WO2022048527A1 - 西达本胺联合雌激素受体下调剂治疗乳腺癌的用途 - Google Patents

西达本胺联合雌激素受体下调剂治疗乳腺癌的用途 Download PDF

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WO2022048527A1
WO2022048527A1 PCT/CN2021/115558 CN2021115558W WO2022048527A1 WO 2022048527 A1 WO2022048527 A1 WO 2022048527A1 CN 2021115558 W CN2021115558 W CN 2021115558W WO 2022048527 A1 WO2022048527 A1 WO 2022048527A1
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breast cancer
estrogen receptor
chidamide
drug
fulvestrant
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English (en)
French (fr)
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周游
王雅楠
张堃
朱静忠
付鑫
宁志强
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深圳微芯生物科技股份有限公司
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Publication of WO2022048527A1 publication Critical patent/WO2022048527A1/zh

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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
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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 relates to the field of biomedicine, in particular to the use of Chidamide combined with an estrogen receptor down-regulator for treating breast cancer.
  • Breast cancer can be divided into estrogen receptor (ER) positive breast cancer, HER2 positive breast cancer and triple negative breast cancer. Among them, ER-positive breast cancer accounts for about 70% of all breast cancers, and ER-positive breast cancer cells have the characteristic of estrogen-dependent growth. Currently, the treatment of breast cancer patients is still dominated by drug therapy.
  • ER estrogen receptor
  • HER2 positive breast cancer triple negative breast cancer.
  • ER-positive breast cancer accounts for about 70% of all breast cancers
  • ER-positive breast cancer cells have the characteristic of estrogen-dependent growth.
  • the treatment of breast cancer patients is still dominated by drug therapy.
  • Endocrine therapy is the treatment of choice for most hormone receptor-positive breast cancers, which inhibits tumor cell growth by reducing estrogen levels in the body or inhibiting the binding activity of estrogen to ER.
  • Commonly used endocrine therapy drugs for breast cancer include tamoxifen, fulvestrant and aromatase inhibitors.
  • Aromatase also known as estrogen synthase, is a complex enzyme of microsomal cytochrome P450, which is composed of hemoglobin P450arom and reduced coenzyme NADPH, and is widely present in ovary, liver, muscle, fat and breast. In cancer cells, it is the key enzyme and rate-limiting enzyme that catalyzes the conversion of androgen to estrogen in organisms. It can aromatize the A ring of androgen, remove the carbon atom at position 19 and convert the carbonyl group at position 1 into a hydroxyl group. It catalyzes the conversion of androgens such as androstenedione and testosterone into estrone and estradiol, which are the main sources of estrogen in postmenopausal women.
  • Estrogen has been implicated in tumor progression, and aromatase plays the ultimate rate-limiting catalytic role in estrogen biosynthesis.
  • Aromatase inhibitors can specifically cause the inactivation of aromatase, block the aromatization reaction, inhibit the production of estrogen, and reduce the level of estrogen in the blood to achieve the purpose of treating breast cancer. It is mostly used in postmenopausal advanced breast cancer patients who have failed anti-estrogen (tamoxifen) therapy.
  • aromatase inhibitors have been developed and marketed as third-generation AR inhibitors.
  • As second- or third-line drugs for breast cancer they have been widely used in clinics in the United States and Japan. Commonly used aromatase inhibitors are exemestane, letrozole, and anastrozole.
  • US4808616 proposes the use of exemacetam for the treatment of advanced hormone-dependent breast cancer.
  • CN01805979.1 discloses the use of exemestane in the first line treatment of metastatic, advanced hormone dependent breast cancer, especially breast cancer in postmenopausal women.
  • CN201710110337.1 discloses the use of a CDK4/6 inhibitor in combination with an aromatase inhibitor in the preparation of a drug for the treatment of breast cancer, and specifically proposes that the aromatase inhibitor is selected from the group consisting of formestane, exemestane, One or more of azole, letrozole, vorozole, and anastrozole.
  • Tamoxifen is used in clinical treatment of breast cancer, and the effective rate is generally 30%.
  • the efficacy of estrogen receptor-positive patients is better (49%), and the efficacy of estrogen receptor-negative patients is poor (7%). It can be used by both premenopausal and postmenopausal patients, while postmenopausal and over 60-year-olds are more effective than premenopausal and younger patients. From the point of view of the lesion site, the skin, lymph nodes and soft tissue have a good effect, and the bone and visceral metastasis have a poor effect.
  • tamoxifen is most commonly prescribed as adjuvant therapy after initial breast cancer treatment. Tamoxifen is also approved for women at high risk of breast cancer, as well as for the treatment of metastatic breast cancer.
  • CN200810117739.5 discloses a pharmaceutical composition containing the active ingredients of tamoxifen (I) and IC-162 (II) or IC-163 (III), using its synergistic mechanism to provide a drug with a good effect of inhibiting breast cancer tumors , and can correspondingly reduce the side effects of tamoxifen.
  • CN00128694.3 discloses the combined administration of tamoxifen and isoflavones for breast cancer prevention in women with breast cancer and women predisposed to breast cancer.
  • Fulvestrant the chemical name is 7- ⁇ -[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5( 10)-Triene-3,17- ⁇ -diol.
  • the molecular formula is C32H47F5O3S.
  • It is a new class of estrogen receptor antagonists - estrogen receptor down-regulator class of anti-breast cancer treatment drugs. For the treatment of metastatic advanced breast cancer in postmenopausal women who do not respond to antiestrogen therapy, have disease progression, or are hormone receptor-positive.
  • Fulvestrant is a "pure" estrogen receptor antagonist without partial estrogen-like agonistic effects.
  • ER inhibits the estrogen signaling pathway by binding, blocking and down-regulating ER. It can compete with ER for binding to ER.
  • the affinity of ER is close to that of estrogen, which is 100 times that of tamoxifen. It is the only anti-estrogen drug that can be widely used clinically after the failure of tamoxifen. Because the drug is endocrine therapy, it will not cause common chemotherapy. Adverse reactions, it has good patient compliance. A number of clinical studies have found that fulvestrant 250mg has a good therapeutic effect and stable safety in the second-line treatment of advanced breast cancer. The currently marketed product is a clear, colorless to yellow viscous solution containing 50 mg/ml fulvestrant for injection.
  • CN201710110542.8 discloses the use of CDK4/6 inhibitor in combination with estrogen receptor antagonist in the preparation of a medicament for the treatment of breast cancer, the estrogen receptor antagonist is selected from one of tamoxifen and fulvestrant or several.
  • Chidamide (Chidamide, Aiposa) is a subtype-selective histone deacetylase (HDAC) inhibitor with the chemical name N-(2-amino-4-fluorophenyl)-4-[ N-[(E)-3-(3-pyridine)acryloyl]aminomethyl]benzamide, a new class 1.1 drug approved for the treatment of relapsed or refractory peripheral T-cell lymphoma (PTCL).
  • HDAC histone deacetylase
  • a total of 16 patients with solid tumors were enrolled in the phase I clinical trial of Chidamide, including non-small cell lung cancer, breast cancer, colon cancer, rectal cancer, gastric cancer, prostate cancer, etc., except for 1 case of submandibular gland adenoid cystic carcinoma In addition to partial remission, the remaining solid tumors have not obtained clear curative effect.
  • the clinical results of this phase I show that Chidamide alone is ineffective for breast cancer. Therefore, in the Phase II clinical trial of Chidamide, no further enrollment in breast cancer was performed. cancer patients.
  • CN03139760.3 discloses chidamide compound, specifically discloses a benzamide-type histone deacetylase inhibitor with differentiation and anti-proliferation activity and the preparation method and application of its medicinal preparation, which discloses the structure The general formula defines the substituents. These compounds, as histone deacetylase inhibitors, can be used to treat differentiation and proliferation-related diseases such as cancer and psoriasis.
  • CN201210489178.8 discloses two crystalline forms of Chidamide, namely Chidamide crystalline form A and Chidamide crystalline form B, and a preparation method of a new crystalline form of Chidamide.
  • the Chidamide crystalline form A and Chidamide crystalline form B have excellent performance in oral absorption and inhibition of cell differentiation and proliferation, and have weak toxicity, good storage and handling stability, and can be used for preparation Drugs to treat disorders related to cell differentiation and proliferation.
  • CN201410136761.X discloses an E-configuration benzamide compound and its medicinal preparation and application, the E-configuration benzamide compound is Chidamide, and its chemical name is N-(2-amino- 4-Fluorophenyl)-4-[N-[(E)-3-(3-pyridine)acryloyl]aminomethyl]benzamide, in its structural formula, the configuration of 3-pyridineacryloyl is E type.
  • the E-configuration Chidamide has subtype-selective histone deacetylase inhibitory activity, mainly inhibiting HDAC1, HDAC2, HDAC3 in class I HDACs and HDAC10 in class IIb HDACs.
  • the E-configuration chidamide can be used to treat diseases related to abnormal activity of histone deacetylase, such as cancer, including lymphoma, solid tumor and hematological tumor.
  • CN201810404747.1 discloses that the combined use of Chidamide and Exemestane can effectively improve the treatment of breast cancer, and obtain a significantly improved effect compared with exemestane monotherapy, especially, it can significantly prolong the progression-free period of patients Survival, improve clinical benefit rate and objective response rate.
  • the object of the present invention is to provide a pharmaceutical composition for the treatment and/or prevention of endocrine therapy-acquired resistant breast cancer.
  • the terms “treating,” “relieving,” and “improving” are used interchangeably. These terms refer to methods for obtaining beneficial or desired effects, including but not limited to therapeutic and/or prophylactic benefits.
  • the present invention provides, in a first aspect, a pharmaceutical composition comprising Chidamide or a derivative thereof and an estrogen receptor down-regulating agent and/or degrading agent.
  • the estrogen receptor down-regulating agent and/or degrading agent include one or more of fulvestrant, elacestrant (RAD1901), brilanestrant (GDC-0810), SAR439859, AZD9496 and OP-1074, preferably For fulvestrant.
  • the chidamide derivatives include pharmaceutically acceptable salts, solvates, hydrates, enantiomers, and crystal forms A and B thereof.
  • pharmaceutically acceptable salts refers to salts derived from various organic and inorganic counterions known in the art.
  • Pharmaceutically acceptable salts include pharmaceutically acceptable acid addition salts and base addition salts.
  • Acid addition salts can be formed using inorganic and organic acids.
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.
  • Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
  • Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, especially such as isopropylamine, trimethylamine amine, diethylamine, triethylamine, tripropylamine and ethanolamine.
  • the pharmaceutical composition further comprises pharmaceutically acceptable excipients and/or carriers.
  • pharmaceutically acceptable excipient and/or carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • solvents dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of these media and agents in pharmaceutically active substances is well known in the art. Except where any conventional medium or agent is incompatible with the active ingredient, it is contemplated for use in the therapeutic compositions of the present invention. Supplementary active ingredients can also be incorporated into the compositions.
  • the dosage of Chidamide or its derivatives is 5-60mg, preferably 10-40mg, more preferably 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg , 45mg, 50mg, 55mg, 65mg;
  • the dosage of estrogen receptor down-regulating agent and/or degrading agent is 50-500mg, preferably 100-400mg, more preferably 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg.
  • the dosage forms of the pharmaceutical composition include tablets, capsules, granules, pills, powders, suppositories and injections.
  • tablette refers to a solid preparation in the form of a tablet or a special-shaped tablet which is uniformly mixed with a drug and excipients and compressed. Tablets are mainly oral ordinary tablets, but also include lozenges, sublingual tablets, oral patches, chewable tablets, dispersible tablets, effervescent tablets, vaginal tablets, immediate-release or sustained-release or controlled-release tablets and enteric-coated tablets.
  • capsule refers to a solid preparation made of a drug or filled with suitable excipients in a hollow hard capsule or sealed in a soft capsule, which can be divided into hard capsules, soft capsules, sustained-release capsules, controlled-release capsules and enteric-coated capsules. Capsules, mainly for oral use.
  • Granule refers to a dry granular preparation with a certain particle size that is prepared by mixing the raw drug and suitable excipients. Granules are a commonly used oral solid dosage form.
  • pill refers to a spherical or spheroid-like tablet-shaped preparation prepared from fine powder or extract of medicinal materials plus suitable binding auxiliary materials.
  • powder refers to a drug or a dry powder preparation prepared by pulverizing and homogeneously mixing with suitable excipients.
  • suppository refers to a solid preparation of a drug and a suitable base having a certain shape for administration into the human body cavity.
  • the suppository is solid at room temperature. After being inserted into the cavity, it can quickly soften and melt or dissolve in the secretion fluid at body temperature, and gradually release the drug to produce local or systemic effects.
  • injectable refers to sterile solutions (including emulsions and suspensions) of pharmaceutical preparations for injection into the body, as well as sterile powders or concentrated solutions for solution or suspension immediately before use.
  • the present invention provides, in a second aspect, the use of the above pharmaceutical composition for preparing a medicament for the treatment and/or prevention of breast cancer, where the breast cancer is endocrine therapy-acquired drug-resistant breast cancer.
  • the endocrine therapy-acquired resistant breast cancer is estrogen receptor (ER)-positive, refractory or relapsed breast cancer after endocrine therapy.
  • the endocrine therapy-acquired resistant breast cancer includes estrogen receptor antagonist/inhibitor (ERA/ERI), estrogen receptor modulator (SERM), estrogen receptor antagonist (SERM), Selective estrogen receptor down-regulator/degrader (SERD), aromatase inhibitor (AI), luteinizing hormone-releasing hormone (LH-RH) analog therapy Refractory or recurrent breast cancer.
  • the endocrine therapy acquired drug-resistant breast cancer includes estrogen receptor modulator (selective estrogen receptor modulator, SERM) (for example tamoxifen), estrogen receptor down-regulator/degrader (selective estrogen receptor modulator, SERM) Down-regulator/degrader, SERD) (such as fulvestrant), aromatase inhibitor (aromatase inhibitor, AI) (such as letrozole) after treatment of refractory or recurrent breast cancer.
  • SERM selective estrogen receptor modulator
  • SERM selective estrogen receptor modulator/degrader
  • SERM selective estrogen receptor modulator/degrader
  • SERD selective estrogen receptor modulator/degrader
  • AI aromatase inhibitor
  • letrozole such as letrozole
  • the present invention provides, in a third aspect, a kit comprising Chidamide or a derivative thereof and an estrogen receptor downregulator and/or degrader in a single formulation or in separate formulations.
  • the estrogen receptor down-regulating agent and/or degrading agent include one or more of fulvestrant, elacestrant (RAD1901), brilanestrant (GDC-0810), SAR439859, AZD9496 and OP-1074, preferably For fulvestrant.
  • the chidamide derivatives include pharmaceutically acceptable salts, solvates, hydrates, enantiomers, and crystal forms A and B thereof.
  • the chidamide or its derivative and the estrogen receptor down-regulating agent and/or degrading agent are unit preparations with the same or different specifications, and can be provided in separate containers.
  • kit refers to a package containing a therapeutic drug. Often the correct proportions of the various drugs are provided in a kit and packaged in a form that enables the best and economical use of those expensive and difficult-to-obtain drugs, with precise doses of individual drugs that can be used as a set in the treatment of a disease.
  • the present invention provides, in a fourth aspect, a method for treating and/or preventing breast cancer, comprising the step of simultaneously, separately or sequentially administering to a patient in need thereof a therapeutically effective amount of the aforementioned pharmaceutical composition or kit,
  • the breast cancer is endocrine therapy acquired drug resistance breast cancer, preferably the endocrine therapy acquired drug resistance breast cancer is estrogen receptor (estrogen receptor, ER) positive, refractory or recurring breast cancer after endocrine therapy , further preferably, the endocrine therapy-acquired resistant breast cancer includes estrogen receptor antagonist/inhibitor (estrogen receptor antagonist/inhibitor, ERA/ERI), estrogen receptor modulator (selective estrogen receptor modulator, SERM) , estrogen receptor down-regulator/degrader (selective estrogen receptor down-regulator/degrader, SERD), aromatase inhibitor (aromatase inhibitor, AI), luteinizing hormone releasing hormone (luteinizing hormone releasing hormone, LH-
  • the endocrine therapy acquired drug-resistant breast cancer includes estrogen receptor modulator (selective estrogen receptor modulator, SERM) (for example tamoxifen), estrogen receptor down-regulator/degrader (selective estrogen receptor modulator, SERM) Down-regulator/degrader, SERD) (such as fulvestrant), aromatase inhibitor (aromatase inhibitor, AI) (such as letrozole) after treatment of refractory or recurrent breast cancer.
  • SERM selective estrogen receptor modulator
  • SERM selective estrogen receptor modulator/degrader
  • SERM selective estrogen receptor modulator/degrader
  • SERD selective estrogen receptor modulator/degrader
  • AI aromatase inhibitor
  • letrozole such as letrozole
  • the present invention firstly establishes acquired tamoxifen-resistant breast cancer cell lines MCF7/TAMR and T47D/TAMR, which are also resistant to fulvestrant.
  • the inhibitory effect of Chidamide and Fulvestrant on drug-resistant cell lines was detected by colorimetric method.
  • the GI50 of . is 274.5nM.
  • the invention also establishes the pathological model of letrozole-acquired drug-resistant breast cancer in nude mice, and based on this, studies the combined drug efficacy of Chidamide and Fulvestrant in the letrozole-acquired drug-resistant animal model.
  • the results showed that from the 14th week after the start of administration, drug resistance was formed and grouping began, and the experiment ended on the 25th week. Showing significant drug resistance, letrozole has been unable to effectively inhibit the growth of tumor volume, and was the first to reach the experimental end point; 4-AD+chida. At the 25th week, the terminal tumor inhibition rate was only 11.09%; the terminal tumor inhibition rate of the 4-AD+FUL.
  • Figure 1 shows the identification of drug resistance to MCF7/TAMR and T47D/TAMR
  • Figure 2 shows the effect of Chidamide on fulvestrant in drug-resistant cell lines
  • Figure 3 shows the establishment of an animal pathological model of letrozole-acquired drug-resistant breast cancer
  • Figure 4 shows the efficacy study of Chidamide combined with Fulvestrant in drug-resistant animal pathological models.
  • Example 1 Inhibitory effect of chidamide and fulvestrant in combination on tamoxifen (or fulvestrant) acquired resistance to breast cancer cell lines
  • Tamoxifen is the most commonly used endocrine therapy drug for estrogen receptor (ER) positive breast cancer, but acquired drug resistance often occurs during long-term medication.
  • Human breast cancer cell line MCF7 and human breast ductal carcinoma cell line T47D are the most commonly used cell lines in preclinical basic research of ER-positive breast cancer, and have certain sensitivity to endocrine therapy drugs such as TAM.
  • tamoxifen active metabolite 4-hydroxytamoxifen (4-OH-TAM) can enable MCF7 and T47D to acquire drug tolerance , the formation of acquired tamoxifen-resistant cell lines.
  • MCF7 and T47D cell lines were purchased from American Type Culture Collection (ATCC), wherein MCF7 was cultured in high glucose DMEM medium (Gibco Company, USA) containing 10% fetal bovine serum (FBS), T47D was cultured in RPMI1640 medium (Gibco Company, USA) containing 10% FBS and 0.2 U/mL bovine insulin, and the culture conditions were all at 37°C and 5% CO 2 .
  • Forma Steri-Cycle cell incubators were purchased from Thermo Fisher Scientific.
  • the method for inducing acquired tamoxifen-resistant cell lines is as follows: the standard medium of MCF7 and T47D is replaced by phenol red-free medium, and 10% FBS is replaced by 5% activated carbon-treated FBS, and the culture system is The final dose of 1000nM 4-OH-TAM was added, and after continuous subculture for about 8 months, a cell line that could still proliferate stably under the culture conditions was obtained, which was an acquired drug-resistant breast cancer cell line, and were named according to the source. for MCF7/TAMR and T47D/TAMR.
  • the concentration that causes 50% growth inhibition, GI 50 ) of the obtained cell line by 4-OH-TAM was determined by gradient dose drug treatment and colorimetric method, which was similar to that of 4-OH-TAM on the original cell line.
  • the drug resistance of the obtained cell lines was identified in comparison to the GI 50 of the cell lines. A lower GI50 value indicates that the test cell line is more sensitive to the test compound, and a higher value indicates that the test cell line is more resistant to the test compound.
  • Alamar blue TM Cell Viability Assay Reagent (ThermoFisher Scientific, Wilmington, USA) is a redox indicator that produces absorbance changes and fluorescent signals based on metabolic activity.
  • alamar blue In the oxidized state, alamar blue is violet-blue and non-fluorescent, while in the reduced state, it is converted into a reduction product with pink or red fluorescence, reflecting the consumption of oxygen molecules by the cells under study, thus showing the Metabolic activity is strong or weak.
  • the proliferating cells exhibited a reducing environment, and the alamar blue TM was reduced and released into the culture medium, so that the culture medium changed from non-fluorescent indigo blue to fluorescent pink; while damaged or Inactive cells are less metabolically active and therefore correspond to lower signals.
  • the maximum absorption wavelengths of alamar blue TM in the reduction state and oxidation state are 570nm and 620nm, respectively, and the OD570 and OD620 values are measured in the microplate reader.
  • the absorbance value obtained by subtracting the latter from the former reflects the relative level of cell proliferation in the experiment. .
  • each cell line reaches 0, 1, 3, 10, 30, 100, 300, 1000, 3000, respectively , 10000nM, each cell line, each drug dose set 3 replicates.
  • alamar Blue TM cell viability detection reagent according to the relevant product operation manual to detect the cell viability of different cell lines cultured under the action of different doses of 4-OH-TAM, and calculate the cells per well with 0 nM dose as the reference. The percentage of growth inhibition was calculated by GraphPad Prism 7.00 software.
  • the GI 50 of 4-OH TAM for MCF7 and T47D were 6.136 and 10.86 nM, respectively, while the GI 50 of the drug-resistant cell lines MCF7/TAMR and T47D/TAMR reached 3581 nM and above 10000 nM, respectively. . It showed that two tamoxifen-acquired drug-resistant cell lines, MCF7/TAMR and T47D/TAMR, were formed under the induction of 4-OH TAM pulse method.
  • the GI 50 of FUL. to the above four cell lines was calculated and compared.
  • the results showed that the GI 50 of FUL. to MCF7 and T47D were 1.671 and 5.638 nM, respectively, while the GI 50 of MCF7/TAMR and T47D/TAMR reached 1.671 and 5.638 nM respectively. 262.7 and 536.1 nM, indicating that the tamoxifen-acquired resistant cell lines were also resistant to fulvestrant.
  • MCF7/TAMR and T47D/TAMR were digested with trypsin to prepare cell suspensions, sampled and counted, and inoculated with 5000 cells/180 ⁇ L per well of FBS and 1nM E2-containing phenol red-free culture medium.
  • FBS FBS
  • 1nM E2-containing phenol red-free culture medium In a 96-well cell culture plate, after culturing overnight, the cells in the 96-well plate were divided into 2 groups with 10 wells in each group, and different concentrations of FUL-phenol red-free culture medium mixture were added at a volume of 10 ⁇ L per well to make each well.
  • the final concentrations of FUL in the medium reached 0, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 nM, respectively, and at the same time, 10 ⁇ L of chidamide (chidamide, chida.)-free phenol red was added to 10 wells of group 1.
  • the culture medium was mixed so that the final concentration of chida. in each well was 300 nM, and 10 ⁇ L of phenol red-free medium was added to 10 wells of group 2. Three replicates were set for each cell line and each combination of test drugs.
  • alamar Blue TM cell viability detection reagent was added to detect the cell viability of different cell lines cultured under different experimental conditions, and the percentage of cell growth inhibition in each well was calculated with the 0 nM dose well as a reference, and then passed the GraphPad Prism 7.00 software. Plot the growth inhibition curve of FUL. on cell lines under different experimental conditions and related GI 50 .
  • the GI 50 of FUL. was 521.8 nM when acting as a single drug, and when adding a non-toxic dose of 300 nM chida. and FUL. together, FUL. reached 63.53 nM. GI50 for this cell line.
  • Chidamide has a significant synergistic sensitization effect on fulvestrant.
  • Letrozole is a representative drug of the third-generation aromatase inhibitor (AI), which can block androstenedione (4-androstenedione, 4-AD) or by inhibiting the activity of aromatase.
  • AI third-generation aromatase inhibitor
  • the conversion of testosterone to estrogen affects the biosynthesis of estrogen and reduces the level of estrogen in the body. It is widely used in the treatment of gynecological tumors and other diseases. Long-term use of aromatase inhibitors such as letrozole can also cause acquired drug resistance.
  • an ER-positive breast cancer cell line that artificially overexpressed the human aromatase gene was inoculated into ovariectomized cells (removal of murine estrogen production). pathway) in female nude mice, and continued to inject 4-AD to establish a tumor growth pathological model in nude mice that relies on the catalyzed production of estrogen by human aromatase.
  • letrozole administration to the above pathological models can effectively inhibit the growth of the transplanted tumor within a certain period of time, but similar to the process of clinical treatment, under the long-term drug action, the transplanted tumor will also develop acquired resistance to the drug. drug to form an animal pathological model of letrozole-acquired-resistant breast cancer. In this resistance model, testing of different drugs and drug combinations can continue.
  • MCF7-CA The ER-positive breast cancer cell line MCF7-CA that can stably express aromatase was established by infecting MCF7 cell line with lentivirus expressing aromatase gene.
  • the 4-AD group continued to subcutaneously inject 100 ⁇ g 4-AD every day, while the 4-AD+letr group received 100 ⁇ g letr subcutaneous injection of 100 ⁇ g 4-AD every day. Instead, the tumor-bearing volume was monitored weekly. When the tumor volume of the test mouse reached or exceeded 2000 mm 3 , the mouse was deemed to have reached the end point of the experiment.
  • the tumor volume of the 4-AD group increased rapidly due to the lack of drug effect, and all the mice in this group reached the end point of the experiment at the 12th week after the start of administration.
  • the 4-AD+letr group tumor proliferation was inhibited due to the drug effect of letr.
  • the tumor volume of each mouse was basically kept below 200mm3 , but from the 11th week, the group averaged
  • the tumor-bearing volume started to exceed 200 mm 3 and increased to 400 mm 3 at the 14th week, so it was considered that a letrozole-acquired drug-resistant breast cancer nude mouse model was formed. The results are shown in FIG. 3 .

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Abstract

属于生物医药领域，具体涉及西达本胺联合雌激素受体下调剂和/或降解剂治疗乳腺癌的用途。通过研究发现，在他莫昔芬(或氟维司群)获得性耐药的乳腺癌细胞系中，西达本胺对氟维司群具有显著的协同增敏作用，同时，西达本胺与氟维司群联合用药在来曲唑获得性耐药动物模型中显现出显著的协同疗效。

Description

西达本胺联合雌激素受体下调剂治疗乳腺癌的用途 技术领域
本发明涉及生物医药领域，具体涉及西达本胺联合雌激素受体下调剂治疗乳腺癌的用途。
背景技术
乳腺癌可分为雌激素受体(estrogen receptor，ER)阳性乳腺癌、HER2阳性乳腺癌和三阴性乳腺癌。其中，ER阳性乳腺癌约占全部乳腺癌的70％，ER阳性乳腺癌细胞具有依赖雌激素生长的特性。目前乳腺癌患者的治疗方式仍然以药物治疗为主。
内分泌治疗是大多数激素受体阳性乳腺癌首选治疗手段，其通过降低体内雌激素水平或抑制雌激素与ER的结合活性抑制肿瘤细胞生长。常用的乳腺癌内分泌治疗药物有他莫昔芬、氟维司群和芳香化酶抑制剂等。
芳香化酶(aromatase,AR)也叫雌激素合成酶，是微粒体细胞色素P450的一种复合酶，它由血红蛋白P450arom和还原型辅酶NADPH组成，广泛存在于卵巢、肝脏、肌肉、脂肪和乳腺癌细胞中，是催化生物体内雄激素向雌激素转化的关键酶和限速酶，可将雄激素的A环芳香化，脱去19位的碳原子并将1位的羰基转化为羟基，这样就将催化雄烯二酮和睾酮等雄激素转化为雌酮和雌二醇，后两者即为绝经后女性雌激素的主要来源。雌激素与肿瘤进展有关，芳香化酶在雌激素生物合成中起最终的限速催化作用。研究表明，有三分之二的乳腺癌是雌激素依赖性的，它们能在雌激素减少后得以消退。芳香化酶抑制剂能特异性导致芳香化酶失活，阻断芳构化反应，抑制雌激素生成，降低血液中雌激素水平从而达到治疗乳腺癌的目的。多用于抗雌激素(他莫昔芬)治疗失败 的绝经后晚期乳腺癌患者。芳香化酶抑制剂作为乳腺癌治疗经近三十年的努力，已开发上市了三代AR抑制剂，作为治疗乳腺癌的二线或三线药物，在美国、日本等已广泛用于临床。常用的芳香化酶抑制剂有依西美坦、来曲唑、阿那曲唑。
US4808616提出了依美西坦用于治疗晚期激素依赖的乳腺癌的用途。CN01805979.1公开了依西美坦在转移的、晚期的激素依赖乳腺癌、特别是绝经后妇女中的乳腺癌的第一线治疗中的用途。
CN201710110337.1公开了一种CDK4/6抑制剂与芳香化酶抑制剂联合在制备治疗乳腺癌的药物中的用途，具体提出了芳香化酶抑制剂选自福美坦、依西美坦、法倔唑、来曲唑、伏氯唑、阿那曲唑中的一种或几种。
他莫昔芬用于临床治疗乳腺癌，有效率一般在30％作用，雌激素受体阳性患者疗效较好(49％)，阴性患者疗效差(7％)。绝经前和绝经后患者均可使用，而绝经后和60岁以上的人较绝经前和年轻患者的效果为好。从病灶部位来看，皮肤、淋巴结和软组织的疗效好，骨和内脏转移的效果差。对于不适合接受芳香酶抑制剂治疗的乳腺癌女性，在乳腺癌初步治疗后，他莫西芬作为辅助治疗是最常用的处方。他莫西芬也被批准用于乳腺癌高危女性，以及转移性乳腺癌的治疗。
CN200810117739.5公开了一种含有他莫昔芬(I)和IC－162(II)或IC－163(III)的活性成分的药物组合物，利用其协同机制，提供具有抑制乳腺癌肿瘤效果好，并能相应减低他莫昔芬的副作用。
CN00128694.3公开了将他莫昔芬和异黄酮联合施用于患有乳腺癌的妇女和预先有乳腺癌倾向的妇女用于预防乳腺癌。
氟维司群(Fulvestrant)，化学名称是7-α-[9-[(4,4,5,5,5-五氟戊基)亚磺酰基] 壬基]雌-1,3,5(10)-三烯-3,17-β-二醇。分子式为C32H47F5O3S。其是一类新的雌激素受体拮抗剂—雌激素受体下调剂类抗乳腺癌治疗药物。用于抗雌激素疗法治疗后无效、病情进展或激素受体呈阳性的绝经后妇女转移性晚期乳腺癌的治疗。氟维司群是一种“纯粹”的雌激素受体拮抗剂，没有部分雌激素样激动效应，它通过结合、阻断并下调ER从而抑制雌激素信号通路，能与ER竞争性结合，与ER的亲和力接近雌激素，是他莫西芬的100倍，是唯一在他莫昔芬作用失败后可广泛用于临床的抗雌激素药物，由于该药为内分泌疗法，不会引起化疗常见的不良反应，故具有较好的患者依从性。多项临床研究发现，氟维司群250mg剂量在晚期乳腺癌的二线治疗中有良好治疗效果且安全性稳定。目前市售的产品是用于注射的含有50mg/ml氟维司群的透明的、无色至黄色的粘稠溶液。
CN201710110542.8公开了CDK4/6抑制剂与雌激素受体拮抗剂联合在制备治疗乳腺癌的药物中的用途，雌激素受体拮抗剂选自他莫昔芬、氟维司群中的一种或几种。
西达本胺(Chidamide，爱谱沙)是一种亚型选择性组蛋白去乙酰化酶(HDAC)抑制剂，化学名为N-(2-氨基-4-氟苯基)-4-[N-[(E)-3-(3-吡啶)丙烯酰基]氨甲基]苯甲酰胺，被批准用于复发或难治的外周T细胞淋巴瘤(PTCL)治疗的1.1类新药。在西达本胺的I期临床试验中共入组了16例实体瘤患者，包括非小细胞肺癌、乳腺癌、结肠癌、直肠癌、胃癌、前列腺癌等，除1例颌下腺腺样囊性癌获得部分缓解外，其余实体瘤均未获得明确疗效，此I期临床结果显示西达本胺单药对于乳腺癌无效，因此，在西达本胺II期临床试验中，也没有继续入组乳腺癌患者。
CN03139760.3公开了西达本胺化合物，具体公开了一种具有分化和抗增殖 活性的苯甲酰胺类组蛋白去乙酰化酶抑制剂及其药用制剂的制备方法与应用，其公开了结构通式，对取代基进行了定义。该类化合物作为组蛋白去乙酰化酶抑制剂，可以用于治疗与分化和增殖相关的疾病如癌症和牛皮癣。
CN201210489178.8公开了西达本胺的两种结晶形式，即西达本胺晶型A和西达本胺晶型B，以及西达本胺新的结晶形式的制备方法。所述西达本胺晶型A和西达本胺晶型B在口服吸收性和抑制细胞的分化和增殖的方面性能优异，并且其毒性弱，具有良好的储存和处理稳定性，可用于制备治疗与细胞分化和增殖相关的疾病的药物。
CN201410136761.X公开了一种E构型苯甲酰胺类化合物及其药用制剂与应用，所述E构型苯甲酰胺类化合物为西达本胺，其化学名称为N-(2-氨基-4-氟苯基)-4-[N-[(E)-3-(3-吡啶)丙烯酰基]氨甲基]苯甲酰胺，在其结构式中，3-吡啶丙烯酰基的构型为E型。所述E构型西达本胺具有亚型选择性组蛋白去乙酰化酶抑制活性，主要抑制第I类HDAC中的HDAC1、HDAC2、HDAC3和第IIb类HDAC中的HDAC10。所述E构型西达本胺可以用于治疗与组蛋白去乙酰化酶活性异常相关的疾病，如癌症，包括淋巴瘤、实体肿瘤和血液系统肿瘤等。
CN201810404747.1公开了将西达本胺与依西美坦联合使用，可以有效改善乳腺癌的治疗，获得相对于依西美坦单药治疗显著提高的效果，特别是可显著延长患者的无进展生存期，提高临床获益率和客观缓解率。
然而，临床在用的抗乳腺癌药物从他莫昔芬到芳香化酶抑制剂等内分泌治疗都存在严重的耐药问题，尤其是内分泌治疗获得性耐药的治疗成为了临床治疗无法走出的困境。
发明内容
本发明的目的在于提供用于内分泌治疗获得性耐药乳腺癌治疗和/或预防的 药物组合物。
本文使用的术语仅是为了描述特定实施方案，而并非意在限制本发明。如本文使用的单数形式“一种”、“一个”及“该”也意在包括复数形式，除非上下文明确另有指明。此外，就术语“包括”、“包含”、“具有”、“含有”或其变化形式在发明详述和/或权利要求中使用来说，这些术语意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包括”、“包含”、“具有”、“含有”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。
如本文使用的术语“治疗”、“缓解”和“改善”可互换使用。这些术语是指用于获得有益的或预期的效果(包括但不限于治疗益处和/或预防益处)的方法。
具体来说，本发明在第一方面提供了一种药物组合物，其包含西达本胺或其衍生物和雌激素受体下调剂和/或降解剂。
优选的，所述雌激素受体下调剂和/或降解剂包括氟维司群、elacestrant(RAD1901)、brilanestrant(GDC-0810)、SAR439859、AZD9496、OP-1074中的一种或几种，优选为氟维司群。
优选的，所述西达本胺衍生物包括其可药用盐、溶剂化物、水合物、对映异构体以及晶型A和晶型B。
术语“可药用盐”是指由本领域公知的多种有机和无机的抗衡离子衍生的盐。可药用盐包括药学上可接受的酸加成盐和碱加成盐。酸加成盐可以利用无机酸和有机酸形成。可由其衍生出盐的无机酸包括例如盐酸、氢溴酸、硫酸、硝酸、磷酸等。可由其衍生出盐的有机酸包括例如乙酸、丙酸、乙醇酸、丙酮酸、草酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉 桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸等。药学上可接受的碱加成盐可利用无机碱和有机碱形成。可由其衍生出盐的无机碱包括，例如，钠、钾、锂、铵、钙、镁、铁、锌、铜、锰、铝等。可由其衍生出盐的有机碱包括，例如，伯胺、仲胺和叔胺、包括天然存在的取代胺在内的取代胺、环胺、碱性离子交换树脂等，特别是例如异丙胺、三甲胺、二乙胺、三乙胺、三丙胺和乙醇胺。
优选的，所述的药物组合物还包含可药用赋形剂和/或载体。
术语“可药用赋形剂和/或载体”包括任何及所有溶剂、分散介质、涂料、抗细菌剂和抗真菌剂、等渗和吸收延迟剂等。这些介质和药剂在药物活性物质中的应用在本领域中是众所周知的。除了任何常规介质或试剂与活性成分不相容的情况以外，考虑将其用于本发明的治疗组合物中。补充的活性成分也可引入组合物中。
优选的，所述的药物组合物中，西达本胺或其衍生物的用量为5-60mg，优选为10-40mg，进一步优选为5mg、10mg、15mg、20mg、25mg、30mg、35mg、40mg、45mg、50mg、55mg、65mg；雌激素受体下调剂和/或降解剂的用量为50-500mg，优选为100-400mg，进一步优选为50mg、100mg、150mg、200mg、250mg、300mg、350mg、400mg、450mg、500mg。
优选的，所述的药物组合物，其剂型包括片剂、胶囊、颗粒剂、丸剂、散剂、栓剂和注射剂。
术语“片剂”是指药物与辅料均匀混合后压制而成的片状或异形片状的固体制剂。片剂以口服普通片为主，也有含片、舌下片、口腔贴片、咀嚼片、分散片、泡腾片、阴道片、速释或缓释或控释片与肠溶片等。
术语“胶囊剂”是指药物或与适宜辅料充填于空心硬胶囊或密封于软质囊 材中制成的固体制剂，可分为硬胶囊、软胶囊、缓释胶囊、控释胶囊和肠溶胶囊，主要供口服用。
术语“颗粒剂”是指原料药和适宜的辅料混合制成具有一定粒度的干燥颗粒状制剂。颗粒剂是常用的一种口服固体剂型。
术语“丸剂”是指药材细粉或药材提取物加适宜的粘合辅料制成的球形或类球片形制剂。
术语“散剂”是指药物或与适宜的辅料经粉碎、均匀混合制成的干燥粉末状制剂。
术语“栓剂”是指药物与适宜基质制成的具有一定形状的供人体腔道内给药的固体制剂。栓剂在常温下为固体，塞入腔道后，在体温下能迅速软化熔融或溶解于分泌液，逐渐释放药物而产生局部或全身作用。
术语“注射剂”是指药物制成的供注入体内的无菌溶液(包括乳浊液和混悬液)以及供临用前配成溶液或混悬液的无菌粉末或浓溶液。
本发明在第二方面提供了上述药物组合物用于制备治疗和/或预防乳腺癌的药物的用途，所述乳腺癌为内分泌治疗获得性耐药乳腺癌。
优选的，所述内分泌治疗获得性耐药乳腺癌为雌激素受体(estrogen receptor,ER)阳性、经内分泌治疗后难治或复发的乳腺癌。所述内分泌治疗获得性耐药乳腺癌包括雌激素受体拮抗剂/抑制剂(estrogen receptor antagonist/inhibitor,ERA/ERI)、雌激素受体调节剂(selective estrogen receptor modulator,SERM)、雌激素受体下调剂/降解剂(selective estrogen receptor down-regulator/degrader,SERD)、芳香化酶抑制剂(aromatase inhibitor,AI)、促黄体生成激素释放素(luteinizing hormone releasing hormone,LH-RH)类似物治疗后难治或复发的乳腺癌。进一步优选的，所述内分泌治疗获得性耐药乳腺癌包括雌激素受体调节 剂(selective estrogen receptor modulator,SERM)(例如他莫昔芬)、雌激素受体下调剂/降解剂(selective estrogen receptor down-regulator/degrader,SERD)(例如氟维司群)、芳香化酶抑制剂(aromatase inhibitor,AI)(例如来曲唑)治疗后难治或复发的乳腺癌。
本发明在第三方面提供了一种药盒，其包含以单一制剂形式存在的或以分开的制剂形式存在的西达本胺或其衍生物和雌激素受体下调剂和/或降解剂。
优选的，所述雌激素受体下调剂和/或降解剂包括氟维司群、elacestrant(RAD1901)、brilanestrant(GDC-0810)、SAR439859、AZD9496、OP-1074中的一种或几种，优选为氟维司群。
优选的，所述西达本胺衍生物包括其可药用盐、溶剂化物、水合物、对映异构体以及晶型A和晶型B。
其中，所述西达本胺或其衍生物和雌激素受体下调剂和/或降解剂为分别具有相同或不同规格的单位制剂，可分别置于单独容器中提供。
术语“药盒”是指包括有治疗用药物的成套包装。通常药盒中提供各种药物的正确比例，并以能使那些昂贵的和难以获得的药物以最好和节省的形式包装，其中个药物具有精确的剂量，在治疗疾病时可成套使用。
本发明在第四方面提供了一种用于治疗和/或预防乳腺癌的方法，其包括向有需要的患者同时、分别或依次施用治疗有效量的前述的药物组合物或药盒的步骤，所述乳腺癌为内分泌治疗获得性耐药乳腺癌，优选为所述内分泌治疗获得性耐药乳腺癌为雌激素受体(estrogen receptor,ER)阳性、经内分泌治疗后难治或复发的乳腺癌，进一步优选为所述内分泌治疗获得性耐药乳腺癌包括雌激素受体拮抗剂/抑制剂(estrogen receptor antagonist/inhibitor,ERA/ERI)、雌激素受体调节剂(selective estrogen receptor modulator,SERM)、雌激素受体下调剂/ 降解剂(selective estrogen receptor down-regulator/degrader,SERD)、芳香化酶抑制剂(aromatase inhibitor,AI)、促黄体生成激素释放素(luteinizing hormone releasing hormone,LH-RH)类似物治疗后难治或复发的乳腺癌。进一步优选的，所述内分泌治疗获得性耐药乳腺癌包括雌激素受体调节剂(selective estrogen receptor modulator,SERM)(例如他莫昔芬)、雌激素受体下调剂/降解剂(selective estrogen receptor down-regulator/degrader,SERD)(例如氟维司群)、芳香化酶抑制剂(aromatase inhibitor,AI)(例如来曲唑)治疗后难治或复发的乳腺癌。
本发明的有益效果：
本发明首先建立了获得性他莫昔芬耐药乳腺癌细胞系MCF7/TAMR和T47D/TAMR，其对氟维司群也发生了耐药。通过比色法检测了西达本胺与氟维司群联用对耐药细胞系的抑制作用，结果显示，在他莫昔芬获得性耐药细胞系MCF7/TAMR中，单药作用时FUL.的GI50为274.5nM，当在体系中加入终剂量为300nM的chida.与FUL.共同作用于MCF7/TAMR时，虽然上述剂量的chida.对细胞系的增殖并不产生抑制作用(即300nM chida.与0nM FUL.共同作用于细胞时的抑制率为0)，但chida.的加入使FUL.在41.6nM时即达到了对MCF7/TAMR的GI50。同样，在本次实验的T47D/TAMR中，单药作用时FUL.的GI50为521.8nM，而加入非毒性剂量的300nM chida.与FUL.共同作用时，使FUL.在63.53nM时达到了对该细胞系的GI50，由此可知，在他莫昔芬(或氟维司群)获得性耐药的乳腺癌细胞系中，西达本胺对氟维司群具有显著的协同增敏作用。
本发明还建立了曲唑获得性耐药乳腺癌裸鼠病理模，以此为基础研究了西达本胺与氟维司群在来曲唑获得性耐药动物模型中的联合用药药效，结果显示，自开始给药后第14周形成耐药并分组开始，至第25周实验结束，在来曲唑获 得性耐药动物模型中，继续给予来曲唑治疗的4-AD+letr组表现出显著的耐药性，来曲唑已无法有效抑制肿瘤体积的增长，率先达到实验终点；4-AD+chida.组即给予西达本胺单药治疗同样未能有效抑制肿瘤体积增长，第25周时，终末抑瘤率仅为11.09％；给予氟维司群单药治疗的4-AD+FUL.组终末抑瘤率为39.76％；而给予西达本胺与氟维司群联合用药的4-AD+(chida.+FUL.)组终末抑瘤达到了97.30％。因此西达本胺与氟维司群联合用药在来曲唑获得性耐药动物模型中显现出显著的协同疗效。
附图说明
图1为对MCF7/TAMR和T47D/TAMR的耐药性鉴定；
图2为西达本胺在耐药细胞系中对氟维司群的作用研究；
图3为来曲唑获得性耐药乳腺癌动物病理模型的建立；
图4为西达本胺与氟维司群联用在耐药动物病理模型中的药效研究。
具体实施方式
以下进一步通过非限制性实例来说明本发明，其不旨在限制本发明所涵盖的范围。
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。
实施例1 西达本胺与氟维司群联用对他莫昔芬(或氟维司群)获得性耐药的乳腺癌细胞系的抑制作用
1.获得性他莫昔芬耐药乳腺癌细胞系的建立
1.1 4-羟他莫昔芬冲击法诱导获得性耐药乳腺癌细胞系
实验原理：他莫昔芬(tamoxifen,TAM)是针对雌激素受体(estrogen receptor,ER)阳性乳腺癌最常使用的内分泌治疗药物，但在长期服药过程中常会发生获得性耐药。人乳腺癌细胞系MCF7和人乳腺导管癌细胞系T47D是ER阳性乳腺癌的临床前基础研究中最常应用的细胞系，对TAM等内分泌治疗药物具有一定敏感性。在培养体系中加入相对高剂量的他莫昔芬活性代谢产物4-羟他莫昔芬(4-OH-TAM)进行数个月的诱导培养，可使MCF7和T47D获得对药物的耐受性，形成获得性他莫昔芬耐药细胞系。
实验步骤：MCF7和T47D细胞系均购于American Type Culture Collection(ATCC)，其中MCF7培养于含有10％胎牛血清(fetal bovine serum,FBS)的高糖DMEM培养液(Gibco Company,USA)中，T47D培养于含有10％FBS、0.2U/mL牛胰岛素的RPMI1640培养液(Gibco Company,USA)中，培养条件均为37℃、5％CO 2。Forma Steri-Cycle细胞培养箱购自Thermo Fisher Scientific。诱导获得性他莫昔芬耐药细胞系的方法如下：将MCF7和T47D的标准培养液替换成无酚红培养液，同时将10％FBS替换成5％的活性炭处理FBS，并在培养体系中加入终剂量为1000nM的4-OH-TAM，持续传代培养约8个月后，获得在该培养条件下仍可稳定增殖的细胞系，即为获得性耐药乳腺癌细胞系，根据源头分别命名为MCF7/TAMR和T47D/TAMR。
1.2通过比色法鉴定细胞系耐药性
实验原理：通过梯度剂量药物处理及比色法测算4-OH-TAM对所获得细胞系的半数生长抑制剂量(the concentration that causes 50％growth inhibition,GI 50)，与4-OH-TAM对原细胞系GI 50相比较，鉴定所获得细胞系的耐药性。GI 50值越低表示受试细胞系对受试化合物越敏感、越高表示受试细胞系对受试化合物越耐受。
Alamar blue TM细胞活力检测试剂(ThermoFisher Scientific,Wilmington,USA)是一种氧化还原指示剂，能根据代谢活性产生吸光度变化和荧光信号。在氧化状态下，alamar blue呈现紫蓝色无荧光性，而在还原状态下，转变为呈粉红或红色荧光的还原产物，反映所研究的细胞对氧分子的消耗，因而可以显示被观察细胞的代谢活动强弱。在测试过程中，增殖当中的细胞内表现为一种还原环境，alamar blue TM被还原后释放到培养液中，使培养液从无荧光的靛青蓝变成有荧光的粉红色；而受损或无活性的细胞代谢活性较低，因此对应的信号较低。alamar blue TM在还原状态和氧化状态的最大吸收波长分别为570nm和620nm，在酶标仪中分别测定OD570和OD620值，前者减去后者得到的吸光度值即反映了试验中细胞增殖的相对水平。
实验步骤：将贴壁培养的细胞系MCF7、MCF7/TAMR、T47D、T47D/TAMR分别经胰酶消化后制备成细胞悬液，取样计数，用含5％活性炭处理FBS及1nM雌二醇(estrogen,E2)的无酚红培养液按每孔5000个/190μL接种于96孔细胞培养板中，5％CO 2、37℃培养箱中培养过夜后，按每孔10μL体积加入不同浓度的4-OH-TAM-无酚红培养液混合物或FUL.-无酚红培养液混合物，使每孔中4-OH-TAM或FUL.终浓度分别达到0,1,3,10,30,100,300,1000,3000,10000nM，每株细胞系、每个药物剂量设置3次重复。继续培养120小时后，按相关产品操作手册加入alamar Blue TM细胞活力检测试剂，检测不同剂量4-OH-TAM作用下培养的不同细胞系的细胞活力，并以0nM剂量孔为参照计算每孔细胞生长抑制百分率，进而通过GraphPad Prism 7.00软件计算各受试化合物对不同受试细胞系的半数生长抑制剂量(GI 50)。
如图1所示，根据计算，4-OH TAM对MCF7和T47D的GI 50分别为6.136和10.86nM，而对耐药细胞系MCF7/TAMR和T47D/TAMR的GI 50分别达到了3581nM 及10000nM以上。表明在4-OH TAM冲击法诱导下，形成了两株他莫昔芬获得性耐药细胞系MCF7/TAMR和T47D/TAMR。
同时，测算比较了FUL.对上述4株细胞系的GI 50，结果表明，FUL.对MCF7和T47D的GI 50分别为1.671和5.638nM，而对MCF7/TAMR和T47D/TAMR的GI 50分别达到了262.7和536.1nM，表明他莫昔芬获得性耐药细胞系对氟维司群也发生了耐药。
2.西达本胺与氟维司群联用对耐药细胞系的抑制作用
实验步骤：将MCF7/TAMR和T47D/TAMR分别经胰酶消化后制备成细胞悬液，取样计数，用含5％活性炭处理FBS及1nM E2的无酚红培养液按每孔5000个/180μL接种于96孔细胞培养板中，培养过夜后，将96孔板中细胞分为2组，每组10个孔，按每孔10μL体积加入不同浓度的FUL-无酚红培养液混合物，使每孔中FUL的最终浓度分别达到0,1,3,10,30,100,300,1000,3000,10000nM，同时，在第1组的10个孔中加入10μL西达本胺(chidamide,chida.)-无酚红培养液混合物，使每孔中chida.的最终浓度为300nM，在第2组的10个孔中加入10μL无酚红培养液。每株细胞系、每组合受试药物设置3次重复。继续培养120小时后，加入alamar Blue TM细胞活力检测试剂，检测不同实验条件下培养的不同细胞系的细胞活力，并以0nM剂量孔为参照计算每孔细胞生长抑制百分率，进而通过GraphPad Prism 7.00软件绘制不同实验条件下FUL.对细胞系的生长抑制曲线及相关GI 50。
结果如图2所示，本次实验中，在他莫昔芬获得性耐药细胞系MCF7/TAMR中，单药作用时FUL.的GI 50为274.5nM，当在体系中加入终剂量为300nM的chida.与FUL.共同作用于MCF7/TAMR时，虽然上述剂量的chida.对细胞系的增殖并不产生抑制作用(即300nM chida.与0nM FUL.共同作用于细胞时的抑制 率为0)，但chida.的加入使FUL.在41.6nM时即达到了对MCF7/TAMR的GI 50。同样，在本次实验的T47D/TAMR中，单药作用时FUL.的GI 50为521.8nM，而加入非毒性剂量的300nM chida.与FUL.共同作用时，使FUL.在63.53nM时达到了对该细胞系的GI 50。
综上，在他莫昔芬(或氟维司群)获得性耐药的乳腺癌细胞系中，西达本胺对氟维司群具有显著的协同增敏作用。
实施例2 西达本胺与氟维司群联用在来曲唑获得性耐药动物模型中的药效研究
1.来曲唑获得性耐药乳腺癌动物病理模型的建立
实验原理：来曲唑是第3代芳香化酶抑制剂(aromatase inhibitor,AI)的代表性药物，可通过抑制芳香化酶的活性阻断雄烯二酮(4-androstenedione,4-AD)或睾酮向雌激素的转化，从而影响了雌激素的生物合成，降低体内的雌激素水平，被广泛用于治疗妇科肿瘤等疾病。长期使用来曲唑等芳香化酶抑制剂同样会产生获得性耐药，本实验通过将人为过表达人源芳香化酶基因的ER阳性乳腺癌细胞系接种至摘除卵巢(去除鼠源雌激素产生途径)的雌性裸鼠体内，并持续注射4-AD，从而在裸鼠体内建立一个依赖于人源芳香化酶催化产生雌激素的肿瘤生长病理模型。
对上述病理模型进行来曲唑给药，在一定时间内，药物可有效抑制移植瘤的生长，但与临床治疗的过程相似，长期的药物作用下，移植瘤也会产生对药物的获得性耐药，从而形成来曲唑获得性耐药乳腺癌动物病理模型。在该耐药模型中，可以继续进行不同药物及药物组合的测试。
实验步骤：通过向MCF7细胞系中感染表达芳香化酶基因的慢病毒，建立可稳定表达芳香化酶的ER阳性乳腺癌细胞系MCF7-CA。将MCF7-CA扩大培养至足 够数量，胰酶消化收集，重悬于无酚红、无血清培养液中，制备成2.5×10 7/100μL的细胞悬液，与等体积无酚红Matrigel(Corning,USA)混合，以200μL/只皮下注射接种于摘除卵巢的6周龄雌性裸鼠腋下(n＝50)。所有接种MCF-7CA的裸鼠每只每天皮下注射100μg 4-AD，并通过游标卡尺监测所接种肿瘤的长径(L)和短径(W)，通过公式V＝0.5×L×W 2计算肿瘤体积。当平均肿瘤体积达到200mm 3时，将受试鼠随机分为两组，即4-AD组(n＝10)和4-AD+letr组(n＝40)。4-AD组继续每只每天皮下注射100μg 4-AD，4-AD+letr组则每只每天皮下注射100μg 4-AD的同时注射10μg letr。改为每周监测荷瘤体积，当受试鼠所荷肿瘤体积达到或超过2000mm 3时，视为该鼠达到实验终点。
在本实验中，4-AD组由于无药物作用，所荷肿瘤体积迅速增大，在开始给药后第12周该组所有受试鼠达到实验终点。4-AD+letr组由于letr的药物作用，肿瘤增殖受到抑制，在开始给药后的10周内，各鼠所荷肿瘤体积基本保持在200mm 3以下，但从第11周开始，该组平均荷瘤体积开始超过200mm 3并增长至第14周达到400mm 3，由此认为来曲唑获得性耐药乳腺癌裸鼠病理模型形成，结果如图3所示。
2.西达本胺与氟维司群在来曲唑获得性耐药动物模型中的联合用药
实验步骤：将上述4-AD+letr组已经形成来曲唑获得性耐药乳腺癌病理模型的40只受试鼠重新随机分为4组，分别为：4-AD+letr组、4-AD+chida.组、4-AD+FUL.组、4-AD+(chida.+FUL.)组，各组n＝10。即各组均继续给予每只每天100μg 4-AD的皮下注射，并各组分别给予每只每天10μg letr皮下注射、每只每天5mg/kg chida.灌胃、每只每7天一次5mg Ful.皮下注射以及每只每天5mg/kg chida.灌胃联合每7天一次5mg FUL.皮下注射。
自开始给药后第14周形成耐药并分组开始，至第25周实验结束，在来曲 唑获得性耐药动物模型中，继续给予来曲唑治疗的4-AD+letr组表现出显著的耐药性，来曲唑已无法有效抑制肿瘤体积的增长，率先达到实验终点；4-AD+chida.组即给予西达本胺单药治疗同样未能有效抑制肿瘤体积增长，第25周时，终末抑瘤率仅为11.09％；给予氟维司群单药治疗的4-AD+FUL.组终末抑瘤率为39.76％；而给予西达本胺与氟维司群联合用药的4-AD+(chida.+FUL.)组终末抑瘤达到了97.30％，结果如图4所示。因此西达本胺与氟维司群联合用药在来曲唑获得性耐药动物模型中显现出显著的协同疗效。
以上对本发明进行了详细的介绍，本文中应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想，包括最佳方式，并且也使得本领域的任何技术人员都能够实践本发明，包括制造和使用任何装置或系统，和实施任何结合的方法。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。本发明专利保护的范围通过权利要求来限定，并可包括本领域技术人员能够想到的其他实施例。如果这些其他实施例具有不是不同于权利要求文字表述的结构要素，或者如果它们包括与权利要求的文字表述无实质差异的等同结构要素，那么这些其他实施例也应包含在权利要求的范围内。

Claims (9)

  1. 包含西达本胺或其衍生物和雌激素受体下调剂和/或降解剂的药物组合物在制备用于治疗和/或预防乳腺癌的药物中的用途，其中所述乳腺癌为内分泌治疗获得性耐药乳腺癌。
  2. 根据权利要求1所述的用途，其中所述内分泌治疗获得性耐药乳腺癌为雌激素受体(estrogen receptor,ER)阳性、经内分泌治疗后难治或复发的乳腺癌。
  3. 根据权利要求1所述的用途，其中所述雌激素受体下调剂和/或降解剂选自氟维司群、elacestrant(RAD1901)、brilanestrant(GDC-0810)、SAR439859、AZD9496、OP-1074中的一种或几种。
  4. 根据权利要求1所述的用途，其中所述西达本胺衍生物包括其可药用盐、溶剂化物、水合物、对映异构体以及晶型A和晶型B。
  5. 根据权利要求1-4任一项所述的用途，其中所述西达本胺或其衍生物的用量为5-60mg，优选为10-40mg，所述雌激素受体下调剂和/或降解剂的用量为50-500mg，优选为100-400mg。
  6. 根据权利要求1-4任一项所述的用途，其中所述药物组合物还包含可药用赋形剂和/或载体。
  7. 根据权利要求1-4任一项所述的用途，其中所述药物组合物的剂型包括片剂、胶囊、颗粒剂、丸剂、散剂、栓剂和注射剂。
  8. 用于治疗和/或预防乳腺癌的方法，其包括向有需要的患者同时、分别或依次施用治疗有效量的权利要求1-7中任一项所述的药物组合物的步骤，所述乳腺癌为内分泌治疗获得性耐药乳腺癌.
  9. 根据权利要求8所述的方法，其中所述内分泌治疗获得性耐药乳腺癌为雌激素受体(estrogen receptor,ER)阳性、经内分泌治疗后难治或复发的乳腺癌。
PCT/CN2021/115558 2020-09-01 2021-08-31 西达本胺联合雌激素受体下调剂治疗乳腺癌的用途 WO2022048527A1 (zh)

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