WO2023138011A1 - 不占用核糖体资源的翻译抑制剂作为抗肿瘤药物 - Google Patents

不占用核糖体资源的翻译抑制剂作为抗肿瘤药物 Download PDF

Info

Publication number
WO2023138011A1
WO2023138011A1 PCT/CN2022/106988 CN2022106988W WO2023138011A1 WO 2023138011 A1 WO2023138011 A1 WO 2023138011A1 CN 2022106988 W CN2022106988 W CN 2022106988W WO 2023138011 A1 WO2023138011 A1 WO 2023138011A1
Authority
WO
WIPO (PCT)
Prior art keywords
translation
tumor
translation inhibitor
inhibitor
ribosome
Prior art date
Application number
PCT/CN2022/106988
Other languages
English (en)
French (fr)
Inventor
张弓
陈洋
余卓
Original Assignee
深圳承启生物科技有限公司
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 深圳承启生物科技有限公司 filed Critical 深圳承启生物科技有限公司
Publication of WO2023138011A1 publication Critical patent/WO2023138011A1/zh
Priority to US18/442,146 priority Critical patent/US20240277742A1/en

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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • 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
    • 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
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • 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
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the invention belongs to the field of medicine, and relates to antineoplastic drugs, in particular translation inhibitor molecules that do not occupy ribosome resources are used for antineoplastic drugs.
  • drugs such as rapamycin, sirolimus, and everolimus are used as anticancer drugs in clinical practice. Their targets are all mTOR, which have certain curative effects, but are prone to failure. The reason is as mentioned above.
  • the classic translation inhibitor cycloheximide was used to treat cancer, but its side effects were severe, it was never used as a first-line drug, and it is no longer used clinically.
  • Homoharringtonine also known as omacetaxine
  • Homoharringtonine also known as omacetaxine
  • Homoharringtonine also known as omacetaxine
  • Homoharringtonine also known as omacetaxine
  • the invention provides a translation inhibitor, specifically a translation inhibitor that does not occupy ribosome resources.
  • the translation inhibitor only prevents the translation initiation process, does not lock ribosomes on mRNA, does not interfere with the translation elongation function of assembled ribosomes, and does not significantly promote dissociation.
  • the translation inhibitor that does not occupy ribosome resources is selected from one or more of methyl aurintricarboxylate, kasugamycin or Hippuristanol, or a prodrug of one of them or a pharmaceutically acceptable salt of one of them.
  • the translation inhibitor that does not occupy ribosome resources is methyl aurintricarboxylate, kasugamycin or Hippuristanol, or a prodrug of one of them or a pharmaceutically acceptable salt of one of them. More preferably, the pharmaceutically acceptable salt is ammonium salt, sodium salt, potassium salt or hydrochloride.
  • the translation inhibitor provided by the present invention that does not occupy ribosome resources can be used to prepare antitumor drugs, preferably, to prepare anticancer drugs, especially drugs for lung cancer, ovarian cancer, liver cancer or breast cancer.
  • the translation inhibitor that does not occupy ribosome resources can also be used in combination with other known anti-tumor drugs.
  • the present invention also provides a pharmaceutical composition, comprising a translation inhibitor that does not occupy ribosome resources and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising a translation inhibitor that does not occupy ribosome resources and a pharmaceutically acceptable carrier.
  • the translation inhibitors in the pharmaceutical composition that do not occupy ribosome resources are selected from methyl aurintricarboxylate, kasugamycin or Hippuristanol, or one or more of their prodrugs or pharmaceutically acceptable salts.
  • pharmaceutically acceptable carriers include, but are not limited to, diluents, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorptive carriers, lubricants.
  • the pharmaceutical composition can be made into liquid preparation forms such as tablets, capsules, powders, granules, lozenges, suppositories, oral liquids or sterile parenteral suspensions, and injection forms such as large or small volume injections and lyophilized powders.
  • the present invention also provides an anti-tumor kit, comprising the above-mentioned pharmaceutical composition, and one or more other anti-tumor drugs.
  • the translation inhibitor provided by the invention does not occupy ribosome resources, can effectively inhibit the malignant phenotype of tumors, and has high safety at the same time, can be used to prepare antitumor drugs, and has broad application prospects in preventing or treating tumors.
  • Figure 1 Chemical structures of several translation inhibitors that do not occupy ribosomal resources.
  • A is methyl aurin tricarboxylate
  • B is Kasugamycin
  • C is Hippuristanol.
  • Figure 3 Anti-tumor activity of methyl aurintricarboxylate on lung cancer cells and its safety test. Among them, A cell migration; B cell invasion; C cell apoptosis; D cell clone formation; E cytotoxicity experiment.
  • Figure 4 Effect of methyl aurintricarboxylate on apoptosis of ovarian cancer cells.
  • Figure 5 The inhibitory effect of methyl aurintricarboxylate on the proliferation of liver cancer cells.
  • Figure 7 Antitumor activity of kasugamycin in nude mouse lung cancer xenograft model.
  • Figure 9 Antitumor activity of Hippuristanol in nude mouse lung cancer xenograft model.
  • the methods and techniques of the present invention are generally performed according to conventional methods known in the art unless otherwise indicated.
  • the nomenclatures associated with biology, pharmacy, medicine and chemistry described herein, and the laboratory methods and techniques are those known and commonly used in the art. Standard techniques are used for cell and tissue-related culture and testing methods, pharmaceutical preparation, formulation and delivery, and patient treatment.
  • Translation in this application refers to the translation of eukaryotic cells (Eukaryotic translation), which is the biological process in which messenger RNA is translated into protein in eukaryotes. It consists of four phases: initiation, extension, termination, and recirculation. Translation initiation is the first and most complex step in the translation process.
  • this process can be divided into three stages: first, a variety of translation initiation factors and related proteins bind to the 40S ribosomal small subunit, and then combine with methionine to initiate tRNA, thereby forming the 43S pre-initiation complex; then, the pre-initiation complex binds to the 5' end of the activated mRNA, and moves along the 5' to 3' direction on the 5' untranslated sequence until the correct start codon (usually the first AUG) is found, and the 48S complex is formed; then, 60 The large S ribosomal subunit binds up, eventually forming the 80S initiation complex, ready to start translation. Each step in the initiation process requires the participation of multiple eukaryotic initiation factors.
  • Translation inhibitor broadly refers to an active substance that inhibits or hinders the translation process or translation factors. In this application, it refers to an active molecule that inhibits translation initiation or elongation.
  • Ribosome is a kind of organelle in the cell, which is formed by the combination of two subunits, one large and one small.
  • the main components are intertwined RNA (called “ribosomal RNA” or “rRNA” for short) and protein (called “ribosomal protein”, or “RP” for short).
  • ribosome is the site of protein synthesis in the cell, which can read the genetic information contained in the messenger RNA nucleotide sequence and convert it into the sequence information of amino acids in the protein to synthesize the protein.
  • Not occupying ribosome resources means that in the process of inhibiting translation, ribosomes are not stuck on mRNA, and the size and size of ribosome subunits are still free, which only affects the assembly of the initiation complex. It has no effect on ribosomes that have entered the state of translation elongation, does not prevent the elongation process, and does not actively dissociate intact ribosomes.
  • Translation inhibitor that does not occupy ribosome resources refers to a translation inhibitor that prevents the process of translation initiation, does not lock ribosomes on mRNA, does not interfere with the translation elongation function of assembled ribosomes, and does not significantly promote its dissociation.
  • Tumor is a new organism formed by the body under the action of various carcinogenic factors, a certain cell in a local tissue loses its normal regulation of its growth at the gene level, resulting in abnormal clonal proliferation. Tumors are generally divided into benign and malignant. Malignant tumors are often called cancers or cancers.
  • malignancies include, but are not limited to, bladder, blood, bone, brain/central nervous system, head and neck, breast, cervix, colon, duodenum, esophagus, eye, gallbladder, heart, kidney, larynx, liver, lung, oral cavity, ovary, thyroid, pancreas, pharynx, prostate, rectum, stomach, testis, uterus, skin, and AIDS-related cancers, Hodgkin's disease, lymphoma Lymphoma (including Hodgkin's lymphoma and non-Hodgkin's lymphoma), multiple myeloma, melanoma, leukemia (including lymphocytic leukemia, hairy cell leukemia, acute myeloid leukemia), choriocarcinoma, rhabdomyosarcoma, neuroblastoma, etc.
  • lymphoma Lymphoma including Hodgkin's lymphoma and non-Hodgkin
  • Anti-tumor refers to resisting, inhibiting or eliminating tumors, which may be replaced by “treating tumors", “anti-cancer” or “treating cancer” in this application.
  • Anti-tumor drugs in this application include drugs for the prevention or treatment of tumor growth and metastasis.
  • Treatment of a subject refers to any type of intervention or treatment of a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the appearance, progression, development, severity or recurrence of symptoms, complications, conditions or biochemical indicators associated with a disease. Antineoplastic drugs promote tumor regression or even elimination in a subject.
  • Promoting tumor regression means that administration of a therapeutically effective amount of the drug, alone or in combination with an antineoplastic agent, results in decreased tumor growth or size, tumor necrosis, decreased severity of at least one disease symptom, increased frequency and duration of disease symptom-free periods, prevention of impairment or disability resulting from the disease, or otherwise ameliorating disease symptoms in a patient.
  • the terms “effective” and “effectiveness” with respect to treatment include both pharmacological effectiveness and physiological safety.
  • Pharmacological effectiveness refers to the ability of a drug to promote tumor regression in a patient.
  • Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and/or organism level due to drug administration.
  • a “therapeutically effective dose” is any amount of a drug that, alone or in combination with another therapeutic agent, promotes regression of disease, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of impairment or disability resulting from the disease.
  • a therapeutically effective amount or dose of a drug includes a "prophylactically effective amount” or a “prophylactically effective dose”, which is any amount of a drug as described below that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of a disease, inhibits the onset or recurrence of a disease.
  • a drug or therapeutic agent to promote disease regression or inhibit disease progression or recurrence can be assessed by various methods known to the skilled artisan, for example, in clinical trials in human subjects, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assay systems.
  • “Pharmaceutically acceptable salt” refers to toxicologically compatible organic or inorganic salts of active molecules.
  • Exemplary salts include, but are not limited to: sulfate, citrate, acetate, oxalate, chlorine bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannin, pantothenate, tartrate, ascorbate, succinate.
  • a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions. If the active molecule is a base, pharmaceutically acceptable salts can be prepared by treating the free base with an acid by conventional chemical methods.
  • Such acids include inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, methanesulfonic, phosphoric, etc., or organic acids such as acetic, maleic, succinic, mandelic, fumaric, malonic, pyruvic, oxalic, glycolic, salicylic, uronic (such as glucuronic or galacturonic), alpha hydroxy acids, citric, tartaric, amino acids (such as aspartic, glutamic), aromatic acids (such as benzoic or cinnamic) , sulfonic acid (such as p-toluenesulfonic acid or ethanesulfonic acid), etc.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, methanesulfonic, phosphoric, etc.
  • organic acids such as acetic, maleic, succinic, mandelic, fumaric, malonic, pyruvic,
  • the desired pharmaceutically acceptable salt can be prepared by appropriate methods from inorganic or organic bases such as ammonia, amines, alkali metal hydroxides or alkaline earth metal hydroxides, and the like.
  • suitable salts include, but are not limited to, amino acid salts (such as glycine and arginine), ammonium salts, primary amine salts, secondary amine salts, tertiary amine salts, cyclic amines (such as piperidine, morpholine, and piperazine), sodium salts, calcium salts, potassium salts, magnesium salts, manganese salts, iron salts, copper salts, zinc salts, aluminum salts, and lithium salts.
  • amino acid salts such as glycine and arginine
  • ammonium salts such as glycine and arginine
  • primary amine salts such as glycine and arginine
  • secondary amine salts such as tertiary amine salts
  • cyclic amines such
  • Active metabolites of the compounds involved in the application or their pharmaceutically acceptable salts, and prodrugs that can be transformed into the structures of the compounds involved in the application and their pharmaceutically acceptable salts in vivo are also included in the claims of the application.
  • the active substances contemplated herein may be administered to mammals, preferably humans, alone or in pharmaceutical compositions in combination with a pharmaceutically acceptable carrier, according to standard pharmaceutical techniques. It can be administered orally or subcutaneously, intramuscularly, intraperitoneally, intravenously, rectally and locally, in the eyes, lungs, nasal cavity, or parenterally.
  • “Pharmaceutically acceptable carrier” refers to one or more excipients, stabilizers, fillers, binders, humectants, disintegrants, solution retarders, absorption accelerators, wetting agents, absorbents, lubricants, colorants, diluents, emulsifiers, preservatives, solubilizers, suspending agents, etc. These carriers can be administered to subjects at dosages and concentrations commensurate with a reasonable benefit/risk ratio without undue adverse side effects (eg, toxicity, irritation, and allergic response).
  • Examples of pharmaceutically acceptable carriers include water, citrate or phosphate buffer, starch, lactose, sucrose, glucose, mannitol, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, glycerin, agar, calcium carbonate, alginic acid, sodium carbonate, paraffin, quaternary ammonium compounds, cetyl alcohol, glyceryl monostearate, kaolin and bentonite, talc, calcium stearate, magnesium stearate, polyethylene glycol, sodium lauryl sulfate, ethanol, isopropanol, ethyl carbonate Esters, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils, tetrahydrofuran alcohol, fatty acid esters, isostearyl alcohol sulphur, polyoxyethylene sorbitol and sorbitol esters
  • the present application relates to active substances that can be used in combination with one or several other drugs known to treat or improve similar conditions.
  • the administration method and dose of the original drug remain unchanged, while the drug prepared from the active substance of the present application is administered simultaneously or subsequently.
  • the drug prepared by the active substance of the present application is taken together with one or several other drugs, it is preferable to use a pharmaceutical composition containing one or several known drugs and the drug prepared by the active substance of the present application.
  • Drug combination also includes taking the drug prepared from the active substance of the present application and one or several other known drugs in overlapping time periods.
  • the dose of the drug prepared by the active substance of the present application or the known drug may be lower than that when they are administered alone.
  • Drugs or active ingredients that can be used in combination to treat tumors include, but are not limited to: estrogen receptor modulators, androgen receptor modulators, retinal-like receptor modulators, cytotoxins/cytostatics, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine/threonine amino acid protein inhibitor, Bcr-Abl inhibitor, c-Kit inhibitor, Met inhibitor, Raf inhibitor, MEK inhibitor, MMP inhibitor, topoisomerase inhibitor, histidine deacetylase inhibitor, proteasome inhibitor, CDK inhibitor, Bcl-2 family protein inhibitor, MDM2 family protein inhibitor, IAP family protein inhibitor, STAT family protein inhibitor, PI
  • Methyl aurintricarboxylate is prepared by using aurintricarboxylic acid as a raw material, reacting with methanol, adopting standard Steglich esterification, and synthesizing under the catalysis of DCC and DMAP, or adopting improved Steglich esterification, and synthesizing under the catalysis of EDC and DMAP.
  • the reaction can be carried out at room temperature, or slightly heated to 45°C to increase the reaction rate. If a high-purity product is required, it can be purified and separated by HPLC.
  • DCC is N,N'-Dicyclohexylcarbodiimide
  • DMAP is 4-Dimethylaminopyridine
  • EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride].
  • Kasugamycin CAS No.: 6980-18-3; Supplier: Beijing Biolab Technology Co., Ltd.; Product No.: Y15224.
  • Hippuristanol CAS number: 80442-78-0; supplier: product of Hangzhou Guangyuan Biotechnology Co., Ltd.; product number: GY05894.
  • the polysome profiling experiment was carried out by sucrose density ultracentrifugation. Normal cell lysates and drug-treated cell lysates were ultracentrifuged using a 15-50% sucrose density gradient, and the solution was slowly drawn out to measure OD260nm (260nm optical density, 260nm Optical Density). The results are shown in Figure 2.
  • Figure 2A black line, gray solid line and gray dashed line are the experimental curves obtained from normal cells, cells treated with methyl aurintricarboxylate (concentration 50 ⁇ g/ml) and cells treated with Harringtonin (concentration 10 ⁇ g/ml);
  • Figure 2B gray solid line, gray dashed line, black dotted line are the cells treated with kasugamycin (concentration 10 ⁇ g/ml), Pateamine A (concentration 2 ⁇ M), Hippuristanol (concentration 1 ⁇ M) The resulting experimental curve.
  • the peak height of the curve is related to the concentration.
  • aurintricarboxylic acid, kasugamycin, Pateamine A or Hippuristanol Under the action of aurintricarboxylic acid, kasugamycin, Pateamine A or Hippuristanol, the peaks of 40S and 60S are dominant, and the peaks of complete ribosomes such as 1x and 2x are very small, indicating that the translation initiation is suppressed as a whole, and the large and small subunits of ribosomes cannot be successfully assembled into complete ribosomes.
  • This result can prove that aurintricarboxylic acid, kasugamycin, Pateamine A or Hippuristanol do not occupy ribosomal resources, and are translation inhibitors that do not occupy ribosomal resources.
  • drugs such as the control drug Harringtonin will block ribosomes at the translation initiation site and cannot enter the translation elongation stage, so the 1x ribosome peak is very high, while the polyribosome peak is very small (ribosomes that have entered the translation elongation stage are not controlled by Harringtonin, given enough time, they will all complete translation and fall off), and the peaks of 40S and 60S subunits are also very small.
  • Harringonin also inhibits translation initiation, it will occupy a large amount of ribosome resources.
  • These assembled 80S ribosomes are stuck at the translation initiation site, cannot perform translation functions, and cannot be simply dissociated into large and small subunits, so ribosome resources will be occupied.
  • the selected cell lines include: HBE normal lung epithelial cells, and two lung adenocarcinoma cells with different malignant degrees, A549 and H1299.
  • the experimental steps are as follows: (a) culture the above-mentioned cells with cell culture medium at 37° C. and 0.5% CO 2 environment; (b) add the active substance to be tested (methyl aurin tricarboxylate or cisplatin) and shake well, and continue to culture the cells in the original environment. (c) Detecting the migration, invasion, formation of apoptotic clones and cytotoxicity of the obtained cells, the results are shown in FIG. 3 .
  • Apoptosis cells were fluorescently labeled with Annexin V, and the occurrence of apoptosis was detected by fluorescence microscopy or flow cytometry.
  • Figure 3C the ratio of cell apoptosis and the concentration curve of aurin tricarboxylate
  • the addition of aurin tricarboxylate does not induce apoptosis in normal cells (HBE), but it can induce apoptosis in cancer cells (A549 and H1299)
  • the increase of the concentration of aurin tricarboxylate final concentration 10 ⁇ g/ml, 20 ⁇ g/ml, 40 ⁇ g/ml
  • the proportion of apoptosis in cancer cells increases, and the proportion of cancer cell apoptosis is positively correlated with the concentration of aurin tricarboxylate.
  • the onset concentration (0.25-0.5 mg/ml) of aurin tricarboxylic acid is 12.5
  • Cytotoxicity 48 hours after adding methyl aurin tricarboxylate (final concentration 20 ⁇ g/ml) and cisplatin (50 ⁇ M) to HBE, A549 and H1299 cells, the cytotoxicity was detected by LDH (lactate dehydrogenase, lactose dehydrogenase) method.
  • Cisplatin concentration determines the reference reference report (fang, C.ET Al.mir-488 Inchibits ProLiferation and Cisplatin Sensibility in Non-Small-Cell Lung Cancer (NSCLC) Cells by Activation T He EIF3A-Mediated Ner Signaling Pathway.sci.rep.7 ,2017,40384; yang x.et al.Actl6a Promotes Repair of Cisplatin-Ina Damage, A New Mechanis OF. Platinum Resistance in Cancer, PNAS, Jan 2021,118 (3), E2015808118), using 80-90 % to inhibit the concentration of lung cancer A549 cells.
  • NSCLC Non-Small-Cell Lung Cancer
  • Example 4 Methyl aurintricarboxylate is tested for the antitumor activity of ovarian cancer cells
  • the steps are as follows (a) culture HCCLM3 and MHCC97H9 with cell culture medium at 37°C and 0.5% CO2 environment; (b) add aurin tricarboxylate solution to the culture medium so that the final concentration of aurin tricarboxylate reaches 10 ⁇ g/ml, 20 ⁇ g/ml, 50 ⁇ g/ml, 80 ⁇ g/ml, and 120 ⁇ g/ml, and continue to culture for 48-84 hours before sampling; (c) CCK-8 ( Cell Counting Kit-8) method was used to detect cell proliferation.
  • the human breast cancer cell line MDA-MB-468 was cultured, and kasugamycin was added at 1 mg/L, and the cell proliferation was measured by the well-known sulforhodamine B assay in the field (reference: Qin et al., BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5, Nature Communications 6:8471).
  • kasugamycin As can be seen in Figure 6, after the addition of kasugamycin, cell proliferation slowed down significantly from the 4th day, indicating that kasugamycin has the ability to inhibit the proliferation of breast cancer cells.
  • Embodiment 8H ippuristanol tests the antitumor activity of breast cancer cells
  • the human breast cancer cell line MDA-MB-468 was cultured, and 50nM Hippuristanol was added to carry out the well-known Transwell migration and invasion assay in the field. 100,000 cells were added to the Transwell chamber, and migration/invasion was carried out for 24 hours, and stained with crystal violet. It can be seen from Figure 8 that 50nM Hippuristanol can effectively reduce the migration and invasion of breast cancer cells.
  • the translation inhibitor of the present invention that does not occupy ribosome resources not only has excellent tumor suppression or killing effect, but also has superior safety, and is an excellent anti-tumor pharmaceutical molecule.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Emergency Medicine (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明提供了一种不占用核糖体资源的翻译抑制剂。该不占用核糖体资源的翻译抑制剂显示良好的抗肿瘤作用，且安全性高，可以用于制备抗肿瘤药物。

Description

不占用核糖体资源的翻译抑制剂作为抗肿瘤药物 技术领域
本发明属于医药领域，涉及抗肿瘤药物，特别是不占用核糖体资源的翻译抑制分子用于抗肿瘤药物。
背景技术
肿瘤无限增殖和迁移侵袭，离不开旺盛的蛋白质合成(即翻译过程)。肿瘤细胞内多条经典促癌通路，如MAPK通路、mTOR通路等，下游均为增强翻译。癌细胞内还会通过上调p90RSK的翻译效率，从而正反馈自激活翻译起始通路，从而维持自身翻译系统的高度活性(Wang et al.,Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific，Nucleic Acids Research，2013，41(9):4743-54)。因此，抑制翻译系统很早就被作为一种可能的抑制肿瘤细胞的方案。目前，雷帕霉素(rapamycin)、西罗莫司(temsirolmus)、依维莫司(everolimus)等药物在临床上被作为抗癌药物使用，其靶点均为mTOR，有一定的疗效，但容易失效，其原因正如上文所述，癌细胞内有多条通路可上调翻译系统，例如p90RSK和p70S6K等即可绕过mTOR，因而可以被肿瘤细胞利用而造成对这些药物的耐药。于是人们希望能够直接抑制翻译系统本身。几十年前，经典的翻译抑制剂放线菌酮(cycloheximide)曾被用于治疗癌症，但其副作用很大，从未被作为一线药物，现在临床上已不再使用。高三尖杉酯碱(harringtonine，又称omacetaxine)近年来也在临床上被尝试用于癌症的治疗，但同样因为副作用很大而无法在实体肿瘤中使用。
因此，十分有必要提供一种新的翻译抑制剂来解决上述技术问题。
发明内容
本发明提供了一种翻译抑制剂，具体为不占用核糖体资源的翻译抑制剂，所述翻译抑制剂，仅阻止翻译起始过程，不将核糖体卡在mRNA上，对已经组装完毕的核糖体不干扰其翻译延伸功能，不对其解离有明显的促进作用。优选地，这种不占用核糖体资源的翻译抑制剂选自金精三羧酸甲酯、春日霉素或Hippuristanol，或它们之一的前药或它们之一的药学上可接受的盐中的一种或多种。较优地，不占用核糖体资源的翻译抑制剂为金精三羧酸甲酯、春日霉素或Hippuristanol，或它们之一的前药或它们之一的药学上可接受的盐。更优地，药学上可接受的盐为铵盐、钠盐、钾盐或盐酸盐。
本发明提供的不占用核糖体资源的翻译抑制剂，可用于制备抗肿瘤药物，优选地， 用于制备抗恶性肿瘤，特别是针对肺癌、卵巢癌、肝癌或乳腺癌的药物。
在一些情况下，所述不占用核糖体资源的翻译抑制剂，还可以与已知的其它药物抗肿瘤药物联用。
本发明还提供了一种药物组合物，包含不占用核糖体资源的翻译抑制剂和药学上可接受的载体。优选地，所述药物组合物中不占用核糖体资源的翻译抑制剂选自金精三羧酸甲酯、春日霉素或Hippuristanol，或它们之一的前药或药学上可接受的盐中的一种或多种。
在一些情况下，药学上可接受的载体包括但不限于稀释剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂。所述药物组合物可以制成片剂、胶囊、粉剂、颗粒、锭剂、栓剂、口服液或无菌胃肠外悬液等液体制剂形式，以及大或小容量注射剂、冻干粉剂等针剂形式。
本发明还提供了一种抗肿瘤药盒，包含上述药物组合物，以及一种或多种其它抗肿瘤药物。
本发明所提供的不占用核糖体资源的翻译抑制剂，能够有效地抑制肿瘤的恶性表型，同时又具有很高的安全性，可用于制备抗肿瘤药物，在预防或治疗肿瘤方面有着广泛的应用前景。
附图说明
图1.几种不占用核糖体资源的翻译抑制剂的化学结构。其中，A为金精三羧酸甲酯；B为春日霉素(Kasugamycin)；C为Hippuristanol。
图2.几种活性分子不占用核糖体资源的实验结果。
图3.金精三羧酸甲酯对肺癌细胞抗肿瘤活性及其安全性测试。其中，A细胞迁移；B细胞侵袭；C细胞凋亡；D细胞克隆形成；E细胞毒性实验。
图4.金精三羧酸甲酯对卵巢癌细胞的细胞凋亡的作用。
图5.金精三羧酸甲酯对肝癌细胞增殖的抑制作用。
图6.春日霉素对乳腺癌细胞增殖的抑制作用。
图7.春日霉素在裸鼠肺癌移植瘤模型中的抗肿瘤活性。
图8.Hippuristanol对乳腺癌细胞增殖的抑制作用。其中，Hipp代表Hippuristanol。
图9.Hippuristanol在裸鼠肺癌移植瘤模型中的抗肿瘤活性。
具体实施方式
本发明的方法与技术通常依据本领域已知的传统方法进行，除非另有说明。与本文中描述的生物学、药学、医学及化学相关的命名法，及实验方法与技术是本领域已知且常用 的。细胞及组织相关的培养及测试方法、医药制法、调配法与传送法，及患者的治疗法均采用标准技术。
除非另有说明，本文中所使用的科学与技术术语应具有本领域技术人员通常理解的含义。但下列术语具有如下定义：
“翻译”在本申请中指真核细胞的翻译(Eukaryotic translation)，是信使RNA在真核生物中翻译成蛋白质的生物学过程。它由四个阶段组成：起始，延伸，终止，和再循环。翻译起始是翻译过程中的第一步，也是最为复杂的一步。在真核生物中，这一过程可以被分为三段：首先，多种翻译起始因子及相关蛋白质结合到40S核糖体小亚基上，再结合甲硫氨酸起始tRNA，从而形成43S前起始复合物；接着，前起始复合物结合到被激活的mRNA的5'端末端，并沿着5'至3'的方向在5'端非翻译序列上移动，直到寻找到正确的起始密码子(一般为第一个AUG)，并形成48S复合物；然后，60S核糖体大亚基结合上来，最终形成80S起始复合物，准备开始翻译。而起始过程中的每一步都需要多个真核起始因子的参与。
“翻译抑制剂”广义上泛指对翻译过程或翻译因子起抑制或阻碍作用的活性物质，在本申请中，指抑制翻译起始或延伸的活性分子。
“核糖体”(ribosome)，是细胞中的一种细胞器，由一大一小两个亚基结合形成，主要成分是相互缠绕的RNA(称为“核糖体RNA”简称“rRNA”)和蛋白质(称为“核糖体蛋白质”，简称“RP”)。核糖体是细胞内蛋白质合成的场所，能读取信使RNA核苷酸序列所包含的遗传信息，并使之转化为蛋白质中氨基酸的序列信息以合成蛋白质。
“不占用核糖体资源”，是指在对翻译起抑制作用的过程中，不将核糖体卡在mRNA上，核糖体大小亚基仍是自由的，仅影响起始复合物组装。对已经进入翻译延伸状态的核糖体不起作用，不阻止其延伸过程，也不对完整核糖体进行主动解离。
“不占用核糖体资源的翻译抑制剂”，是指一种阻止翻译起始过程，不将核糖体卡在mRNA上，对已经组装完毕的核糖体不干扰其翻译延伸功能，不对其解离有明显的促进作用的翻译抑制剂。
“肿瘤”(tumor)是机体在各种致癌因素作用下，局部组织的某一个细胞在基因水平上失去对其生长的正常调控，导致其克隆性异常增生而形成的新生物。一般将肿瘤分为良性和恶性两大类。恶性肿瘤通常称为癌症或癌肿(cancer)。恶性肿瘤的例子包括但不限于膀胱癌、血癌、骨癌、脑癌/中枢神经系统癌、头颈癌、乳腺癌、宫颈癌、结肠癌、十二指肠癌、食道癌、眼癌、胆囊癌、心脏癌、肾癌、喉癌、肝癌、肺癌、口腔癌、卵巢癌、甲状腺癌、胰腺癌、咽癌、前列腺癌、直肠癌、胃癌、睾丸癌、子宫癌、皮肤癌，以及艾滋病相关 的癌症、何杰金氏病、淋巴瘤(包括霍奇金淋巴瘤和非霍奇金淋巴瘤)、多发性骨髓瘤、黑色素瘤、白血病(包括淋巴细胞白血病、毛细胞白血病、急性髓细胞白血病)、绒毛膜癌、横纹肌肉瘤、神经母细胞瘤等。
“抗肿瘤”指抵抗、抑制或消除肿瘤，在本申请中也可用“治疗肿瘤”、“抗癌”或“治疗癌症”替代。“抗肿瘤药物”，在本申请中包含预防或治疗肿瘤生长与转移的药物。对受试者的“治疗”是指以逆转、减轻、改善、抑制、减缓或防止与疾病有关的症状、并发症、病症或生化指标的出现、进展、发展、严重程度或复发为目的对受试者进行任何类型的干预或处理。抗肿瘤药物促进受试者体内的肿瘤消退甚至消除。“促进肿瘤消退”意思是单独施用或与抗肿瘤剂组合施用治疗有效量的药物导致肿瘤生长减少或大小减小、肿瘤坏死、至少一种疾病症状的严重程度降低，无疾病症状期的频率和持续时间增加，防止由患病导致的障碍或失能，或者以其他方式改善患者的疾病症状。此外，术语关于治疗的“有效”和“有效性”包括药理学有效性和生理学安全性二者。药理学有效性是指药物促进患者肿瘤消退的能力。生理学安全性是指由于药物施用导致的细胞、器官和/或生物体水平上的毒性或者其它不良生理效果(不良作用)的水平。
“治疗有效剂量”是药物的任何如下所述的量，当单独使用或与另一种治疗剂组合使用该量的药物时，可促进疾病消退，疾病消退表现为疾病症状的严重度降低、无疾病症状期的频率和持续时间增加、或者防止由患病导致的障碍或失能。药物的治疗有效量或剂量包括“预防有效量”或“预防有效剂量”，“预防有效量”或“预防有效剂量”是药物的任何如下所述的量，当将该量的药物单独施用或者与另一种治疗剂组合施用于具有发生疾病的风险或者遭受疾病复发的受试者时，可抑制疾病的发生或复发。药物或治疗剂促进疾病消退或抑制疾病发展或复发的能力可以用技术人员已知的各种方法进行评估，例如在人类受试者的临床试验中，在可预测在人类中的效力的动物模型系统中，或者通过在体外测定系统中测定试剂的活性。
“药学上可接受的盐”，指活性分子在毒理学上兼容的有机盐或无机盐。示例盐包括但不限于：硫酸盐、柠檬酸盐、醋酸盐、草酸盐、溴化氯、碘化物、硝酸盐、硫酸氢盐、磷酸盐、酸磷酸盐、异烟酸盐、乳酸盐、水杨酸盐、柠檬酸盐、酒石酸盐、油酸盐、单宁酸盐、泛酸盐、酒石酸盐、抗坏血酸盐、琥珀酸盐。马来酸盐、龙胆酸盐、富马酸盐、葡萄糖酸盐、葡萄糖醛酸盐、甲酸盐、苯甲酸盐、谷氨酸盐、甲磺酸“甲磺酸盐”、乙磺酸盐、苯磺酸盐、碱金属(如:钠和钾)盐、碱土金属(如:镁)盐和铵盐。一种药学上可接受的盐可以有一个或多个带电原子和/或一个或多个平衡离子。如果活性分子是碱，药学上可接受的 盐可以通过常规化学方法用酸处理游离碱来制备。这样的酸包括无机酸，如盐酸、氢溴酸、硫酸、硝酸、甲基磺酸、磷酸等，或有机酸,如醋酸、顺丁烯二酸、琥珀酸、扁桃酸、富马酸、丙二酸、丙酮酸、草酸、乙醇酸、水杨酸、糖醛酸(如葡萄糖醛酸或半乳糖醛酸)、阿尔法羟基酸、柠檬酸、酒石酸、氨基酸(如天冬氨酸、谷氨酸)、芳香酸(如苯甲酸或肉桂酸)、磺酸(如对甲苯磺酸或乙烷磺酸)等。如果活性分子是酸，所需的药学上可接受的盐可以通过合适的方法，用无机碱或有机碱来制备，这样的碱如氨、胺、氢氧化碱金属或氢氧化碱土金属等。合适盐的示例包括但不限于，氨基酸盐(如甘氨酸和精氨酸)、铵盐、伯胺盐、仲胺盐、叔胺盐、环胺(如哌啶、吗啉和哌嗪)、钠盐、钙盐、钾盐、镁盐、锰盐、铁盐、铜盐、锌盐、铝盐和锂盐。
本申请所涉及的化合物或其药学可接受的盐的活性代谢产物，以及可以在体内转变为本申请所涉及的化合物及其药学可接受的盐的结构的前药，也包含在本申请的权利要求中。
根据标准药学技术，本申请涉及活性物质可单独或在药用组合物中与药学上可接受的载体组合给予哺乳动物，优选人。可口服或皮下、肌注、腹膜内、静脉、直肠及局部、眼睛、肺部、鼻腔、胃肠外给药。
“药学上可接受的载体”，是指一种或多种辅料、稳定剂、填料、粘结剂、保湿剂、崩解剂、溶液缓凝剂、吸收促进剂、润湿剂、吸收剂、润滑剂、着色剂、稀释剂、乳化剂、防腐剂、增溶剂、悬浮剂等。这些载体可施用于受试者，其剂量和浓度与合理的效益/风险比相称，没有不当的不良副作用(如毒性、刺激和过敏反应)。药物可接受的载体的例子包括水、柠檬酸盐或磷酸盐缓冲液、淀粉、乳糖、蔗糖、葡萄糖、甘露醇、羧甲基纤维素、海藻酸盐、明胶、聚乙烯醇吡咯烷酮、甘油、琼脂、碳酸钙、海藻酸、碳酸钠、石蜡、季铵盐化合物、十六醇、甘油单硬脂酸酯、高岭土和膨润土、滑石粉、硬脂酸钙、硬脂酸镁、聚乙二醇、十二烷基硫酸钠、乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁二醇、油、四氢呋喃醇、脂肪酸酯、硫氧化异硬脂醇、聚氧乙烯山梨醇和山梨醇酯、微晶纤维素、偏氢氧化铝、黄蓍胶及其混合物和本行业技术人员所熟知的其他成分。
本申请涉及活性物质可以与已知的治疗或改进相似病状的其它一种或几种药物联用。联合给药时，原来药物的给药方式和剂量保持不变，而同时或随后施用本申请活性物质制备的药物。当本申请活性物质制备的药物与其它一种或几种药物同时服用时，优选使用同时含有一种或几种已知药物和本申请活性物质制备的药物的药用组合物。药物联用也包括在重叠的时 间段服用本申请活性物质制备的药物与其它一种或几种已知药物。当本申请活性物质制备的药物与其它一种或几种药物进行药物联用时本申请活性物质制备的药物或已知药物的剂量可能比它们单独用药时的剂量较低。
可以进行联用以治疗肿瘤的药物或活性成分包括但不局限为：雌激素受体调节剂、雄激素受体调节剂、视网膜样受体调节剂、细胞毒素/细胞抑制剂、抗增殖剂、蛋白转移酶抑制剂、HMG-CoA还原酶抑制剂、HIV蛋白激酶抑制剂、逆转录酶抑制剂、血管生成抑制剂、细胞增殖及生存信号抑制剂、干扰细胞周期关卡的药物和细胞凋亡诱导剂、细胞毒类药物、酪氨酸蛋白抑制剂、EGFR抑制剂、VEGFR抑制剂、丝氨酸/苏氨酸蛋白抑制剂、Bcr-Abl抑制剂、c-Kit抑制剂、Met抑制剂、Raf抑制剂、MEK抑制剂、MMP抑制剂、拓扑异构酶抑制剂、组氨酸去乙酰化酶抑制剂、蛋白酶体抑制剂、CDK抑制剂、Bcl-2家族蛋白抑制剂、MDM2家族蛋白抑制剂、IAP家族蛋白抑制剂、STAT家族蛋白抑制剂、PI3K抑制剂、AKT抑制剂、整联蛋白阻滞剂、干扰素-α、白介素-12、COX-2抑制剂、p53、p53激活剂、VEGF抗体、EGF抗体等。
金精三羧酸甲酯、春日霉素及Hippuristanol的化学结构式见图1。
金精三羧酸甲酯，以金精三羧酸为原料，与甲醇反应，采用标准的施特格利希(Steglich)酯化反应，在DCC和DMAP的催化下合成，或采用改良的Steglich酯化反应，在EDC和DMAP的催化下合成，均可制得。反应可在室温进行，或稍加热至45℃以提升反应速率。若需要高纯度的产物，可用HPLC进行纯化分离。DCC为二环己基碳二亚胺(N,N′-Dicyclohexylcarbodiimide)；DMAP为4-二甲氨基吡啶(4-Dimethylaminopyridine)；EDC为1-乙基-3-(3-二甲基胺丙基)碳化二亚胺盐酸盐【1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride】。
春日霉素和Hippuristanol均由外购获得。
春日霉素：CAS号：6980-18-3；供应商：北京百奥莱博科技有限公司；产品货号：Y15224。
Hippuristanol：CAS号：80442-78-0；供应商：杭州光远生物技术有限公司产品；货号：GY05894。
下面将结合本申请具体的实施例，对本申请技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
实施例1金精三羧酸甲酯的制备
Figure PCTCN2022106988-appb-000001
在15ml乙腈中，加入1mmol金精三羧酸(CAS号：4431-00-9；供应商：国药集团化学试剂有限公司；货号：LA1590501),2.55mmol DMAP,1.28mmol EDC,0.85mmol甲醇。混合均匀。室温反应过夜。反应完成后，减压，使乙腈挥发，粗生成物为固体。加入20mL乙醚(也可以用乙酸乙酯)进行萃取。20mL 1M HCl洗涤2次。20mL饱和碳酸钠溶液洗涤两次。无水硫酸钠干燥。在旋转蒸发仪中减压去除乙醚(或乙酸乙酯)。得金精三羧酸甲酯0.77mmol，收率77％。LC-MS(m/z)：437.38[M] +。
实施例2金精三羧酸甲酯、春日霉素及Hippuristanol均为不占用核糖体资源的翻译抑制剂的证明
运用本领域技术人员公知的方法，进行蔗糖密度超速离心测定多聚核糖体(polysome profiling)实验。使用15-50％的蔗糖密度梯度对正常细胞溶解产物及药物处理过的细胞溶解产物进行超速离心，缓慢抽出溶液，测定OD260nm(260nm光密度，260nm Optical Density)，结果如图2所示。图2A，黑色线、灰色实线及灰色虚线分别为正常细胞、金精三羧酸甲酯(浓度50μg/ml)处理的细胞及Harringtonin(浓度10μg/ml)处理的细胞所得到的实验曲线；图2B，灰色实线、灰色虚线、黑色点划线分别为经春日霉素(浓度10μg/ml)、Pateamine A(浓度2μM)、Hippuristanol(浓度1μM)处理的细胞所得到的实验曲线。曲线峰高与浓度有关。
正常细胞内进行着正常的翻译，因此能看到40S,60S,80S(单个核糖体，图上标为1x)，2x核糖体，3x核糖体等峰。正常状况下，细胞内80％以上的核糖体都在翻译中，因此40S 60S的峰相对较低。金精三羧酸、春日霉素、Pateamine A或Hippuristanol作用下，40S、60S峰占主导地位，1x,2x等完整核糖体峰很小，说明翻译起始被整体压制，核糖体的大小亚基并不能成功组装成完整的核糖体。这个结果可以证明金精三羧酸、春日霉素、Pateamine A或Hippuristanol并不占用核糖体资源，是不占用核糖体资源的翻译抑制剂。通过透析、蔗糖密度梯度离心等方式去掉药物后，加入合适的缓冲液成分，翻译又可以再次正常进行。
而对照药物Harringtonin等药物会将核糖体卡在翻译起始位点上而不能进入翻译延伸阶段，因此1x核糖体峰很高，而多核糖体峰很小(已进入翻译延伸阶段的核糖体不受 Harringtonin控制，给予足够的时间，其都将完成翻译而脱落)，40S、60S大小亚基峰也很小。这说明Harringonin虽然也抑制翻译起始，但会大量占用核糖体资源，这些已组装好的80S核糖体被卡在翻译起始位点上，不能行使翻译功能，也无法简单地解离成大小亚基，因此会占用核糖体资源。
实施例3金精三羧酸甲酯对肺癌细胞的抗肿瘤活性测试
运用本领域技术人员公知的方法，分别进行了细胞迁移、细胞侵袭、细胞凋亡、细胞克隆形成和细胞毒性实验。
所选取的细胞系为括：HBE正常肺上皮细胞，及A549和H1299两种恶性程度不同的肺腺癌细胞。实验步骤如下：(a)用细胞培养液于37℃、0.5％CO 2环境中培养上述细胞；(b)加入待测试的活性物质(金精三羧酸甲酯或顺铂)摇匀，继续在原环境中培养细胞。(c)检测所得到的细胞的迁移、侵袭、凋亡克隆形成及细胞毒性情况，结果如图3所示。
(1)细胞迁移：通过Transwe11实验检测HBE、A549和H1299细胞用药前后穿过小孔的迁移能力，金精三羧酸甲酯的处理终浓度为20μg/ml，如图3A所示，与空白对照相比，金精三羧酸甲酯对正常细胞(HBE)影响不大，对癌细胞(A549和H1299)转移能力显著抑制。
(2)细胞侵袭：对是否经金精三羧酸甲酯(终浓度20μg/ml)的处理的HBE、A549和H1299细胞进行Transwe11侵袭实验。如图3B所示，与空白对照相比，金精三羧酸甲酯对癌细胞(A549和H1299)侵袭能力显著抑制。
(3)细胞凋亡：使用Annexin V对细胞进行荧光标记，利用荧光显微镜或流式细胞仪检测细胞凋亡的发生。如图3C(细胞凋亡比例与金精三羧酸甲酯的加药浓度关系曲线)所示，金精三羧酸甲酯的加入不会诱导正常细胞(HBE)凋亡，但是会诱导癌细胞(A549和H1299)凋亡，且随着金精三羧酸甲酯浓度的增加(终浓度10μg/ml、20μg/ml、40μg/ml)，癌细胞的凋亡比例上升，癌细胞凋亡比例与金精三羧酸甲酯的浓度呈正相关。同样的实验条件下，金精三羧酸的起效浓度(0.25～0.5mg/ml)是金精三羧酸甲酯的12.5～25倍。
(4)细胞克隆形成：对未经处理的和金精三羧酸甲酯(终浓度20μg/ml)处理的A549和H1299细胞分别进行琼脂克隆形成实验，从图3D可以看出，金精三羧酸甲酯处理后癌细胞(A549和H1299)每1000个细胞的克隆数显著降低，说明癌细胞的干性和转移后的定植能力被抑制。
(5)细胞毒性：对HBE、A549和H1299细胞分别加入金精三羧酸甲酯(终浓度20μg/ml)、顺铂(50μM)48小时后，通过LDH(lactate dehydrogenase，乳糖脱氢酶)法， 检测细胞毒性。
顺铂(Cisplatin)浓度确定参考文献报道(Fang,C.et al.MiR-488 inhibits proliferation and cisplatin sensibility in non-small-cell lung cancer(NSCLC)cells by activating the eIF3a-mediated NER signaling pathway.Sci.Rep.7,2017,40384；Yang X.et al.ACTL6A promotes repair of cisplatin-induced DNA damage,a new mechanism of platinum resistance in cancer,PNAS,Jan 2021,118(3),e2015808118)，采用能80-90％抑制肺癌A549细胞的浓度。
该实验测定490nm与630nm处光密度之差，差值越大说明细胞毒性越大。由图3E可见顺铂在50μM浓度下，虽可以有效杀伤A549和H1299两种肺癌细胞，但同样也能有效杀伤HBE正常细胞，因而显示出很大的毒性。相比之下金精三羧酸甲酯则毒性很小，因此可知其安全性高。
以上(1)～(5)实验结果显示金精三羧酸甲酯不但对癌细胞具有良好的抑制和杀灭作用，而且对正常细胞几乎无影响，安全性高。
实施例4金精三羧酸甲酯对卵巢癌细胞抗肿瘤活性测试
采用人正常成纤维细胞(原代培养)作为正常对照，ES-2人卵巢癌细胞系作为癌细胞，进行了细胞凋亡实验。步骤如下：(a)用细胞培养液于37℃、0.5％CO 2环境中培养上述细胞：(b)加入金精三羧酸甲酯溶液，摇匀，继续在原环境中培养细胞48小时。(c)Annexin V流式细胞法检测凋亡。结果如图4所示，其中，Q1区为未凋亡细胞，Q3区的细胞为早期凋亡(观测凋亡的重点区域)，Q2区为晚期凋亡，包括一部分坏死的细胞。
从图4可以看到，金精三羧酸甲酯加入量20～50μg/ml时，对正常细胞凋亡基本没有影响。其加入量为5μg/ml时，即对ES-2细胞系有明显的促进凋亡的作用，20μg/ml时早期凋亡已达67.6％，更高浓度下几乎所有细胞凋亡殆尽。
另外，金精三羧酸甲酯加入量20μg/ml及以上时，晚期凋亡+坏死细胞并没有大幅度增加，且没有明显的量效关系(具体数据见表1)。说明金精三羧酸甲酯不会明显引发坏死，这对避免加剧肿瘤炎症微环境有重要作用。
表1金精三羧酸甲酯加药量与晚期凋亡及坏死细胞比例
Figure PCTCN2022106988-appb-000002
实施例5金精三羧酸甲酯对肝癌细胞抗肿瘤活性测试
取中国人的肝癌细胞系HCCLM3和MHCC97H，金精三羧酸甲酯加入的情况下检测细胞增 殖情况。步骤如下(a)用细胞培养液于37℃、0.5％CO2环境中培养HCCLM3和MHCC97H9；(b)向培养基中加入金精三羧酸甲酯溶液，使金精三羧酸甲酯的终浓度分别达到10μg/ml、20μg/ml、50μg/ml、80μg/ml、120μg/ml，继续培养48-84h后取样；(c)CCK-8(Cell Counting Kit-8)法检测细胞增殖。
HCCLM3和MHCC97H细胞用药前后增殖结果如图5所示。从图中可以看出，随着药物浓度的增加，癌细胞增殖速率明显降低，说明金精三羧酸甲酯对两株肝癌细胞系的增殖的抑制作用均呈现量效关系，在50μg/ml浓度及以上有明显的抑制作用。
实施例6春日霉素对乳腺癌细胞抗肿瘤活性测试
培养人乳腺癌细胞系MDA-MB-468，加入1mg/L的春日霉素，通过领域内周知的磺酰罗丹明B法(sulforhodamine B assay)测定细胞增殖(参考文献：Qin et al.,BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5,Nature Communications 6:8471)。如图6可见，加入春日霉素后，细胞增殖从第4天开始明显放缓，说明春日霉素具备抑制乳腺癌细胞增殖的能力。
实施例7春日霉素裸鼠肺癌移植瘤模型抗肿瘤活性测试
运用本领域公知的技术，在裸鼠移植瘤模型上进行春日霉素抑制肿瘤的实验。取裸鼠，皮下注射肺癌细胞H1299，待肿瘤成型后一次性给药(灌胃)春日霉素10μg/kg剂量，在第1、3、5天取上述裸鼠活体测量其肿瘤直径。肿瘤直径变化结果详见图7，可见肿瘤明显缩小，说明春日霉素的抗肿瘤作用明显。
实施例8H ippuristanol对乳腺癌细胞抗肿瘤活性测试
培养人乳腺癌细胞系MDA-MB-468，加入50nM的Hippuristanol，进行领域内周知的Transwell迁移侵袭实验，Transwell小室中加入10万个细胞，迁移/侵袭进行24小时，结晶紫染色。从图8可知，50nM的Hippuristanol可以有效降低乳腺癌细胞的迁移和侵袭。
实施例9 Hippuristanol裸鼠肺癌移植瘤模型抗肿瘤活性测试
运用本领域公知的技术，在裸鼠移植瘤模型上进行Hippuristanol抑制肿瘤的实验。取裸鼠，皮下注射肺癌细胞A549，待肿瘤成型后开始给药Hippuristanol每日10mg/kg剂量，每天灌胃一次，在第1、3、5天取上述裸鼠活体测量其肿瘤直径。肿瘤直径变化结果详见图9，可见肿瘤明显缩小，说明Hippuristanol的抗肿瘤作用明显。
本发明的不占用核糖体资源的翻译抑制剂不但具有优良的肿瘤抑制或杀灭作用，而且具有优越的安全性，是优异的抗肿瘤成药分子。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任 何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。

Claims (10)

  1. 一种翻译抑制剂，其特征在于，所述翻译抑制剂为不占用核糖体资源的翻译抑制剂。
  2. 如权利要求1所述的翻译抑制剂，选自金精三羧酸甲酯、春日霉素和Hippuristanol，及它们之一的前药和它们之一的药学上可接受的盐中的一种或多种。
  3. 如权利要求1所述的翻译抑制剂，其特征在于，所述翻译抑制剂为金精三羧酸甲酯、春日霉素或Hippuristanol，或它们之一的前药或它们之一的药学上可接受的盐。
  4. 如权利要求3所述的翻译抑制剂，其特征在于，所述药学上可接受的盐为铵盐、钠盐、钾盐或盐酸盐。
  5. 权利要求1～4任一项所述的翻译抑制剂用于制备抗肿瘤药物的用途。
  6. 如权利要求5所述的用途，其特征在于，所述肿瘤为恶性肿瘤。
  7. 如权利要求6所述的用途，其特征在于，所述恶性肿瘤选自肺癌、卵巢癌、肝癌和卵巢癌中的一种或多种。
  8. 一种药物组合物，包含权利要求1～4任一项所述翻译抑制剂，以及药学上可接受的载体。
  9. 如权利要求8所述的药物组合物，其特征在于，所述药物组合物中，还包含一种或多种权利要求1～4所述翻译抑制剂以外的其它抗肿瘤的活性成分。
  10. 一种抗肿瘤药盒，包含权利要求8～9任一项所述的药物组合物，以及一种或多种其它抗肿瘤药物。
PCT/CN2022/106988 2022-01-24 2022-07-21 不占用核糖体资源的翻译抑制剂作为抗肿瘤药物 WO2023138011A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/442,146 US20240277742A1 (en) 2022-01-24 2024-02-15 Translation inhibitor without occupying ribosome resources as anti-tumor drug

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210082654.8 2022-01-24
CN202210082654.8A CN114404400A (zh) 2022-01-24 2022-01-24 不占用核糖体资源的翻译抑制剂作为抗肿瘤药物

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/442,146 Continuation US20240277742A1 (en) 2022-01-24 2024-02-15 Translation inhibitor without occupying ribosome resources as anti-tumor drug

Publications (1)

Publication Number Publication Date
WO2023138011A1 true WO2023138011A1 (zh) 2023-07-27

Family

ID=81278238

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/106988 WO2023138011A1 (zh) 2022-01-24 2022-07-21 不占用核糖体资源的翻译抑制剂作为抗肿瘤药物

Country Status (3)

Country Link
US (1) US20240277742A1 (zh)
CN (1) CN114404400A (zh)
WO (1) WO2023138011A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114404400A (zh) * 2022-01-24 2022-04-29 深圳承启生物科技有限公司 不占用核糖体资源的翻译抑制剂作为抗肿瘤药物
CN115737619A (zh) * 2022-11-21 2023-03-07 深圳承启生物科技有限公司 一种用于抑制肿瘤的药物靶点、用途及口服药物

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4007270A (en) * 1975-11-18 1977-02-08 American Cyanamid Company Complement inhibitors
US20100130359A1 (en) * 2007-05-02 2010-05-27 Basf Se Fungicidal Pyridazines, Processes for Their Preparation and Their Use for Controlling Harmful Fungi, and Compositions Comprising Them
US20190211339A1 (en) * 2015-10-16 2019-07-11 Children's Medical Center Corporation Modulators of telomere disease
CN110638802A (zh) * 2019-10-22 2020-01-03 深圳承启生物科技有限公司 Atca在制备治疗肿瘤的药物中的用途
WO2021001743A1 (en) * 2019-07-02 2021-01-07 Effector Therapeutics, Inc. Translation inhibitors and uses thereof
CN114404400A (zh) * 2022-01-24 2022-04-29 深圳承启生物科技有限公司 不占用核糖体资源的翻译抑制剂作为抗肿瘤药物

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4007270A (en) * 1975-11-18 1977-02-08 American Cyanamid Company Complement inhibitors
US20100130359A1 (en) * 2007-05-02 2010-05-27 Basf Se Fungicidal Pyridazines, Processes for Their Preparation and Their Use for Controlling Harmful Fungi, and Compositions Comprising Them
US20190211339A1 (en) * 2015-10-16 2019-07-11 Children's Medical Center Corporation Modulators of telomere disease
WO2021001743A1 (en) * 2019-07-02 2021-01-07 Effector Therapeutics, Inc. Translation inhibitors and uses thereof
CN110638802A (zh) * 2019-10-22 2020-01-03 深圳承启生物科技有限公司 Atca在制备治疗肿瘤的药物中的用途
CN114404400A (zh) * 2022-01-24 2022-04-29 深圳承启生物科技有限公司 不占用核糖体资源的翻译抑制剂作为抗肿瘤药物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAINEI SAI KIRAN, ITOUA MAÏGA RAYELLE, CENCIC REGINA, PUTNAM ANDREA A, AMADOR LUIS A, RODRIGUEZ ABIMAEL D, JANKOWSKY ECKHARD, PELL: "A Comparative Study of Small Molecules Targeting eIF4A", RNA, COLD SPRING HARBOR LABORATORY PRESS, US, vol. 26, no. 5, 3 February 2020 (2020-02-03), US , pages 541 - 549, XP093079845, ISSN: 1355-8382, DOI: 10.1261/rna *
SHEN LEO, PELLETIER JERRY: "Selective targeting of the DEAD-box RNA helicase eukaryotic initiation factor (eIF) 4A by natural products", NATURAL PRODUCT REPORTS, ROYAL SOCIETY OF CHEMISTRY, GB, vol. 37, no. 5, 27 May 2020 (2020-05-27), GB , pages 609 - 616, XP093079841, ISSN: 0265-0568, DOI: 10.1039/C9NP00052F *

Also Published As

Publication number Publication date
CN114404400A (zh) 2022-04-29
US20240277742A1 (en) 2024-08-22

Similar Documents

Publication Publication Date Title
WO2021129820A1 (zh) 含螺环的喹唑啉化合物
US11291667B2 (en) Combination therapy involving diaryl macrocyclic compounds
WO2021190467A1 (zh) 含螺环的喹唑啉化合物
US20240277742A1 (en) Translation inhibitor without occupying ribosome resources as anti-tumor drug
CN101998951B (zh) 酰基硫脲化合物或其盐及其用途
KR101668931B1 (ko) 항종양제의 효과 증강제
CN113527335A (zh) 作为egfr抑制剂的大环类化合物及其应用
EP4192826A1 (en) Small molecules for the treatment of autoimmune diseases and cancer
WO2015117551A1 (zh) 吡咯取代吲哚酮类衍生物、其制备方法、包含该衍生物的组合物、及其用途
CN116375707A (zh) Menin抑制剂及其用途
US20090209537A1 (en) Aurora inhibitors
EP2036893A1 (en) Abl KINASE INHIBITOR
AU2019265011B2 (en) Inhibitors of the RAS oncoprotein, methods of making and methods of use thereof
CN104327097A (zh) 雷帕霉素的三氮唑衍生物和用途
WO2024140798A1 (zh) Menin抑制剂及其用途
TW202039423A (zh) 戊烷脒之類似物及其用途
TW202346293A (zh) 含氮雜環衍生物及其组合物和藥學上的應用
CN113620974A (zh) 大环类酪氨酸激酶抑制剂的晶型及其制备方法
CN116473970A (zh) 用于非小细胞肺癌治疗的激酶抑制剂
CN116836161A (zh) Parp1抑制剂
WO2024174949A1 (zh) 作为cdk7激酶抑制剂的杂芳环化合物及其制备和应用
CN115716829A (zh) 一种喹啉并咪唑酮联氘代吡唑类化合物及其应用
CN120097974A (zh) 一种非糖苷类山荷叶素衍生物及其制备方法和应用
CN118119393A (zh) 一种cdk4/6抑制剂的医药用途
WO2024008083A1 (zh) 用作cdk7激酶抑制剂的化合物及其应用

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: 22921441

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 02.01.2025)