WO2017067454A1 - Utilisation pharmaceutique et produit médicamenteux utilisant un inhibiteur de lsd1 pour la prévention et le traitement du cancer du sein triplement négatif - Google Patents

Utilisation pharmaceutique et produit médicamenteux utilisant un inhibiteur de lsd1 pour la prévention et le traitement du cancer du sein triplement négatif Download PDF

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WO2017067454A1
WO2017067454A1 PCT/CN2016/102559 CN2016102559W WO2017067454A1 WO 2017067454 A1 WO2017067454 A1 WO 2017067454A1 CN 2016102559 W CN2016102559 W CN 2016102559W WO 2017067454 A1 WO2017067454 A1 WO 2017067454A1
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lsd1
drug
ory
gene
partially
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PCT/CN2016/102559
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Chinese (zh)
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孙英丽
王均云
常双
王冬
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中国科学院北京基因组研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • 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

Definitions

  • the invention belongs to the field of biomedicine and relates to the medical use and pharmaceutical products of the LSD1 inhibitor for preventing and treating triple negative breast cancer.
  • TNBC Triple negative breast cancer
  • ER estrogen receptor
  • PR progesterone receptor
  • HER-2 human epidermal growth factor receptor 2
  • ER estrogen receptor
  • PR progesterone receptor
  • HER-2 human epidermal growth factor receptor 2
  • TNBC Triple negative breast cancer
  • ER estrogen receptor
  • PR progesterone receptor
  • HER-2 human epidermal growth factor receptor 2
  • ER estrogen receptor
  • PR progesterone receptor
  • HER-2 human epidermal growth factor receptor 2
  • HER-2 human epidermal growth factor receptor 2
  • LSD1 Human lysine specific demethylase
  • LSD1A Human lysine specific demethylase
  • FAD flavin adenine dinucleotide
  • LSD1 specifically removes the dimethyl modification (me2) and the ninth lysine residue (H3K9) on the fourth lysine residue (H3K4) on histone H3.
  • Base modification [3] LSD1
  • LSD1 also removes methylation modifications from other non-histone substrates such as p53, DNMT1 or E2F1 [4,5] .
  • LSD1 is highly expressed in a variety of tumors, including prostate cancer, undifferentiated neuroblastoma, estrogen-negative breast cancer [6] , bladder cancer, and colorectal cancer.
  • inhibitors of LSD1 mainly include phenylcyclopropylamine inhibitors, substrate polypeptide analogs, and polyamine LSD1 small molecule inhibitors. Since LSD1 belongs to the family of amine oxidases, inhibitors of amine oxidase were first used to investigate whether or not to inhibit LSD1. The earliest discovered LSD1 inhibitor was tranylcypromine, also known as tranylcypromine (TCP). TCP is clinically used to treat depression, which inhibits LSD1 activity, but its choice for the amine oxidase family. Poor sex. The structure of TCP is as shown in Equation A below.
  • ORY-1001 Developed by ORYZON Genomics SA, the highly selective, robust LSD1 inhibitor ORY-1001 has an IC 50 ⁇ 20nM and is 100 times more powerful than LSD1.
  • ORY-1001 is a LSD1 selective inhibitor with no significant inhibitory activity against other FAD-dependent oxidases (MAO-A/B, IL4I1, KDM1B > 100 ⁇ M, SMOX 7 ⁇ M).
  • MAO-A/B, IL4I1, KDM1B > 100 ⁇ M, SMOX 7 ⁇ M.
  • ORY-1001 can inhibit the proliferation and colonization of acute chronic leukemia MV(4;11) cells, and can also significantly inhibit tumor growth in the MV(4;11) mouse model [7] .
  • the structure of ORY-1001 is as shown in the following formula I.
  • ORY-1001 for Phase I/II Phase A clinical trials for the treatment of acute myeloid leukemia (AML) [8] .
  • EMA European Medicines Agency
  • the inhibition of LSD1 by drugs can affect the expression of multiple genes in cells, which can transform leukemia mother cells into normal differentiated cells, and also reduce the proliferation and survival of leukemia stem cells.
  • ORY-1001 can significantly reduce the load of tumor cells and prolong the survival time of mice in an acute lymphoblastic leukemia (ALL) mouse model.
  • ALL acute lymphoblastic leukemia
  • LSD1 can promote the growth of triple negative breast cancer cells and can slow down the G2/M phase arrest caused by ionizing radiation.
  • LSD1 Inhibitors such as ORY-1001
  • ORY-1001 especially in combination with paclitaxel, are effective in inhibiting the proliferation of triple-negative breast cancer cells, significantly inhibiting the growth of triple-negative breast cancer tumors, and have potential for use in the prevention and treatment of triple-negative breast cancer.
  • One aspect of the invention relates to the use of any of the following items (1) to (4) for the manufacture of a medicament for the treatment and/or prevention of triple negative breast cancer in a mammal:
  • nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the LSD1 gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
  • the nucleic acid construct is a recombinant vector, preferably a recombinant expression vector, more preferably a recombinant lentiviral expression vector;
  • the present inventors have found in experiments that overexpression of LSD1 enhances the growth of triple negative breast cancer cells and promotes DNA double-strand break repair.
  • Another aspect of the invention relates to the use of any one of the following items 1 to 4 for the manufacture of a medicament for the treatment and/or prevention of triple negative breast cancer in a mammal:
  • 4LSD1 is involved in the inhibition of the DNA damage repair pathway
  • the mammal is a human.
  • the use, wherein the drug that inhibits or blocks the activity of the LSD1 protein is selected from the group consisting of a tranylcypromine inhibitor, a substrate polypeptide analog, a polyamine LSD1 small molecule inhibitor, Any one or more of an antibody against LSD1 and ORY-1001.
  • the use, wherein the drug that completely knocks out or partially knocks out the LSD1 gene is a polynucleotide for completely knocking out or partially knocking out LSD1; preferably, More
  • the nucleotide is an siRNA such as shRNA or a guide RNA for the CRISPR/Cas9 system.
  • the present inventors used ORY-1001 in combination with paclitaxel to act on triple-negative breast cancer cells, and through CCK-8 cell proliferation assay and cell colony formation assay, it was found that the combination of the two drugs significantly inhibited the proliferation and colony formation of breast cancer cells in vitro; In situ xenograft experiments revealed that the combination of the two drugs significantly inhibited tumor growth in a mouse model in vivo.
  • a further aspect of the invention relates to a pharmaceutical product comprising a separately packaged pharmaceutical preparation 1 and a separately packaged pharmaceutical preparation 2, wherein:
  • the pharmaceutical preparation 1 contains (as an active ingredient) any one of the following items 1-4 as follows:
  • 4LSD1 is involved in the inhibition of the DNA damage repair pathway
  • the drug for inhibiting or blocking the activity of LSD1 protein is selected from the group consisting of a tranylcypromine inhibitor, a substrate polypeptide analog, a polyamine LSD1 small molecule inhibitor, an anti-LSD1 antibody, and any of ORY-1001.
  • a tranylcypromine inhibitor a substrate polypeptide analog
  • a polyamine LSD1 small molecule inhibitor an anti-LSD1 antibody
  • the drug that completely knocks out or partially knocks out the LSD1 gene is a polynucleotide for completely knocking out or partially knocking out LSD1; preferably, the polynucleotide is siRNA such as shRNA, or is used Guide RNA for the CRISPR/Cas9 system;
  • the pharmaceutical preparation 2 comprises (as an active ingredient) any one or more selected from the group consisting of paclitaxel, an anthracycline chemotherapeutic drug, a platinum chemotherapeutic drug, and epirubicin; preferably, the anthracycline chemotherapeutic drug Any one or more selected from the group consisting of doxorubicin (doxorubicin), epirubicin, and pirarubicin; preferably, the platinum-based chemotherapeutic drug is carboplatin or cisplatin;
  • the pharmaceutical formulation 1 and/or pharmaceutical formulation 2 further comprises one or several pharmaceutically acceptable excipients.
  • the pharmaceutical product wherein, when paclitaxel is contained, the pharmaceutical preparation 2 is an injection preparation; preferably, the concentration of paclitaxel is 1-10 mg/mL; for example, 4-8 mg/mL Or 6mg/mL.
  • the pharmaceutical product wherein
  • the amount of the active ingredient in the pharmaceutical preparation 1, such as ORY-1001 is from 10 mg to 500 mg, preferably from 300 mg to 500 mg, for example, 400 mg; and/or
  • the amount of the active ingredient such as paclitaxel in the pharmaceutical preparation 2 is 100 to 200 mg, preferably 135 to 175 mg, for example, 150 mg.
  • the pharmaceutical product is for use in the treatment and/or prevention of triple negative breast cancer.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of active ingredient 1 and an effective amount of active ingredient 2, wherein:
  • the active ingredient 1 is selected from any one of the following items 1-4:
  • 4LSD1 is involved in the inhibition of the DNA damage repair pathway
  • the drug for inhibiting or blocking the activity of LSD1 protein is selected from the group consisting of a tranylcypromine inhibitor, a substrate polypeptide analog, a polyamine LSD1 small molecule inhibitor, an anti-LSD1 antibody, and any of ORY-1001.
  • a tranylcypromine inhibitor a substrate polypeptide analog
  • a polyamine LSD1 small molecule inhibitor an anti-LSD1 antibody
  • the drug that completely knocks out or partially knocks out the LSD1 gene is a polynucleotide for completely knocking out or partially knocking out LSD1; preferably, the polynucleotide is siRNA such as shRNA, or is used Guide RNA for the CRISPR/Cas9 system; an LSD1 inhibitor; preferably ORY-1001;
  • the active ingredient 2 is any one or more selected from the group consisting of paclitaxel, an anthracycline chemotherapeutic drug, a platinum chemotherapeutic drug, and epirubicin; preferably, the anthracycline chemotherapeutic drug is selected from the group consisting of doxorubicin Any one or more of (doxorubicin), epirubicin and pirarubicin; preferably, the platinum-based chemotherapeutic drug is carboplatin or cisplatin;
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition wherein
  • the amount of active ingredient 1 such as ORY-1001 is from 10 mg to 500 mg, preferably from 300 mg to 500 mg, for example 400 mg; and/or
  • the amount of active ingredient 2 such as paclitaxel is from 100 to 200 mg, preferably from 135 to 175 mg, for example 150 mg.
  • the pharmaceutical composition is for use in the treatment and/or prevention of triple negative breast cancer.
  • the invention also relates to a method of treating and/or preventing triple negative breast cancer comprising administering an effective amount to a subject From any of the following 1-4 items:
  • 4LSD1 is involved in the inhibition of the DNA damage repair pathway
  • the drug for inhibiting or blocking the activity of LSD1 protein is selected from the group consisting of a tranylcypromine inhibitor, a substrate polypeptide analog, a polyamine LSD1 small molecule inhibitor, an anti-LSD1 antibody, and any of ORY-1001.
  • a tranylcypromine inhibitor a substrate polypeptide analog
  • a polyamine LSD1 small molecule inhibitor an anti-LSD1 antibody
  • the drug that completely knocks out or partially knocks out the LSD1 gene is a polynucleotide for completely knocking out or partially knocking out LSD1; preferably, the polynucleotide is siRNA such as shRNA, or is used Guide RNA for the CRISPR/Cas9 system;
  • the method further comprises the step of administering to the subject an effective amount of any one or more selected from the group consisting of paclitaxel, an anthracycline-based chemotherapeutic drug, a platinum-based chemotherapeutic drug, and epirubicin; preferably, further comprising the test Perform radiotherapy and/or surgery.
  • ORY-1001 is administered orally or by injection; and/or
  • the route of administration of paclitaxel is injection.
  • the amount of paclitaxel administered and the number of administrations are based on the amount of paclitaxel administered and the number of doses currently administered in the treatment of TNBC.
  • Paclitaxel dose is 135-175mg / m 2; preferably 150mg / m 2; preferably, once every three weeks.
  • m 2 refers to the body surface area of the subject.
  • the dose of paclitaxel or ORY-1001 can be calculated according to the body surface area of the patient.
  • the body surface area is based on the body and body weight of the patient.
  • the intersection of body and body weight on the body surface area calculation table represents the body. Surface area.
  • ORY-1001 is administered in an amount of 10-30 ⁇ g/kg/day, administered orally; paclitaxel is administered in an amount of 10-30 mg/kg, administered
  • the method is intravenous, twice a week.
  • the method the dose of ORY-1001 is 20 ⁇ g/kg/day, and the administration mode is oral; the dosage of paclitaxel is 20 mg/kg, and the administration method is intravenous injection. , twice a week.
  • the dosage administered will depend on a number of factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, as well as the condition and prior medical history of the patient to be treated. It is common practice in the art to start with a dose that is lower than that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved.
  • a further aspect of the invention relates to a method of inhibiting triple negative breast cancer cells in vivo or in vitro, comprising administering to the cells an effective amount of any one of the following 1-4 items:
  • 4LSD1 is involved in the pathway inhibitor of DNA damage repair
  • the drug for inhibiting or blocking the activity of LSD1 protein is selected from the group consisting of a tranylcypromine inhibitor, a substrate polypeptide analog, a polyamine LSD1 small molecule inhibitor, an anti-LSD1 antibody, and any of ORY-1001.
  • a tranylcypromine inhibitor a substrate polypeptide analog
  • a polyamine LSD1 small molecule inhibitor an anti-LSD1 antibody
  • the drug that completely knocks out or partially knocks out the LSD1 gene is a polynucleotide for completely knocking out or partially knocking out LSD1; preferably, the polynucleotide is siRNA such as shRNA, or is used Guide RNA for the CRISPR/Cas9 system;
  • the method further comprises the step of applying to the cell an effective amount of any one or more selected from the group consisting of paclitaxel, an anthracycline chemotherapeutic drug, a platinum chemotherapeutic drug, and epirubicin.
  • the cells When in vitro, preferably, the cells have a ORY-1001 concentration of 5-20 ⁇ M and a paclitaxel concentration of 100 nM; more preferably, the ORY-1001 concentration is 10 ⁇ M and the paclitaxel concentration is 100 nM.
  • a further aspect of the invention relates to a method of screening for a medicament for preventing and/or treating triple negative breast cancer, comprising:
  • the step of detecting inhibition of the test drug or blocking the activity level of the LSD1 protein is a step of detecting inhibition of the test drug or blocking the activity level of the LSD1 protein.
  • test drug can inhibit or reduce the LSD1 gene expression level, or inhibit or block the LSD1 protein activity level, it can be used as a drug candidate.
  • test drug can inhibit or reduce the LSD1 gene expression level, or inhibit or block the LSD1 protein activity level, it can be used as a drug candidate.
  • the test drug is added to a triple negative breast cancer cell, such as a human, in a mammal, such as a cell without the test drug.
  • the test drug is administered to a mammal positive for triple negative breast cancer, such as a human Or rodents, observe or test whether the symptoms or indicators of triple-negative breast cancer improve.
  • the LSD1 protein when referring to the amino acid sequence of the LSD1 protein, it includes the full length of the LSD1 protein, and also includes an active fragment which retains the function of LSD1, and a fusion protein containing the full length or the active fragment.
  • the LSD1 protein is a human LSD1 protein.
  • amino acid sequence of the human LSD1 protein is as follows: (852AA)
  • the LSD1 gene when mentioned, it includes not only a nucleic acid sequence encoding the LSD1 protein but also a degenerate sequence thereof; and further, a regulatory sequence other than the reading frame may be included.
  • the LSD1 gene is the human LSD1 gene.
  • TNBC triple negative breast cancer
  • ER estrogen receptor
  • PR progesterone receptor
  • Her-2 human epidermal growth factor receptor 2
  • nucleic acid construct is a single- or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule.
  • the nucleic acid construct further comprises one or more regulatory sequences operably linked.
  • operably linked refers to the functionality of two or more nucleotide regions or nucleic acid sequences.
  • the "operably linked” can be achieved by means of genetic recombination.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide inhibiting a certain protein can be inserted.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC); phage such as lambda phage or M13 phage And animal viruses, etc.
  • the animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papilloma polyves Virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as papilloma polyves Virus (such as SV40).
  • a vector may contain a variety of elements that control expression.
  • the term "host cell” refers to a cell into which a vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, such as fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Insect cells such as Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • S2 Drosophila cells such as S2 Drosophila cells or Insect cells
  • Sf9 fibroblasts
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the term "effective amount” refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • subject can refer to a patient or other animal that receives the composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, a cow, Horse and so on.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition described herein.
  • the concentration unit ⁇ M represents ⁇ mol/L
  • mM represents mmol/L
  • nM represents nmol/L unless otherwise specified.
  • the dosage in a cell when mentioned, unless otherwise specified, it generally means the final concentration of the drug after the administration.
  • knockdown of DNA or RNA includes, but is not limited to, complete knockout and partial knockout.
  • Complete knockout refers to reducing the level of target DNA or target RNA or the level of expressed protein to an almost undetectable level (in fact, it is generally difficult to knock out the target DNA or target RNA 100%).
  • Partial knockout means that the degree of knockout is greater than zero, less than the case of complete knockout.
  • the present invention also encompasses any one or more selected from the following 1 to 8 items:
  • a pharmaceutical composition for treating triple negative breast cancer comprising: a therapeutically effective amount of paclitaxel or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an inhibitor of LSD1,
  • the LSD1 The inhibitor is preferably selected from the group consisting of a tranylcypromine inhibitor, a substrate polypeptide analog, and a polyamine LSD1 small molecule inhibitor, more preferably selected from the LSD1 inhibitor ORY-1001 of Formula I:
  • composition for treating triple negative breast cancer according to item 1, wherein the therapeutically effective amount of paclitaxel or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of an inhibitor of LSD1 are sequentially given in order
  • the drug is administered to the subject in need.
  • composition for treating triple negative breast cancer according to item 2, wherein the administration of the therapeutically effective amount of paclitaxel or a pharmaceutically acceptable salt thereof occurs in a therapeutically effective amount of an inhibitor of LSD1 Before the administration.
  • composition for treating triple negative breast cancer according to any one of items 1 to 3, wherein the composition has a therapeutic synergistic effect.
  • the inhibitor of LSD1 is preferably selected from the group consisting of a tranylcypromine inhibitor, a substrate polypeptide analog and a polyamine LSD1 small molecule inhibitor, more preferably selected from the group I LSD1 inhibitor ORY-1001:
  • the present invention provides LSD1 inhibitors such as ORY-1001, especially in combination with paclitaxel in the treatment of triple-negative breast cancer, and has a good application prospect.
  • Figure 1 shows the methylation changes of breast cancer cell lines before and after ORY-1001 treatment. After treatment with ORY-1001, LSD1 demethylase activity was inhibited and H3K4 dimethylation levels were elevated in MDA-MB-231 cells.
  • Figure 2 shows that ORY-1001 reduces the enrichment ratio of cancer stem cells in breast cancer cell lines caused by continuous low dose radiation.
  • Figure 2A not processed with ORY-1001;
  • Figure 2B ORY-1001 processed.
  • FIG. 3 CCK method was used to detect the absorbance values of the MDA-MB-231 cells in the four groups, namely the ORY-1001 group, the paclitaxel group, the ORY-1001+ paclitaxel group, and the double-negative control group at 450 nm.
  • Figure 4 Bar graph of clone numbers of 4 groups of MDA-MB-231 cells, namely ORY-1001 group, paclitaxel group, ORY-1001 + paclitaxel group, and double negative control group.
  • Figure 5 A graph of tumor size versus time for different treatments.
  • Figure 6 Figure 6A, Western blot results of LSD1 overexpression, in which flag tag proved to be foreign protein expression rather than endogenous protein; 6B, CCK-8 method to detect cell viability, abscissa, control: not transferred Draw blank control; mock: control with only transfection reagent added; vector: transfection, addition of no load; LSD1: transfection, insertion sequence is LSD1 sequence; Figure 6C, flow cytometry for detection of LSD1 overexpression and ionizing radiation Effect on cell cycle; Figure 6D, qRT-PCR method to detect the expression level of LSD1 expression after Knock-down of LSD1 in cells; Figure 6E, cell survival rate by CCK-8 method, abscissa, control: not Transfection; mock: vector negative control with random sequence added; vector: transfection, addition of no load; siLSD1: transfection, inserts are siLSD1-1 and siLSD1-2.
  • Figure 7 Flow chart of sample processing of gene chip for gene expression profiling.
  • Figure 8 Figure 8A immunofluorescence experiment, after ionizing radiation, overexpression of LSD1 compared to the control H3K4me2 levels decreased.
  • Figure 8B immunofluorescence experiments, after ionizing radiation, overexpression of LSD1 increased ⁇ -H2AX expression compared to controls.
  • Figure 8C western blot analysis, after ionizing radiation, overexpressing LSD1, the expression level of DNA damage double-strand break related proteins p-ATM, p-P53, ⁇ -H2AX increased accordingly.
  • Figure 8D LSD1 overexpression and control Z score ratio, the ratio > 1 indicates that the promotion Activation of the signaling pathway, and vice versa.
  • FIG. 8E the effect of LSD1 inhibitor treatment on cellular signaling pathways following IR irradiation.
  • a Z-score above 0 indicates accelerated pathway activation and vice versa. From top to bottom, the signaling pathway is followed by PPPR ⁇ /RXR ⁇ activation, vitamin C antioxidant, HIPPO signal, PTEN signal, PPAR signal, Rac signal, Calcium signal, p53 signal, B cell activating factor signal, IL-1 signal, sheath.
  • Amino alcohol-1-phosphate signaling Rho-mediated actin-dependent motility regulation, HIV1-mediated apoptosis, CXCR4 signaling, ceramide signaling, Renin-Angiotensin signaling, 4-1BB signaling in T lymphocytes, Wnt /Ca+ signal, regulation of cell cycle G2/M DNA damage checkpoint, ARP-WSAP complex-mediated actin nucleation, TNFR1 signal, role of RIG1-like receptor in antiviral innate immunity, phospholipase C signal, RhoA signal, Rho family GTPase signal, NRF2-mediated oxidative stress response, angiopoietin signal, thrombin signal, NGF signal, Wnt/ ⁇ -ctenin signal.
  • the primary antibody used in western blot is as follows:
  • anti-p-ATM antibody (1:5000, ab81292, abcam, MN, USA),
  • Anti-ATM antibody (1:3000, GTX70103, GeneTex, San Diego, CA, USA),
  • Anti- ⁇ -H2AX antibody (1:1000; 05636, Millipore, CA, USA),
  • Anti-H2AX antibody (1:1000, 2595, Millipore, CA, USA),
  • Anti-p-P53 antibody (1:1000, 9284, Cell signaling),
  • Anti-P53 antibody (1:1000,2524,Cell Signaling),
  • Anti-LSD1 antibody (1:1000, ab90966, abcam, MN, USA),
  • Anti-H3K4me2 and H3 antibodies were purchased from Millipore.
  • Secondary antibody Biyuntian anti-mouse/anti-rabbit secondary antibody.
  • Example 1 Methylation changes of breast cancer cell lines before and after ORY-1001 treatment
  • Cell line human triple negative breast cancer cell line MDA-MB-231, purchased from ATCC (US).
  • ORY-1001 Presented by the Shanghai Institute of Medicine, Chinese Academy of Sciences.
  • the MDA-MB-231 cells were inoculated into a 6-well plate culture until the cell density reached 60%, and the ORY-1001 drug was applied to a final concentration of 10 nmol/well, and cultured for one week after the addition, during which the medicated medium was changed depending on the cell growth.
  • the corresponding cell sample extract protein was collected and used for the following Western blot assay.
  • Example 2 Flow cytometry analysis of the enrichment ratio of breast cancer stem cells
  • the breast cancer cells administered by ORY-1001 were subjected to continuous low-dose radiation irradiation for biological identification.
  • the first is to identify the breast cancer stem cell surface specific antigen CD44 + CD24 -/low by antibody, using flow cytometry.
  • cell lines CD44 + CD24 - ratio / low were analyzed, when cell lines CD44 + CD24 - / low ratio decreased, then compared with the control group (non-ORY-1001 treated the same cell line), after ORY -1001 decreased the proportion and number of breast cancer stem cells in the cells after administration.
  • the cells obtained in the step (1) (3 cells irradiated by 1*2Gy, 2*2Gy, and 3*2Gy) were passed through CD24-APC (eBioscience Dx, 17-0247) and CD44-FITC (BD, respectively). Pharmigen, 555742) Both antibodies were incubated with cells and analyzed by flow cytometry.
  • the flow cytometry experiment was carried out as follows: by flow cytometry, the cells were treated with different batches, washed with phosphate buffer PBS, and then treated with CD24-APC and CD44-FITC.
  • Incubation was performed with excitation light at 635 nm and 488 nm, respectively, and a negative control (no antibody incubation) and a single positive control (incubated with CD24-APC antibody alone and with CD44-FITC antibody only) for quadrant analysis.
  • ORY-1001 reduced the enrichment ratio of tumor stem cells in the MDA-MB-231 cell line caused by continuous low dose radiation (Fig. 2B), indicating that ORY-1001 can be used to treat triple negative breast cancer.
  • CCK-8 Cell Counting Kit-8, Dojindo
  • ORY-1001, paclitaxel, ORY-1001 + paclitaxel, and double negative control treatment solvent DMSO in an equal volume of paclitaxel and OTY-1001).
  • MDA-MB-231 cells were resuscitated, and the cells were in good growth state and passed to a 6-well plate. After the cells were adherent and stable, they were replaced with medicated medium. The following four experimental groups were set up:
  • control group "--" red blood cell plate count (the other groups were not counted, take the same volume of cell suspension as the control group), planted in a 96-well plate at a density of 1000 cells/well, and set 6
  • CCK-8 reagent was added after 24 hours, 48 hours, 72 hours, and 96 hours, and the absorbance value was measured at 450 nm wavelength after incubation for 4 hours, and a growth curve was drawn.
  • the inventors used the method of cell colony formation to detect the number of clones formed by ORY-1001, paclitaxel, ORY-1001+ paclitaxel and double negative control cells. .
  • MDA-MB-231 cells were resuscitated, and the cells were in good growth state.
  • the cells were transferred to a 6-well plate at a density of 500 cells/well. After the cells were adherent and stable, the cells were replaced with medicated medium, and 4 were set up. The same 4 experimental groups.
  • Example 5 Analysis of mouse body model by in situ xenograft mouse model ORY-1001 combined with chemotherapy drug paclitaxel Intratumor growth
  • mice Bal/bc nude mice were divided into 4 groups: double negative control group, ORY-1001 experimental group, paclitaxel experimental group, ORY-1001 + paclitaxel group, 5 mice in each group.
  • human triple-negative breast cancer MDA-MB-231 cells were injected into the mammary gland of the mice to form tumors.
  • Each mouse was injected with two breast pads (fourth pair of breast pads), and each breast pad was injected with 10 5 One cell, the injection volume was 10 ⁇ l.
  • the tumor volume was calculated to be about 20 mm 3 (ORY-1001, 20 ⁇ g/kg/day, intragastric administration; paclitaxel, 20 mg/kg, twice a week, tail Intravenous injection, the cells were stopped after 7 weeks of injection, and the mice were sacrificed to take tumor measurements, and the tumor size curve was plotted over time.
  • mice in the combination group grew the slowest, and the tumors that grew out were the lightest of the other three groups.
  • the results indicate that ORY-1001 combined with paclitaxel can effectively inhibit tumor growth in triple-negative breast cancer.
  • Example 6 LSD1 promotes the growth of triple negative breast cancer cells and slows down the G2/M phase arrest caused by ionizing radiation Test
  • the triple negative breast cancer cell line MDA-MB-231 was purchased from ATCC (US).
  • LSD1 The sequence of LSD1 is shown in SEQ ID NO: 2, and a suitable restriction site can be added, for example, restriction enzymes using NheI and XhoI.
  • the vector uses the lentiviral vector pCDH-MSCV-MCS-EF1-copGFP-T2A-Puro, (SBI, Cat. No. CD713B-1), and then performs routine molecular biological operations such as digestion, ligation, transformation of competent cells. Screening and identification of positive clones, plasmid extraction, and the like. Finally, sequencing was performed and the results were correct. For the following cell transfection.
  • the 293T cells were digested and inoculated, and the cells were cultured in 2 ml of medium, so that the cell density reached about 50% on the day of transfection;
  • plasmid DNA 2.5 ⁇ g was diluted with 50 ⁇ l of OPTI-MEM, wherein the ratio of the core plasmid to the packaging plasmid was 2:1:1:1, gently mixed, and allowed to stand at room temperature for 5 minutes;
  • Lipofectamine 2000 was gently mixed before use, diluted 7.5 ⁇ l Lipofectamine 2000 with 50 ⁇ l OPTI-MEM, gently mixed, and allowed to stand at room temperature for 5 minutes;
  • the diluted plasmid DNA and the diluted Lipofectamine 2000 were gently mixed, and allowed to stand at room temperature for 20 minutes;
  • the transfection complex is added dropwise to the cell culture dish, and the culture dish is gently shaken back and forth to make it evenly dispersed; after 4-6 hours of transfection, it is replaced with complete medium;
  • the virus supernatant was collected, 1600 g, and centrifuged for 8 minutes.
  • the concentrate was filtered and gently dropped into MDA-MB-231 cell culture medium, 10 ⁇ l of 10 mg/ml poly-brene was added dropwise, mixed, and cultured at 37 °C. After 24 hours, the medium was changed to normal medium, and the gene expression was detected after 18-48.
  • LDA1 overexpressing MDA-MB-231 cells and MDA-MB-231 cells transfected with empty vector (vector control cells) were obtained.
  • the prepared cells were divided into two groups according to whether or not ionizing radiation was performed: IR(+)/IR(-).
  • the ionizing radiation is carried out in accordance with the following step (3).
  • MDA-MB-231 cells were passaged into a six-well plate at a suitable concentration.
  • siRNA transfection was performed, and the transfection reagent was Lipofectamine RNAiMAX; the medium was aspirated and 1 ml was added. Medium without double antibody;
  • siRNA 4 ⁇ l of 20 mM (mmol/L) siRNA was diluted with 250 ⁇ l of Opti-MEM, then 4 ⁇ l of transfection reagent was added, gently mixed with a gun, and allowed to stand at room temperature for 20 min; the mixture was carefully added to a six-well plate. Medium, and gently shake it evenly, and then placed in a constant temperature incubator; after 6 hours, replace 2 ml of complete medium, and after 48 hours of culture, collect the cells for the next experiment.
  • siRNA primer sequences are as follows:
  • siLSD1 negative control primer 5'-UUCUUCGAACGUGUCACGUTT-3' (SEQ ID NO: 3)
  • siLSD1 Primer 1 5'-ATGACTAAGGTAAGATGTAGC-3' (SEQ ID NO: 4)
  • siLSD1 Primer 2 5'-TAAGGTGCTTCTAATTGTTGG-3' (SEQ ID NO: 5)
  • the prepared cells were divided into two groups according to whether or not ionizing radiation was performed: IR(+)/IR(-).
  • the ionizing radiation is carried out in accordance with the following step (3).
  • the cells were subjected to a 2 Gy dose of Co-60 radiation treatment in the case of normal adherent culture, and then returned to the incubator for normal culture for subsequent experiments.
  • the extracted RNA was reverse transcribed into cDNA according to the reverse transcription PCR kit instructions, and the cDNA concentration was determined using a NanoDrop instrument;
  • LSD1 forward primer and LSD1 reverse primer, cDNA template, SYBR premix and pure water were added and thoroughly mixed.
  • LSD1 forward primer 5'-TGACCGGATGACTTCTCAAGA-3', (SEQ ID NO: 6)
  • LSD1 reverse primer 5'-GTTGGAGAGTAGCCTCAAATGTC-3' (SEQ ID NO: 7)
  • the Ct value comparison method was used to analyze the quantification, and the histogram was drawn by using GAPDH or ACTIN as the internal reference.
  • the LSD1 overexpression system was constructed in MDA-MB-231, and the expression of LSD1 was increased by Western blot and the level of H3K4me2 methylation was decreased (see Figure 6A).
  • Ionizing radiation can cause DNA damage in cells and lead to G2/M phase arrest, which can effectively prevent DNA-damaged cells from entering the mitosis phase. It can be seen from the statistical graph (Fig. 6C) that as the radiation dose increases, the proportion of cells in the G1 and S phases decreases, while the cells in the G2/M phase gradually increase, after the LSD1 is overexpressed, the G2/M phase The increase in the proportion of cells slows down. This suggests that overexpression of LSD1 in breast cancer cells can slow the G2/M phase arrest caused by DNA damage, suggesting that LSD1 may promote rapid repair of DNA damage.
  • LSD1 can promote the growth of triple-negative breast cancer cells and slow down the G2/M phase arrest caused by ionizing radiation.
  • Example 7 LSD1 promotes repair of DNA double-strand breaks in triple-negative breast cancer cells and inhibition of ORY-1001 DNA damage repair experiment
  • the cells overexpressing LSD1 were treated with ionizing radiation, and the DNA damage and repair pathway induced by LSD1 were studied by immunoblotting (also known as western blot), immunofluorescence assay and gene chip method. Specific steps are as follows:
  • LSD1 cells and control cells were subjected to 2Grey ionizing radiation and cultured for 1-2 hours for immunofluorescence experiments;
  • the anti-fluorescent quencher is dropped on the slide glass, and each slide is dropped with two drops of about 20 ⁇ l each;
  • the cover slip is clamped on the absorbent paper and the anti-fluorescent quencher is placed on the slide after being sucked up with water, facing downward;
  • the nail polish should be in moderation, not too much or too little;
  • the film was placed in a cassette using a laser confocal microscope, and the film was taken and stored at 4 ° C.
  • the gene chip method was used to study the DNA damage repair of MDA-MB-231 cells after LSD1 overexpression and inhibitor treatment in the case of ionizing radiation.
  • RNA extraction is then subjected to RNA extraction, sample quality inspection, chip experiment operation, chip image scanning, data processing and analysis, and the like.
  • the chip used is Illumina HT-12, and the software used to analyze the signal path is Ingenuity Pathway Analysis (IPA) software. See Figure 7 for sample processing.
  • IPA Ingenuity Pathway Analysis
  • LSD1 was involved in DNA damage repair process, activated a series of DNA damage-related pathways and promoted DNA damage repair.
  • ORY-1001 inhibitor many pathways that promote tumor cell proliferation and cell cycle DNA damage checkpoints were inhibited, and the inhibitory PTEN signaling pathway was activated.
  • Nicholson TB Chen T. LSD1 demethylates histone and non-histone proteins. Epigenetics, 2009, 4(3): 129-132.
  • LSD1 Lysine-specific demethylase 1

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Abstract

L'invention concerne l'utilisation d'un inhibiteur de LSD1 dans la préparation d'un médicament visant à prévenir ou traiter un cancer du sein triplement négatif chez un mammifère. L'inhibiteur de LSD1 est ORY-1001. Le mammifère est, de préférence, un être humain. L'invention concerne un produit médicamenteux et une composition pharmaceutique utilisés pour traiter le cancer du sein triplement négatif. L'invention concerne également un procédé de criblage, de prévention et/ou de traitement du cancer du sein triplement négatif.
PCT/CN2016/102559 2015-10-19 2016-10-19 Utilisation pharmaceutique et produit médicamenteux utilisant un inhibiteur de lsd1 pour la prévention et le traitement du cancer du sein triplement négatif WO2017067454A1 (fr)

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CN110295239A (zh) * 2019-08-19 2019-10-01 四川农业大学 达氏鲟三个内参基因、引物开发及稳定性评价技术方法
CN114223614A (zh) * 2021-12-10 2022-03-25 广东省实验动物监测所 乳腺癌细胞株原位移植模型的构建方法及应用
CN114223614B (zh) * 2021-12-10 2023-08-15 广东省实验动物监测所 乳腺癌细胞株原位移植模型的构建方法及应用
CN115068610A (zh) * 2022-01-27 2022-09-20 中国农业大学 抑制乳腺癌细胞中muc1表达的物质在降低抗乳腺癌药物耐药性中的应用
CN115068610B (zh) * 2022-01-27 2024-04-16 中国农业大学 抑制乳腺癌细胞中muc1表达的物质在降低抗乳腺癌药物耐药性中的应用
WO2023217784A1 (fr) * 2022-05-09 2023-11-16 Oryzon Genomics, S.A. Méthodes de traitement de tumeurs mutantes nf1 à l'aide d'inhibiteurs de lsd1
CN117264020A (zh) * 2023-09-25 2023-12-22 北京春冠利亚生物技术有限公司 一种nk细胞的制备方法及其在治疗癌症中的用途
CN117264020B (zh) * 2023-09-25 2024-03-22 爱特康(江苏)生物科技有限公司 一种nk细胞的制备方法及其在治疗癌症中的用途

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