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CN118593509A - Application of non-gotinib as sensitizer of anti-triple negative breast cancer drug BBDI - Google Patents

Application of non-gotinib as sensitizer of anti-triple negative breast cancer drug BBDI Download PDF

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Publication number
CN118593509A
CN118593509A CN202410809482.9A CN202410809482A CN118593509A CN 118593509 A CN118593509 A CN 118593509A CN 202410809482 A CN202410809482 A CN 202410809482A CN 118593509 A CN118593509 A CN 118593509A
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China
Prior art keywords
breast cancer
negative breast
triple negative
bbdi
cancer drug
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Inventor
王丽虹
姜伟
袁梦琴
刘海洲
尚依妮
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First Affiliated Hospital of Fujian Medical University
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First Affiliated Hospital of Fujian Medical University
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Abstract

The invention discloses application of non-goiter as a sensitizer of a triple negative breast cancer resistant medicament BBDI, and discovers that the non-goiter can be used as a sensitizer of a triple negative breast cancer resistant medicament BBDI (JQ 1) for the first time, enhances cytotoxicity and killing effect of the triple negative breast cancer resistant medicament JQ1 on tumor cells, has good potential of developing the triple negative breast cancer resistant medicament JQ1 sensitizer, and provides a brand-new thought and strategy for developing the triple negative breast cancer treatment medicament, thereby having good clinical application prospect.

Description

Application of non-gotinib as sensitizer of anti-triple negative breast cancer drug BBDI
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of non-gotinib as a sensitizer of a triple negative breast cancer resistant drug BBDI, wherein the triple negative breast cancer resistant drug BBDI is JQ1.
Background
Triple negative breast cancer (TRIPLE NEGATIVE breast cancer, TNBC) accounts for about 15% -20% of all breast cancer patients, is the most invasive subtype in breast cancer, has high mortality and is easy to relapse. Characterized by the lack of expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) and by the lack of human epidermal growth factor receptor 2 (HER 2) amplification. Cancer genomic sequencing studies focused on triple negative breast cancers fail to find new repeatedly mutated oncogenes, and thus cannot immediately develop targeted therapies. Furthermore, triple negative breast cancer is also a heterogeneous disease, indicating that one treatment may not be appropriate for all patients and that multiple new treatment strategies are required. Thus, there is a need for new treatment regimens for triple negative breast cancer.
Currently, chemotherapy is one of the most common treatments for triple negative breast cancer, however, 60% -70% of triple negative breast cancer patients do not show a good response to chemotherapy. BET bromodomain inhibitors (BBDI) are considered potential candidate therapeutic agents for the treatment of triple negative breast cancers in preclinical studies and clinical trials. JQ1, as classical BBDI, can shorten BRD4 communication and transcription of downstream oncogenes, thereby generating selective transcriptional responses and antiproliferative effects in solid tumor and hematological malignancy models, killing tumor cells. However, the clinical use of JQ1 is greatly limited by resistance. Thus, understanding the underlying mechanisms of BBDI resistance contributes to the effective treatment of triple negative breast cancers.
Disclosure of Invention
In view of the above, in order to solve the technical problems faced in the prior art, the invention aims to provide the application of the regorafenib as the sensitizer of the anti-triple negative breast cancer drug BBDI, the regorafenib is firstly adopted as the sensitizer of the anti-triple negative breast cancer drug BBDI (JQ 1), the sensitivity of triple negative breast cancer cells to the JQ1 drug is improved, and the curative effect of the triple negative breast cancer can be remarkably improved by the combination of the regorafenib and the JQ 1.
The above object of the present invention is achieved by the following technical solutions:
the first aspect of the invention provides the use of regorafenib in the preparation of a sensitizer for an anti-triple negative breast cancer drug BBDI.
Further, the anti-triple negative breast cancer drug BBDI is JQ1, and the structural formula of JQ1 is shown in formula (I):
Formula (I).
Further, the non-goiter is capable of increasing the sensitivity of triple negative breast cancer cells to the anti-triple negative breast cancer drug BBDI.
Further, overexpression of GPX4 in triple negative breast cancer cells results in its resistance to the triple negative breast cancer drug BBDI.
Further, the concentration ratio of the regorafenib and the anti-triple negative breast cancer drug BBDI is (40 μm-320 μm): (5. Mu.M-50. Mu.M).
In the present invention, the anti-triple negative breast cancer drug BBDI refers to a BET bromodomain inhibitor (BET bromodomain inhibitor, BBDI), which is a candidate therapeutic agent for triple negative breast cancer and other types of cancer, but the inherent and acquired resistance to BBDI limits its potential clinical application, so that research on the drug resistance mechanism of BBDI and finding an optimal combination drug regimen (for example, a combination drug regimen for increasing BBDI drug sensitivity) are technical problems that need to be solved when BBDI is applied to clinical treatment of triple negative breast cancer and other types of cancer at present.
In some embodiments, the anti-triple negative breast cancer drug BBDI includes, but is not limited to: JQ1, TEN-010, OTX-015, I-BET762, CPI-0610, wherein JQ1 is the first reported BET inhibitor with a short half-life; TEN-010 and OTX-015 are both five-membered and seven-membered and five-membered tricyclic skeletons, and the molecular structure is characterized by different substituents on the amide nitrogen; both I-BET762 and CPI-0610 are five-membered and seven-membered and six-membered tricyclic backbones, the five-membered ring of I-BET762 is triazole, and the five-membered ring of CPI-0610 is isoxazole. In a specific embodiment of the invention, the anti-triple negative breast cancer drug BBDI is JQ1.
In the invention, JQ1 is BET bromodomain inhibitor, the molecular formula is C 23H25ClN4O2 S, the molecular weight is 456.988, the structural formula is shown as the formula (I), and the IC50 for inhibiting BRD4 (1/2) is 77 nM and 33 nM respectively. The corresponding Chinese names are: (S) - (+) -2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-f ] [1,2,4] triazolo [4,3-a ] [1,4] diazepin-6-yl) acetic acid tert-butyl ester corresponding to the English name :(S)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate.
In the present invention, the non-golitinib (Filgotinib) is an oral, ATP-competitive, reversible JAK1 inhibitor for the treatment of inflammatory autoimmune diseases, including inflammatory arthritis and inflammatory bowel disease. JAK-STAT signaling pathway is involved in the pathogenesis of inflammatory and autoimmune diseases, and regoratinib regulates this pathway by preventing STAT phosphorylation and activation. Clinical trial indications for non-golitinib include crohn's disease, ankylosing spondylitis, psoriatic arthritis, cutaneous lupus erythematosus and the like. There is no study or report on the use of regorafenib as a sensitizer for the anti-triple negative breast cancer drug BBDI (JQ 1).
In a specific embodiment of the invention, the experiment proves that GPX4 over-expression leads to the drug resistance of triple negative breast cancer cells to JQ1, the non-gotinib alone has no obvious inhibition effect on the triple negative breast cancer cells, and when the non-gotinib is used in combination with JQ1, GPX4 over-expression cells which are resistant to the JQ1 restore the sensitivity to the JQ1, and the result shows that the non-gotinib can enhance the sensitivity of the triple negative breast cancer cells to the JQ1 and can be used as a sensitizer of the JQ 1.
A second aspect of the invention provides any one of the following applications:
(1) Use of non-golitinib for the preparation of a medicament for increasing the sensitivity of a triple negative breast cancer cell against a triple negative breast cancer medicament BBDI, said triple negative breast cancer medicament BBDI being JQ1 described in the first aspect of the invention;
(2) Use of a combination of non-golitinib and an anti-triple negative breast cancer drug BBDI for the preparation of a medicament for the treatment and/or prophylaxis of triple negative breast cancer, said anti-triple negative breast cancer drug BBDI being JQ1 as described in the first aspect of the invention;
(3) Use of an agent that inhibits the expression level of GPX 4in the preparation of a sensitizer for an anti-triple negative breast cancer drug BBDI, wherein the anti-triple negative breast cancer drug BBDI is JQ1 described in the first aspect of the present invention.
In the present invention, the information of GPX4 is as follows: gene ID of Gene GPX4 (glutathione peroxidase 4) is 2879, details of which are available in NCBI database (https:// www.ncbi.nlm.nih.gov/Gene /) based on the above Gene ID.
In some embodiments, the agent that inhibits the expression level of GPX4 includes, but is not limited to: agents that inhibit the expression of or reduce the expression level of a gene encoding a GPX4 protein, reduce the activity of a GPX4 protein, e.g., agents that inhibit the expression of or reduce the expression level of a GPX4 gene, include, but are not limited to: an agent that inhibits the transcriptional activity of the GPX4 gene, an agent that inhibits the transcriptional level of GPX4 mRNA, an agent that promotes the degradation of GPX4 mRNA, an siRNA directed against the GPX4 gene, an shRNA directed against the GPX4 gene, an agent that inhibits the translation of GPX4 mRNA, an agent that specifically recognizes and cleaves a targeting nucleic acid of the GPX4 gene to reduce its expression level, a dsRNA directed against the GPX4 gene, a microRNA directed against the GPX4 gene, an antisense nucleic acid directed against the GPX4 gene.
In other embodiments, inhibition or reduction of GPX4 gene expression may be achieved by knockdown of the GPX4 whole gene by administration of a targeting vector. In other embodiments, the agent that reduces GPX4 protein activity may be, for example, a specific antibody that targets GPX4 or a small molecule compound that has the activity of inhibiting GPX4 protein.
In a third aspect, the invention provides a pharmaceutical composition for combating triple negative breast cancer.
Further, the pharmaceutical composition comprises non-gotinib and an anti-triple negative breast cancer drug BBDI, wherein the anti-triple negative breast cancer drug BBDI is JQ1 described in the first aspect of the invention.
Further, the concentration ratio of the regorafenib and the anti-triple negative breast cancer drug BBDI is (40 μm-320 μm): (5. Mu.M-50. Mu.M).
Further, the pharmaceutical composition also comprises pharmaceutically acceptable carriers and/or auxiliary materials.
Further, the pharmaceutical composition is a single compound preparation or a combination of two independent single preparations.
Further, the compound preparation is a compound preparation containing the non-goiter and an anti-triple negative breast cancer drug BBDI;
Further, the combination of the single formulation is a combination of a single formulation comprising the non-golitinib and a single formulation comprising the anti-triple negative breast cancer drug BBDI.
In some embodiments, pharmaceutically acceptable carriers and/or excipients described herein include, but are not limited to: diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, disintegrants.
In particular embodiments, the diluents include, but are not limited to: lactose, sodium chloride, glucose, urea, starch, water, etc. The adhesive includes, but is not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methylcellulose, hydroxypropyl methylcellulose, and the like. The surfactants include, but are not limited to: polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc. The wetting agents include, but are not limited to: glycerol, starch, and the like. The adsorbent carrier includes, but is not limited to: starch, lactose, bentonite, silica gel, kaolin, bentonite, etc. The lubricant includes, but is not limited to: zinc stearate, glyceryl monostearate, polyoxyethylene monostearate, monolauryl saccharate, sodium lauryl sulfate, magnesium lauryl sulfate, etc. The filler includes, but is not limited to: mannitol, xylitol, sorbitol, maltose, erythrose, glucose, lactose, sucrose. Such disintegrants include, but are not limited to: crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.
In some embodiments, the pharmaceutical compositions of the present invention may further comprise additives such as stabilizers, buffers, bactericides, isotonic agents, pH controlling agents, surfactants, and chelating agents. The choice of the above-mentioned agents is not particularly limited in the present invention, and any pharmaceutical composition containing the regorafenib and the anti-triple negative breast cancer drug BBDI as main active ingredients is within the scope of the present invention.
In some embodiments, the pharmaceutical compositions of the present invention may also be used in combination with other therapeutic compounds that can be used to treat triple negative breast cancer, which may be administered simultaneously with the primary active ingredient in the pharmaceutical compositions of the present invention, even in the same composition. Other therapeutic compounds may also be administered alone, in separate compositions or in a dosage form different from the primary active ingredient. A partial dose of the main ingredient may be administered simultaneously with the other therapeutic compounds, while the other doses may be administered separately. The dosage of the pharmaceutical composition of the present invention may be adjusted during the course of treatment according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.
In some embodiments, the other therapeutic compounds include, but are not limited to: chemotherapeutic agents, in particular, include, but are not limited to: taxane (paclitaxel, docetaxel, cabazitaxel), antibiotic (doxorubicin, epirubicin, daunorubicin, pirarubicin, etoposide, irinotecan, mitoxantrone), antimetabolite (cisplatin, carboplatin, lobaplatin, nedaplatin, oxaliplatin, gemcitabine, 5-fluorouracil), alkylating agent (cyclophosphamide, ifosfamide).
In a fourth aspect, the invention provides a pharmaceutical formulation for combating triple negative breast cancer.
Further, the pharmaceutical formulation comprises the pharmaceutical composition according to the third aspect of the present invention.
Further, the dosage forms of the pharmaceutical preparation include injection dosage forms, respiratory tract dosage forms, mucosa dosage forms and/or skin dosage forms.
Further, the injection administration type includes: intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and intracavitary injection.
Further, the respiratory tract administration dosage form includes: spray, aerosol, powder spray and other forms.
Further, the mucosal administration form includes: eye drops, nasal drops, eye ointments, gargle, sublingual tablet, adhesive tablet, patch, etc.
Further, the skin administration form includes: various dosage forms such as solution, lotion, liniment, ointment, plaster, paste, patch, etc.
In some embodiments, the dosage forms of the pharmaceutical formulations of the present invention are dosage forms that are convenient for administration prepared by conventional methods, including, but not limited to: parenteral dosage forms, specific examples include, but are not limited to: aqueous injection, powder for injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder fog agent, sustained release agent, controlled release agent, etc.
In some embodiments, the pharmaceutical formulation includes pharmaceutical excipients that are conventionally used in a variety of formulations, including, but not limited to: isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, lubricants, and the like; may also be selected for use in response to the substance, including but not limited to: emulsifying agent, solubilizer, bacteriostat, analgesic, antioxidant, etc.
The auxiliary materials can effectively improve the stability and the solubility of the active ingredients contained in the composition or change the release rate, the absorption rate and the like of the active ingredients, thereby improving the metabolism of various active ingredients in organisms and further enhancing the administration effect of the composition. In addition, specific purposes or modes of administration may be achieved, such as: sustained release administration, controlled release administration, pulsatile administration, and the like, and adjuvants used include, but are not limited to: gelatin, albumin, chitosan, polyether and polyester polymer materials (such as polyethylene glycol, polyurethane, polycarbonate and copolymers thereof). The main manifestations of the beneficial drug administration are: improving the treatment effect, improving the bioavailability, reducing toxic and side effects, improving the compliance of patients and the like.
In some embodiments, the pharmaceutical composition or pharmaceutical formulation of the present invention may be suitably administered in various amounts depending on the formulation method, the administration mode, the age, weight, sex, disease state, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and the like, and the skilled practitioner will generally be able to easily determine the prescription and the desired therapeutically effective dose, as long as it is capable of producing the desired therapeutic and/or prophylactic effect on triple negative breast cancer, and such doses are within the scope of the present invention.
In a fifth aspect, the invention provides a method for increasing sensitivity of a triple negative breast cancer cell to an anti-triple negative breast cancer drug BBDI in an in vitro non-therapeutic setting.
Further, the method comprises the following steps: adopting non-gotinib to treat a triple negative breast cancer cell culture system containing a triple negative breast cancer resistant drug BBDI;
the anti-triple negative breast cancer drug BBDI is JQ1 described in the first aspect of the present invention.
The invention also provides a method for treating triple negative breast cancer, which comprises the following steps: administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition or pharmaceutical formulation provided herein.
In some embodiments, the pharmaceutical composition or pharmaceutical formulation may be administered to the subject by injection, topical administration, or oral administration. For example, the method may comprise administering the pharmaceutical composition or pharmaceutical formulation to the subject three times a day, once every two days, and the like. In some embodiments, the administration by injection may include subcutaneous injection, intramuscular injection, intravenous injection, and the like. In some embodiments, injection administration may include injecting the pharmaceutical composition directly into the lesion or an area near the lesion, and in some embodiments, topical administration may include rectal administration, nasal administration, aural administration, intramedullary administration, intra-articular administration, intrapleural administration, and the like, or any combination thereof. In some embodiments, the pharmaceutical composition or pharmaceutical formulation may be administered to a subject via a combination of different modes of administration.
Compared with the prior art, the invention has the advantages and beneficial effects that:
According to the invention, the regorafenib can be used as a sensitizer for the anti-triple-negative breast cancer drug BBDI (JQ 1) for the first time, experiments prove that the regorafenib can obviously increase the sensitivity of triple-negative breast cancer cells to the anti-triple-negative breast cancer drug JQ1, reduce the drug resistance of the triple-negative breast cancer cells to the anti-triple-negative breast cancer drug JQ1, enhance the cytotoxicity and the killing effect of the anti-triple-negative breast cancer drug JQ1 to tumor cells, has good potential of developing into the sensitizer for the anti-triple-negative breast cancer drug JQ1, provides a brand-new thought and strategy for the field of developing the triple-negative breast cancer therapeutic drug, and has good clinical application prospect.
Drawings
Fig. 1: overexpression of GPX4 resulted in JQ1 resistance, wherein, panel a: relative mRNA expression levels of GPX4 in the GPX4 over-expression group and the empty vector control group; b, drawing: relative mRNA expression levels of GPX4 in the GPX4 silencing group and the empty vector control group; c, drawing: western Blotting results for GPX4 overexpression and silencing groups; d, drawing: western Blotting quantification of GPX4 overexpression and silencing groups; e, drawing: CCK8 assay results (24 hours of treatment) for JQ1 inhibition in GPX4 over-expression and empty vector control groups; f, drawing: IC50 values for JQ1 in GPX4 over-expression and empty vector control; graph G: CCK8 test results of JQ1 inhibition in GPX4 silencing group and empty vector control group; drawing H: IC50 values of JQ1 in GPX4 silenced and empty vector control, data expressed as mean ± standard deviation, significance in panels a and E-F were calculated using unpaired sample T-test (n=3), significance in panels B, D and G-H were calculated using one-way analysis of variance (n=3);
Fig. 2: JQ1 and regatinib in combination reverse JQ1 resistance, wherein, figure A: relative mRNA expression levels of GPX4 in the non-golitinib (160 μm,24 h) treated group and untreated Vector group; b, drawing: relative mRNA expression levels of GPX4 in the non-golitinib (160 μm,24 h) treated group and the untreated GPX4 overexpressed group; c, drawing: western Blotting results of GPX4 in the non-golitinib (160. Mu.M and 320. Mu.M, 24 h) treated Vector and GPX4 overexpressing group; d, drawing: western Blotting quantification of GPX4 in non-golitinib (160. Mu.M and 320. Mu.M, 24 h) treated Vector and GPX4 overexpressing groups; e, drawing: cell viability of Vector and GPX4 overexpressed SUM159 cells 24 hours after non-golitinib treatment; F-G diagram: CCK8 assay (24 hours) of JQ1 inhibition in Vector and GPX4 overexpressing groups (treated or untreated non-gotinib, respectively), data representing mean ± standard deviation, significance in a-B, E panels was calculated using unpaired sample T test (n=3), significance in D, F-G panels was calculated using one-way analysis of variance (n=3);
Fig. 3: JQ1 and regatinib in combination reverse JQ1 resistance, wherein, figure A: colony formation experiments of SUM159 cells in Vector and GPX4 overexpressing groups (with or without JQ1, regoratinib, or a combination of both); b, drawing: quantitative comparison of colony formation experiments in GPX4 over-expression group and Vector group (JQ 1 5. Mu.M, 10. Mu.M and 15. Mu.M, respectively); c, drawing: quantitative comparison of colony formation experiments in Vector group with non-golitinib treated (40. Mu.M), D-F panels: quantitative comparisons of colony formation experiments in the combination of JQ1 (5 μm,10 μm and 15 μm) or JQ 1-non-golitinib (40 μm) were treated separately in GPX4 overexpressing cells, data representing mean ± standard deviation, significance in C-F panels was calculated using unpaired sample T-test (n=3), significance in B panels was calculated using one-way analysis of variance (n=3).
Detailed Description
The inventor of the invention has conducted intensive research on the drug resistance of the triple negative breast cancer drug JQ1 through a single cell RNA sequencing technology, revealed dynamic changes of triple negative breast cancer cells, wherein the changes comprise transcriptome reprogramming, difference of cell composition and change of cell cycle state, the research has found that the correlation between JQ1 drug resistance and iron death inhibition is achieved, and further has found that the regoratinib can remarkably increase the sensitivity of the triple negative breast cancer cells to the triple negative breast cancer drug JQ1, enhance the cytotoxicity and killing effect of the triple negative breast cancer drug JQ1 to tumor cells, and have good potential of developing sensitizer of the triple negative breast cancer drug JQ 1. In order to facilitate an understanding of the present invention, the following terms referred to in the present invention are explained herein:
as used herein, the term "JQ1" is a BET bromodomain inhibitor having the molecular formula C 23H25ClN4O2 S and a molecular weight of 456.988, and the structural formula is shown in formula (I) above, and the term "JQ1" includes salts, esters, analogs, prodrugs and derivatives thereof.
As used herein, the term "regoratinib" is an oral, ATP-competitive, reversible JAK1 inhibitor, and the term "JQ1" includes salts, esters, analogs, prodrugs and derivatives thereof.
As used herein, the term "pharmaceutically acceptable carrier and/or adjuvant" is well documented in Remington's Pharmaceutical Sciences (19 th ed, 1995) which is used as needed to aid stability of a drug or to aid in enhancing the activity of an active ingredient in a drug, which may be used in such a pharmaceutical composition in the form of its original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof, and the pharmaceutical composition so formulated may be administered as needed by any suitable means known to those skilled in the art.
In some embodiments, the pharmaceutically acceptable carrier and/or adjuvant may additionally contain liquids such as water, physiological saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in the composition. These carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by a patient.
As used herein, the term "treatment" refers to the inhibition, containment, alleviation, amelioration, slowing, stopping, delaying or reversing of the progression or exacerbation of a disease in order to prevent or reduce the occurrence or progression of the disease, and the various indicators of the disease, disorder or pathological state described as maintaining and/or administering include alleviation or reducing the symptoms or complications of a particular disease, or curing or eliminating the disease, disorder or condition. In a specific embodiment of the invention, the disease is preferably triple negative breast cancer.
As used herein, the term "prevention" refers to various means or measures, including medical, physical or chemical methods, for preventing the occurrence or progression of a disease, preferably triple negative breast cancer, prior to the disease not being recognized by clinical criteria, to prevent and reduce the occurrence or progression of various symptoms of the disease.
As used herein, the term "therapeutically effective amount" refers to an amount that produces a therapeutic effect on and is acceptable to a human and/or animal. For example, a pharmaceutically or pharmaceutically effective amount refers to the amount of drug required to produce a desired therapeutic effect, which can be reflected by the results of a clinical trial, a model animal study, and/or an in vitro study. The pharmaceutically effective amount depends on several factors, including but not limited to: the characteristic factors of the subject (such as the subject's height, weight, sex, age, and history of administration), the severity of the disease.
The pharmaceutical active ingredients (non-gotinib, anti-triple negative breast cancer drug JQ 1) described in the present invention and pharmaceutically acceptable carriers and/or excipients (carriers for therapeutic administration and/or carriers, which are not themselves essential active ingredients and which have no excessive toxicity after administration) may constitute a pharmaceutical composition or pharmaceutical formulation. In particular embodiments of the invention, the "therapeutically effective amount" refers to an amount sufficient to inhibit, slow or prevent triple negative breast cancer occurrence and progression in a subject.
As used herein, the term "active ingredient" refers to the component of a pharmaceutically acceptable composition that is responsible for the therapeutic effect of the composition, while the other components of the composition (e.g., excipients, carriers, and diluents) are not responsible for the therapeutic effect of the composition, even though they have other functions in the composition that are necessary or desirable as part of a formulation (e.g., lubrication, flavoring, pH control, emulsification, stabilization, preservation, and other functions in addition to the therapeutic effect of the composition as described herein).
As used herein, the term "administering," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous drug, pharmaceutical composition, or pharmaceutical formulation with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of the cell includes contacting the reagent with the cell and contacting the reagent with a fluid. "administration" also means the in vitro and ex vivo treatment of cells by an agent, composition or by another cell, and when the "treatment" is applied to a human, veterinary or research subject, means therapeutic treatment, prophylactic or preventative measures, research and diagnostic application.
As used herein, the term "subject" refers to any animal, and also refers to human and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic animals or pets; and non-mammals such as chickens, amphibians, reptiles, and the like. In a specific embodiment of the invention, the subject is preferably a human.
The invention is further illustrated below in conjunction with specific examples, which are provided solely to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
The drugs, reagents and raw materials used in the present invention are readily available to those of ordinary skill in the art, and unless otherwise indicated, are commercially available, and the experimental methods of the present invention without specifying the specific conditions are usually carried out according to conventional conditions or according to conditions suggested by the manufacturer, and in particular, the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention in any way.
Example use of regatinib as sensitizer for anti-triple negative breast cancer drug BBDI (JQ 1)
1. Experimental materials
Human triple negative breast cancer cell lines SUM159 were purchased from Gibco (America) with Ham's F medium, from Millipore (Israel), from Selleck (America), from Sigma (America), from insulin and hydrocortisone, from Shanghai Ji Ma (China), from SiRNA packages, from Shangzhou Ruibo (China), from FastPure cells/tissue Total RNA extraction kit V2, HISCRIPTEIII RT reverse transcription reagent, from SYBR Green, from Novozan (China), from BCA protein quantification kit from Biyuna (China), from Millipore (America) with PVDF film, from Abcam (America) with GPX4 antibody, from GAPDH (America) from Proteintech (America), from MCE (America) with anti-triple negative breast cancer drug JQ1, and from HY-13030.
2. Experimental method
(1) Cell culture
SUM159 cells were cultured in a combined medium (Ham's F) containing 5% FBS, 5. Mu.g/mL insulin and 1. Mu.g/mL hydrocortisone at 37℃in a 5% carbon dioxide incubator.
(2) CCK8 experiment
SUM159 cells were seeded in 96-well plates and cultured for 24 hours. The original medium was replaced with a medium containing the indicated drug mixture. After 24 or 48 hours, CCK-8 solution was added to the wells and the 96-well plates incubated at 37℃for 3.5 hours. Optical Density (OD) values were measured using an ELISA plate reader and the system analysis determined the cell inhibition rate.
(3) Cell transfection
SUM159 cells were seeded at a density of 2 x 10 5 cells per well in 6-well plates and incubated overnight. The culture medium is replaced by a fresh culture medium, and GPX4 over-expression lentivirus (Shanghai Ji Ma company) with the titer of 10 9 is added, the control lentivirus with the titer of 10 8 is added with the concentration of 10 [ mu ] L, and the culture is carried out in a 5% carbon dioxide box at 37 ℃ for 48 hours with the concentration of the virus infection reagent HITRANSG A [ mu ] L. The transfection was transferred into a cell culture flask, screened with puromycin under pressure for two weeks, and the effect of transfection was evaluated by RT-qPCR and Western blotting.
SUM159 cells were seeded at a density of 1 x 10 5 cells per well in 24 well plates and incubated overnight. The medium was replaced with fresh medium and three GPX4 knockdown siRNAs (Hibiscus sabdariffa, ruibo Biotechnology Co., ltd.) were transfected individually with riboFECT TM CP REAGENT in 24 well plates to a final transfection concentration of 50 nM. The transfection effect is evaluated by RT-qPCR and Western blotting after 24 hours of culture in a 5% carbon dioxide box at 37 ℃, and three GPX4 knockdown siRNAs have better knockdown efficiency, and in the embodiment, the siRNA-1 and siRNA-2 with the best knockdown efficiency are selected for subsequent experiments. Wherein the sequences of the three GPX4 knockdown siRNAs are shown below, respectively.
siRNA-1:5'-GCTACAACGTCAAATTCGA-3'(SEQ ID NO:5);
siRNA-2:5'-GTAACGAAGAGATCAAAGA-3'(SEQ ID NO:6);
siRNA-3:5'-GAGGCAAGACCGAAGTAAA-3'(SEQ ID NO:7)。
(4) Colony formation experiments
SUM-159 cells were packed in 6-well plates at a density of 800 cells per well and cultured for 10 days. Subsequently, cells were fixed with 1 mL methanol per well and stained with 0.1% crystal violet solution. Photographs of the stained colonies were taken and the colony results were quantitatively analyzed using Image J software.
(5) RT-qPCR analysis
Total RNA was extracted and reverse transcribed into synthetic cDNA using 1. Mu.g RNA. Fluorescence quantitative PCR was performed using SYBR Green. The primers for GPX4 are respectively:
F:5'-CCCAGTGAGGCAAGACCGAAG-3'(SEQ ID NO:1);
R:5'-GGCTCCTGCTTCCCGAACTG-3'(SEQ ID NO:2)。
The primers for the reference gene GAPDH were:
F:5'-CGGAGTCAACGGATTTGGTCG-3'(SEQ ID NO:3);
R:5'-TCTCGCTCCTGGAAGATGGTGAT-3'(SEQ ID NO:4)。
PCR results were recorded as cycle threshold (Ct) and normalized to internal control (GAPDH).
(6)Western blot
The cells were collected and lysed and the supernatant centrifuged and stored frozen at-80℃until further use. The BCA kit was used to quantify total protein concentration. SDS-PAGE gel electrophoresis, proteins were transferred to PVDF membrane. 5% nonfat milk powder was incubated overnight at 4℃in a dilution containing primary antibody. Then, the mixture was incubated with the secondary antibody at room temperature for 1 hour. The protein bands were visualized using chemiluminescent substrates and the results were analyzed by Image J software.
3. Experimental results
(1) Overexpression of GPX4 leads to JQ1 resistance
The inventors of the present invention have found a correlation between JQ1 resistance and iron death inhibition through a large number of studies in the early stage. GPX4 as an important iron death regulator, we examined the correlation of its expression level with JQ1 resistance. First, a SUM159 cell line was established for GPX4 overexpression and silencing (FIGS. 1A-D). Subsequently, the sensitivity of these cell lines to JQ1 was assessed. The GPX4 over-expression group significantly reduced the JQ1 inhibition rate (fig. 1E) and the half maximal inhibitory concentration (IC 50) of JQ1 was increased at 24 hours and 48 hours (fig. 1F) compared to the empty vector group. In contrast, GPX 4-silenced cells exhibited higher JQ1 inhibition and reduced IC50 (fig. 3G-H), indicating increased sensitivity of the cells to JQ 1. These results demonstrate a key role for GPX4 in mediating JQ1 resistance, demonstrating its potential as a target for therapeutic intervention.
(2) Combination of regatinib and JQ1 can reverse JQ1 resistance
We examined the effect of regorafenib on GPX4 mRNA and protein levels and found that regorafenib significantly reduced GPX4 mRNA and protein levels in the control and GPX4 over-expression groups (fig. 2A-D). Next we explored the cytotoxic effect of regorafenib and found that after 24 hours and 48 hours of high dose regorafenib treatment, there was no significant difference in the activity of the experimental group compared to the control group cells (fig. 2E), indicating that the cytotoxic effect of regorafenib on the triple negative breast cancer cells was minimal, while indicating that regorafenib alone had no significant inhibitory effect on the triple negative breast cancer cells. GPX4 over-expressing cells resistant to JQ1 restored sensitivity to JQ1 when non-golitinib was used in combination with JQ 1. After 24 hours and 48 hours of the joint treatment of regoratinib (160 μm and 320 μm) with JQ1, the IC50 value of JQ1 in the GPX4 over-expression group was reduced, and the cell growth inhibitory effect was remarkable (fig. 2F-G). Colony formation experiments showed that the GPX4 over-expressed group had more cell colonies at different doses of JQ1 (5. Mu.M, 10. Mu.M and 15. Mu.M) (FIGS. 3A-B), which also indicated that GPX4 over-expression resulted in JQ1 resistance. The colony numbers were not significantly altered by regoratinib alone (fig. 3C), however, the combination therapy of JQ1 and regoratinib significantly reduced the cell colony numbers in the GPX4 over-expression group compared to JQ1 alone (fig. 3D-F). The results show that the regorafenib can enhance the sensitivity of the triple negative breast cancer cells to JQ1, in conclusion, the research of the research confirms that the small molecular regorafenib is used as a potential enhancer of the sensitivity of JQ1 in the JQ1 resistant cells, and provides new insight for developing an alternative treatment method to reduce the resistance of the triple negative breast cancer cells to JQ 1.

Claims (10)

1. The application of the non-gotinib in preparing the sensitizer of the anti-triple negative breast cancer drug BBDI is characterized in that the anti-triple negative breast cancer drug BBDI is JQ1, and the structural formula of the JQ1 is shown as the formula (I):
Formula (I).
2. The use according to claim 1, wherein the non-goitinib is capable of increasing the sensitivity of triple negative breast cancer cells to the anti-triple negative breast cancer drug BBDI.
3. The use according to claim 1, wherein overexpression of GPX4 in triple negative breast cancer cells results in its resistance to the triple negative breast cancer drug BBDI.
4. The use according to claim 1, characterized in that the concentration ratio of the regorafenib and the anti-triple negative breast cancer drug BBDI is (40 μΜ -320 μΜ): (5. Mu.M-50. Mu.M).
5. An application according to any one of the following, characterized in that the application comprises:
(1) Use of non-golitinib for the preparation of a medicament for increasing the sensitivity of a triple negative breast cancer cell against a triple negative breast cancer drug BBDI, said triple negative breast cancer drug BBDI being JQ1 as described in claim 1;
(2) Use of a combination of non-golitinib and an anti-triple negative breast cancer drug BBDI for the preparation of a medicament for the treatment and/or prophylaxis of triple negative breast cancer, said anti-triple negative breast cancer drug BBDI being JQ1 as defined in claim 1;
(3) Use of an agent that inhibits the expression level of GPX4 in the preparation of a sensitizer for an anti-triple negative breast cancer drug BBDI, wherein the anti-triple negative breast cancer drug BBDI is JQ1 as described in claim 1.
6. A pharmaceutical composition for anti-triple negative breast cancer, comprising non-gotinib and an anti-triple negative breast cancer drug BBDI, wherein the anti-triple negative breast cancer drug BBDI is JQ1 as defined in claim 1.
7. The pharmaceutical composition according to claim 6, characterized in that the concentration ratio of the regorafenib and the anti-triple negative breast cancer drug BBDI is (40 μΜ -320 μΜ): (5. Mu.M-50. Mu.M).
8. A pharmaceutical formulation against triple negative breast cancer, characterized in that it comprises the pharmaceutical composition of claim 6 or 7.
9. The pharmaceutical formulation of claim 8, wherein the dosage form of the pharmaceutical formulation comprises an injectable dosage form, a respiratory administration dosage form, a mucosal administration dosage form, and/or a dermal administration dosage form.
10. A method of increasing sensitivity of a triple negative breast cancer cell to an anti-triple negative breast cancer drug BBDI at an in vitro non-therapeutic destination, the method comprising the steps of: adopting non-gotinib to treat a triple negative breast cancer cell culture system containing a triple negative breast cancer resistant drug BBDI;
The anti-triple negative breast cancer drug BBDI is JQ1 described in claim 1.
CN202410809482.9A 2024-06-21 2024-06-21 Application of non-gotinib as sensitizer of anti-triple negative breast cancer drug BBDI Pending CN118593509A (en)

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