JP6975720B2 (ja) 2015-11-30 2015-11-30 腫瘍の診断および治療におけるＡｋｔ２の使用
JP2021181430A JP7303407B2 (ja) 2015-11-30 2021-11-05 腫瘍の診断および治療におけるＡｋｔ２の使用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/096011 WO2017091952A1 (zh) 2015-11-30 2015-11-30 Akt2在诊断和治疗肿瘤中的用途

Publications (1)

Publication Number Publication Date
WO2017091952A1 true WO2017091952A1 (zh) 2017-06-08

Family

ID=58796011

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/096011 WO2017091952A1 (zh) 2015-11-30 2015-11-30 Akt2在诊断和治疗肿瘤中的用途

Country Status (5)

Country Link
US (1) US10849906B2 (zh)
EP (1) EP3375445A4 (zh)
JP (1) JP6975720B2 (zh)
CN (2) CN108472296A (zh)
WO (1) WO2017091952A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109212006A (zh) * 2018-08-24 2019-01-15 北京艾普希隆生物科技有限公司 一种单细胞琼脂糖凝胶电泳试剂盒

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115414484A (zh) * 2022-08-08 2022-12-02 中国医学科学院基础医学研究所 靶向预防或治疗β-catenin活化突变肿瘤的药物及方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993001288A1 (de) 1991-07-08 1993-01-21 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Phagemid zum screenen von antikörpern
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
WO1994002602A1 (en) 1992-07-24 1994-02-03 Cell Genesys, Inc. Generation of xenogeneic antibodies
US5877293A (en) 1990-07-05 1999-03-02 Celltech Therapeutics Limited CDR grafted anti-CEA antibodies and their production
WO1999032619A1 (en) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Genetic inhibition by double-stranded rna
WO1999049029A1 (en) 1998-03-20 1999-09-30 Benitec Australia Ltd Control of gene expression
WO1999053050A1 (en) 1998-04-08 1999-10-21 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
WO2001034815A1 (en) 1999-11-05 2001-05-17 Cambria Biosciences, Llc Dsrna as insect control agent

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6649341B1 (en) * 2000-04-19 2003-11-18 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Human glucocorticoid receptor 1A promoter and splice variants

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5877293A (en) 1990-07-05 1999-03-02 Celltech Therapeutics Limited CDR grafted anti-CEA antibodies and their production
WO1993001288A1 (de) 1991-07-08 1993-01-21 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Phagemid zum screenen von antikörpern
WO1994002602A1 (en) 1992-07-24 1994-02-03 Cell Genesys, Inc. Generation of xenogeneic antibodies
WO1999032619A1 (en) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Genetic inhibition by double-stranded rna
WO1999049029A1 (en) 1998-03-20 1999-09-30 Benitec Australia Ltd Control of gene expression
WO1999053050A1 (en) 1998-04-08 1999-10-21 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
WO2001034815A1 (en) 1999-11-05 2001-05-17 Cambria Biosciences, Llc Dsrna as insect control agent

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"National Advanced Medicine School Planning Teaching Material: Laboratory Animal Science"
BIRD ET AL.: "Science", vol. 242, 1988, pages: 423
HARLOW ET AL.: "Antibodies: A Laboratory Manual", 1988, CSH PRESS
HARLOW, E.; LANE, D.: "Antibodies: A Laboratory Manual", 1999, COLD SPRINGHARBOR LABORATORY PRESS
MILHAVET O; GARY DS; MATTSON MP, PHARMACOL REV., vol. 55, no. 4, December 2003 (2003-12-01), pages 629 - 48
OPALINSKA JB; GEWIRTZ AM, SCI STKE, vol. 206, 28 October 2003 (2003-10-28), pages e47
SABBINENI, HARIKA ET AL.: "Genetic deletion and pharmacological inhibition of Aktl isoform attenuates bladder cancer cell proliferation, motility and invasion", EUROPEAN JOURNAL OF PHARMACOLOGY, 3 July 2015 (2015-07-03), pages 208 - 214, XP029295129, ISSN: 0014-2999 *
SAMBROOK; FRITSCH; MANIATIS: "Molecular Cloning", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3375445A4
WANG, FENG-ZE ET AL.: "CCT 128930 INDUCESCELLCYCLEARREST,DNA damage, and autophagy independent of Akt inhibition", BIOCHIMIE, vol. 103, 1 May 2014 (2014-05-01), pages 118 - 125, XP028857741, ISSN: 0300-9084 *
WARD ET AL.: "Nature", vol. 341, 1989, pages: 544

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109212006A (zh) * 2018-08-24 2019-01-15 北京艾普希隆生物科技有限公司 一种单细胞琼脂糖凝胶电泳试剂盒

Also Published As

Publication number Publication date
CN115109850A (zh) 2022-09-27
CN108472296A (zh) 2018-08-31
JP2019501959A (ja) 2019-01-24
JP6975720B2 (ja) 2021-12-01
US20200000809A1 (en) 2020-01-02
EP3375445A1 (en) 2018-09-19
US10849906B2 (en) 2020-12-01
EP3375445A4 (en) 2019-08-07

Similar Documents

Publication Publication Date Title
Jin et al. Neddylation blockade diminishes hepatic metastasis by dampening cancer stem-like cells and angiogenesis in uveal melanoma
WO2014046617A1 (en) Compositions and methods for treating cancer
EP3336548B1 (en) Method for providing information on chronic myeloid leukemia
JP2010508277A (ja) 癌を検出および抑制するための方法
JP2019527037A (ja) がんのための診断及び治療方法
EP3220908B1 (en) Compositions and methods for treating endometriosis
JP2023504786A (ja) 抗がん効果増進のためのＥＲＲγ抑制剤を有効成分として含む組成物の用途
Zhang et al. GANT61 plays antitumor effects by inducing oxidative stress through the miRNA‐1286/RAB31 axis in osteosarcoma
Chen et al. miR-205 inhibits neuroblastoma growth by targeting cAMP-responsive element-binding protein 1
WO2017091952A1 (zh) Akt2在诊断和治疗肿瘤中的用途
US8288357B2 (en) Use of inhibitors of leukotriene B4 receptor BLT2 for treating human cancers
US20130089563A1 (en) Method of diagnosing and treating cancer
US11510911B2 (en) Method for prediction of susceptibility to sorafenib treatment by using SULF2 gene, and composition for treatment of cancer comprising SULF2 inhibitor
JP5611953B2 (ja) 癌の処置における治療標的としてのチロシンキナーゼ受容体ｔｙｒｏ３
WO2020113877A1 (zh) E2f6抑制剂的功能与用途
JP7303407B2 (ja) 腫瘍の診断および治療におけるＡｋｔ２の使用
US10865415B2 (en) Prevention, diagnosis and treatment of cancer overexpressing GPR160
US20230390279A1 (en) Composition and method for treating cancer
US9255272B2 (en) Use of inhibitors of leukotriene B4 receptor BLT2 for treating asthma
US11679099B2 (en) Use of HIF-2α inhibitors for treating chondrosarcoma, or preventing recurrence and metastasis thereof
US20220155303A1 (en) Use of tctp as biomarker for predicting efficacy, prognosis of immunotherapy or resistance thereto, and target of immunotherapy for enhancing efficacy
JP7472080B2 (ja) メラノーマの処置および診断
JP6846808B2 (ja) Ｃａｒｄ１４を用いた治療、診断およびスクリーニング
US8518889B2 (en) Method of treating cancer with antibodies against long-form leukotriene B4 receptor BLT2
US8361984B2 (en) Small interfering RNAs and methods for prevention, inhibition and/or treatment of malignant progression of breast cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15909469

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2018546727

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2015909469

Country of ref document: EP