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WO2024230090A1 - Utilisation d'un inhibiteur de sialyl-transférase dans la préparation d'un médicament pour neutraliser un microenvironnement tumoral acide - Google Patents

Utilisation d'un inhibiteur de sialyl-transférase dans la préparation d'un médicament pour neutraliser un microenvironnement tumoral acide Download PDF

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WO2024230090A1
WO2024230090A1 PCT/CN2023/128090 CN2023128090W WO2024230090A1 WO 2024230090 A1 WO2024230090 A1 WO 2024230090A1 CN 2023128090 W CN2023128090 W CN 2023128090W WO 2024230090 A1 WO2024230090 A1 WO 2024230090A1
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drug
cancer
optionally
brca1
tumor
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徐晓玲
舒晓东
邓初夏
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University of Macau
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    • 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
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the technical field of tumor treatment, and in particular, to the use of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment.
  • Cancer metastasis is an extremely complex process that is affected by many factors, including acidity, which is generally believed to be mainly caused by lactate/H + produced by glycolysis. Acidity is one of the main characteristics of many cancers. It is currently believed that acidity is caused by the accumulation of metabolic waste caused by cancer cells with high metabolic activity and insufficient blood perfusion.
  • the acidic tumor microenvironment (ATME) is believed to be caused by the removal of protons and H + produced by lactate/H + , bicarbonate and CO2 hydrogenation from the cytosol to the interstitial space.
  • the pH value of the acidic tumor microenvironment (ATME) is generally 5.6-7.0.
  • the acidic microenvironment not only helps cancer cells migrate through the extracellular matrix (ECM) to blood vessels with a relatively high pH (7.35-7.45), but also hinders the infiltration of active immune cells into tumor tissues by reducing the activity of T cells and NK cells, thereby achieving tumor metastasis. To date, no other mechanism that can cause the generation of an acidic tumor microenvironment (ATPME) has been found.
  • ECM extracellular matrix
  • ATPME acidic tumor microenvironment
  • breast cancer has the highest incidence rate among all cancers, with 2.26 million new cases of breast cancer and approximately 685,000 cancer deaths in 2020.
  • breast cancers are carcinomas that originate from mammary epithelial cells, which are arranged in a double-layer structure between the lobules and the terminal ducts.
  • the inner layer contains secretory luminal epithelial cells
  • the outer layer is composed of contractile myoepithelial cells (or basal cells) surrounded by a basement membrane (BM).
  • BM basement membrane
  • the spread of malignant cells to secondary organs depends on their ability to destroy the basement membrane and basal layer, which mainly contain laminin and collagen IV, their ability to invade the extracellular matrix, their ability to infiltrate the circulation, extravasate from blood vessels, and grow in distant organs. Therefore, the study of tumor growth and metastasis plays a key role in the treatment and prevention of cancer.
  • the present disclosure provides a use of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment.
  • the sialyltransferase inhibitor neutralizes the acidic tumor microenvironment by downregulating the expression of Vegfa and/or VegfIl6.
  • the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic.
  • the sialyltransferase inhibitor inhibits the expression of polysialyltransferase and/or polysialyltransferase receptor.
  • the polysialyltransferase comprises St8sia4.
  • the polysialyltransferase receptor comprises at least one of soluble E-selectin and L-selectin.
  • the drug for neutralizing the acidic tumor microenvironment also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.
  • the present disclosure provides an application of a sialyltransferase inhibitor in preparing a drug for treating Brca1 defect-related tumors.
  • the drug for treating Brca1 deficiency-related tumors also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.
  • the Brca1 deficiency-associated tumor is Brca1 deficiency-associated breast cancer.
  • the present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for preventing or treating cancer.
  • the drug for preventing or treating cancer includes drugs for preventing or treating any one of the occurrence, metastasis or growth of cancer.
  • the drug for preventing or treating cancer also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.
  • the cancer includes any one of breast cancer, liver cancer, and spleen cancer.
  • the present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for treating the destruction of the double-layer structure of mammary epithelium.
  • the drug for treating destruction of the double-layer structure of mammary epithelium also includes a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.
  • the present disclosure provides a pharmaceutical composition, comprising the aforementioned sialyltransferase inhibitor and a pharmaceutically acceptable excipient.
  • the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic.
  • the dosage form of the drug includes any one of injection, injection, tablet, granule, granule or capsule.
  • the drug is in the form of nanoparticles.
  • the present disclosure provides a method of neutralizing an acidic tumor microenvironment, the method comprising administering to a subject in need thereof an effective amount of a sialyltransferase inhibitor.
  • the present disclosure provides a method for treating a tumor associated with Brca1 deficiency, the method comprising administering an effective amount of a sialyltransferase inhibitor to a subject in need thereof;
  • the Brca1 deficiency-associated tumor is Brca1 deficiency-associated breast cancer.
  • the present disclosure provides a method for preventing or treating cancer, comprising administering an effective amount of a sialyltransferase inhibitor to a subject in need thereof; wherein the drug for preventing or treating cancer includes drugs for preventing or treating any one of the occurrence, metastasis or growth of cancer.
  • the cancer includes any one of breast cancer, liver cancer, and spleen cancer.
  • the present disclosure provides a method for treating disruption of the bilayer structure of mammary epithelium, the method comprising administering an effective amount of a sialyltransferase inhibitor to a subject in need thereof.
  • the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic.
  • the above method comprises the combined use of a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.
  • the present disclosure provides a sialyltransferase inhibitor for the following purposes:
  • the Brca1 deficiency-related tumor is Brca1 deficiency-related breast cancer
  • Preventing or treating cancer includes preventing or treating any of the occurrence, metastasis or growth of cancer; optionally, the cancer includes any of breast cancer, liver cancer, spleen cancer;
  • the sialyltransferase inhibitor includes at least one of 3Fax-Peracetyl Neu5Ac or Stattic.
  • the use includes a sialyltransferase inhibitor combined with a PD-1 drug; optionally, the PD-1 drug is a PD-1 antibody.
  • FIG1 is a result of the destruction of the double-layer structure of the mammary gland associated with the increase of sialyltransferase provided by the embodiments of the present disclosure
  • FIG2 is a result diagram of the increase of sialyltransferase in mouse mammary tissue provided by the embodiments of the present disclosure
  • FIG3 is a graph showing the increase in polysialic acid content in mouse Brca1-deficient cells and human Brca1-deficient breast cancer samples provided by the embodiments of the present disclosure
  • FIG4 is a graph showing changes in sialyltransferases in Brca1-deficient mice and non-Brca1-deficient mice provided in an embodiment of the present disclosure
  • FIG5 is a diagram showing the effect of sialyltransferase on breast cancer metastasis provided by an embodiment of the present disclosure
  • FIG6 is a diagram showing the effect of sialyltransferase expression on tumor growth and metastasis provided by an embodiment of the present disclosure
  • FIG. 7 is a diagram showing the results of detecting the expression of sialyltransferase in mammary epithelial cells provided by an embodiment of the present disclosure
  • FIG8 is a diagram showing that the Vegfa/Il6 signaling pathway upregulates TGF- ⁇ signals in Brca1-deficient mammary epithelial cells and macrophages according to an embodiment of the present disclosure
  • FIG9 is a result of the tumor immunosuppressive microenvironment induced by high sialylation in breast tissues of mice and human breast cancer patients provided by the embodiments of the present disclosure.
  • FIG10 is a graph showing the relationship between the increase in the number of MDSCs in Brca1-deficient mice and the effect of Vegfa/Il6 provided in an embodiment of the present disclosure
  • FIG. 11 shows the results of inhibiting breast tumor growth and metastasis by using a sialyltransferase inhibitor and a combination of the sialyltransferase inhibitor and a drug provided in an embodiment of the present disclosure
  • FIG. 12 shows the results of inhibiting breast tumor growth and metastasis after intraperitoneal injection of sialyltransferase inhibitors and drugs used in combination therewith provided in the embodiments of the present disclosure
  • FIG. 13 is a diagram showing the results of treating breast tumors with high sialyltransferase expression using a sialyltransferase inhibitor in combination with ⁇ PD-1 according to an embodiment of the present disclosure
  • FIG. 14 shows the effects of the sialyltransferase inhibitor provided in the embodiments of the present disclosure in combination with ⁇ PD-1 on different cells during breast tumor growth and metastasis.
  • Breast1 stands for breast cancer associated gene 1, a tumor suppressor gene whose germline mutations cause familial breast cancer.
  • EMT epithelial-mesenchymal transition
  • epithelial-mesenchymal transition refers to the transformation of epithelial to mesenchymal cells, which endows cells with the ability to metastasize and invade, including stem cell characteristics, reduces apoptosis and senescence, and promotes immunosuppression. It not only plays a key role in the development process, but also participates in processes such as tissue healing, organ fibrosis, and cancer occurrence.
  • MDSCs refers to myeloid-derived suppressor cells, which are precursors of dendritic cells, macrophages and/or granulocytes and have the ability to significantly suppress immune cell responses.
  • ATPME acidic tumor microenvironment
  • Vegfa and "VegfIL6” are both transcriptional regulators involved in angiogenesis and tumor cell invasion, where Vegfa is vascular endothelial growth factor A (VEGF-A).
  • VEGF-A vascular endothelial growth factor A
  • VegfIL6 is vascular endothelial growth factor (VEGF) and IL-6, IL-6 is interleukin-6, a cytokine belonging to the chemokine family.
  • sialyltransferase genes St8sia4 and St3gal1 are responsible for the production of polysialic acid (PSA) and Sialyl-Lewis A (sLeA), respectively.
  • 3Fax-Peracetyl Neu5Ac can be converted to CMP-Neu5Ac and inhibit silyl transferase. It is used as a sialyltransferase inhibitor in this application.
  • Stattic is a STAT3 inhibitor, with a molecular formula of C 8 H 5 NO 4 S and a structural formula as shown below:
  • the present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment.
  • the sialyltransferase inhibitor can neutralize the acidic tumor microenvironment and establish an ecological barrier to tumor metastasis, providing a new approach for the prevention and treatment of tumors.
  • the sources of human tissue microarrays and human breast cancer samples in the following experiments are as follows: 95 tissue microarray human breast cancer samples were kindly provided by the Third affiliated Hospital of Sun Yat-sen University. 25 human breast cancer samples for immunofluorescence staining were kindly provided by the First affiliated Hospital of Sun Yat-sen University, and BRCA mutations were sequenced and identified. Among these 25 primary tumor samples, 4 were BRCA1 mutation carriers and 21 were non-BRCA1 mutation carriers. Human tissues were fixed in formalin, embedded in paraffin, and sectioned for experiments.
  • mice and experimental cell lines in the following experiments are as follows: All mouse experiments performed in this study were approved by the Animal Ethics Committee of the University of Macau.
  • the Brca1 conditional knockout mouse model (Brca1co/co; MMTV-Cre) has been established in the inventors' laboratory.
  • Brca1WT Brca1WT
  • Brca1-MT Brca1-MT mammary epithelial cells
  • different drugs were given, including intraperitoneal injection of sialyltransferase inhibitor 3Fax-P-Neu5Ac (20 mg/kg) for 7 consecutive days and intraperitoneal injection of Stattic (10 mg/kg) 3 times a week.
  • Stattic (10 mg/kg) and 3Fax-P-Neu5Ac (20 mg/kg) were injected intravenously once every three days for a total of 3 times.
  • the ratiometric fluorescent probe was kindly provided by Professor Zhang Xuanjun of the University of Macau, and the Stattic and 3Fax-P-Neu5Ac nanoparticles were kindly provided by Professor Dai Yunlu of the University of Macau.
  • the PD1 antibody i.e., ⁇ PD-1 used in the examples of this application, was purchased from Bio X Cell InVivoMab, catalog number: BE0273-5mg.
  • RNA-seq data and human datasets in the following experiments are as follows: Bone marrow, blood, spleen, peritoneal cells, and mammary tissues from 10-month-old mice and breast tumors collected from Brca1co/co tumor mice were isolated in Trizol, and total RNA was subjected to mRNA isolation and library construction, followed by RNA sequencing (Hiseq, paired-end, 6GB raw data per sample). The data were analyzed using HISAT, StringTie, and Ballgown to obtain differentially expressed genes. FPKM values were extracted for each relevant gene to generate a matrix, and heat maps were drawn for different important genes in different organs by RStudio.
  • the immune cell composition in breast and breast tumors was calculated by ImmuCC and plotted by heat maps.
  • the human dataset of BRCA samples was obtained from TISIDB (http://cis.hku.hk/TISIDB/index.php).
  • the data on the transcription or protein levels of St8sia4 and St3gal1 genes were obtained from breast cancer patients in the TCGA database, and the graphs were obtained from Linkedomics (http://www.linkedomics.org/login.php).
  • the Kaplan-Meier plotter https://kmplot.com/analysis/ was used to obtain the survival curves of breast cancer patients with high or low gene expression.
  • the sources of immunofluorescence staining of tissue sections in the following experiments are as follows: All human and mouse paraffin slides were dewaxed and rehydrated according to standard protocols. The slides were washed with PBS and then heated in R-Buffer-A (10 mL in 90 mL water). After the treatment was completed (or overnight incubation), the slides were washed with PBS and then treated with 0.5% TritonX-100 and 0.5 mg/ml sodium borohydride (in PBS) for 10 minutes at room temperature, respectively. The slides were then incubated with blocking solution (50% 3% BSA and 50% Animal-FreeBlocker) at room temperature overnight or at least 1 hour, and incubated with primary antibodies from different sources overnight at 4°C. Secondary antibodies and DAPI were incubated for 1 hour at room temperature, anti-fading reagents were covered on the tissues, and coverslips were placed on each slide, and images were scanned by CarlZeissLSM880 super-resolution microscope.
  • R-Buffer-A 10 m
  • the source of cell immunofluorescence staining in the following experiments is as follows: cells were seeded in a 4-well chamber, washed twice with PBS, and fixed with 4% formaldehyde for 15 minutes. Then, cells were thoroughly washed with PBS and treated with 0.5% TritonX-100 for 10 minutes, and then incubated with blocking solution at room temperature for at least 1 hour. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature using a Carl Zeiss LSM880 ultrasonoscope. High-resolution microscope scan image.
  • the sources of the cytokine antibody array in the following experiments are as follows: serum from 6-month-old mice and supernatants from different cell lines were diluted with 1 ⁇ blocking buffer. The diluted samples were added to the membrane in a 4-well tray and pre-incubated with 1 ⁇ blocking buffer for 30 minutes at room temperature and incubated overnight at 4°C. The membranes were placed in a clean container and washed with 20-30mL of 1 ⁇ wash buffer I for 30-45 minutes per membrane. They were then returned to the 4-well tray and washed twice with 2ml wash buffer II at room temperature. 2ml 1 ⁇ Biotin-Conjugated Anti-Cytokines was pipetted into each well and the membrane was incubated overnight at 4°C.
  • Detection buffer C and detection buffer D were mixed in equal volumes (1:1) and covered with the membranes in the tray. After incubation for 2 min at room temperature, the membrane was immediately exposed and images were captured using an immunoblot imaging system.
  • the source of tissue pH detection in the following experiments is as follows: Different cell lines were seeded into 24-well plates at the same initial concentration, and after incubation for 48 hours or 72 hours, the pH of the supernatant from different wells was measured using a cell culture biochemical analyzer. To determine the intracellular pH, the ratiometric fluorescent probe was diluted in culture medium (10 ⁇ M) and then added to different cultured cells, followed by incubation at 37°C for 1 hour. The cells were washed with PBS in a 4-well confocal chamber, and green and red fluorescence was captured under a Carl Zeiss LSM880 super-resolution microscope.
  • the sources of protein extraction and immunoblotting in the following experiments are as follows: RIPA buffer (150mM NaCl, 50mMTris-HCl, pH7.4, 10% glycerol, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1mM EDTA) supplemented with protease inhibitors and phosphatase inhibitor cocktails.
  • RIPA buffer 150mM NaCl, 50mMTris-HCl, pH7.4, 10% glycerol, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1mM EDTA
  • the membranes with different target proteins were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 hour at RT. Images were acquired by a dual-color infrared laser imaging system ODYSSEYCLx.
  • the mass spectrometry analysis method in the following experiments was as follows: Single cells from mouse tumor-adjacent mammary tissue and tumor tissue were collected and digested for 2 hours at 37°C with digestion medium containing 5% fetal bovine serum, 5 ⁇ g/ml insulin (I-1882; Sigma-Aldrich), 500 ng/ml hydrocortisone (H0888; Sigma-Aldrich), 300 U/ml collagenase III (S4M7602S, Worthington, Lakewood, NJ) and 100 U/ml hyaluronidase (H3506; Sigma-Aldrich), and then washed with HPSS (14170-112; Life Technologies).
  • digestion medium containing 5% fetal bovine serum, 5 ⁇ g/ml insulin (I-1882; Sigma-Aldrich), 500 ng/ml hydrocortisone (H0888; Sigma-Aldrich), 300 U/ml collagenase III (S4M7602S, Worthington, Lakewood,
  • the cells were washed twice with 1 ml Maxpar Cell Staining Buffer and 1 ml Maxpar water.
  • the cell concentration was adjusted to 2.5-5 x 10 5 / ml with a positive control bead buffer (EQTM four-element calibration, Cat. # 201078), and then data was collected on a mass spectrometer (Fluidigm).
  • the flow cytometry analysis method in the following experiments was as follows: Single cells were collected from nude mouse breast tumors, bone marrow, blood, spleen, and peritoneum, then treated with RBC lysis buffer and washed with PBS. The cells were resuspended in 100 ⁇ L PBS and incubated with the conjugated antibody on ice for 30 minutes. The cells were then washed twice and resuspended in PBS, and the data were acquired using a flow cytometer and analyzed using flow data processing software.
  • the preparation and treatment methods of nanoparticles in the following experiments are as follows:
  • the materials used to prepare the nanoparticle drugs were provided by Professor Dai Yunlu's laboratory. Polyethylene glycol and drugs were dissolved in methanol, and the mixture was added to a solution of deionized water/tetrahydrofuran and sonicated for 5 minutes. After the organic phase was removed by vacuum suction, the unencapsulated components were filtered using a 220 nm filter membrane, and the remaining solution was concentrated and stored in a dark bottle at 4 °C. The morphology of the nanoparticles was studied by transmission electron microscopy, and the UV-visible absorption spectrum was measured by a spectrophotometer.
  • Breast cancer associated gene 1 is a tumor suppressor gene whose germline mutations can lead to familial breast cancer.
  • Brca1 has many important functions, including transcriptional regulation, DNA damage repair, centrosome duplication, cell cycle checkpoints, chromatin remodeling, and protein ubiquitination.
  • Current studies have found that approximately 25% of mice with Brca1 deficiency develop breast tumors at 1.5 years of age, and approximately 20% of these mice have tumor metastases to distant organs.
  • Brca1 deficiency leads to many abnormalities in mammary gland development, such as blunted ductal morphogenesis, dysregulated genome-wide gene expression, enhanced discharge of DNA replication forks, and increased apoptosis; this can be partially supported by the loss of p53, Atm, Chk2, and 53PB1 function or the activation of certain oncogenes.
  • Breast cancer patients with Brca1 mutations also experience a high frequency of multi-organ metastasis. Therefore, the inventors believe that relevant research on Brca1 mutations is of great significance for the prevention and treatment of breast cancer and multi-organ metastasis of tumors.
  • the inventors first used immunofluorescence staining to label luminal epithelial cells (CK18), basal or myoepithelial cells (CK14) and basement membrane (collagen IV) in human breast cancer patients with or without Brca1 mutation and Brca1-MSK mouse model to detect the mammary double-layer structure composed of luminal and basal layers and surrounded by basement membrane, and obtained the results shown in Figure 1.
  • the marks in the figure are: WT indicates non-BRCA1 mutation, and MT indicates BRCA1 mutation.
  • Figure 1 A is a representative image of the tumor-adjacent breast of a breast cancer patient
  • Figure 1 C is a representative image of the tumor tissue of a breast cancer patient
  • Figure 1 A-D compared with non-BRCA1 mutations, the basal layer in the tumor-adjacent breast tissue and tumor tissue of breast cancer patients with BRCA1 mutations is severely missing.
  • Figure 1E is a representative image of the tumor-adjacent mammary gland of Brca1-MSK mice
  • Figure 1G is a representative image of the tumor tissue of Brca1-MSK mice
  • the experiment used CK18/Collagen IV co-staining, and calculated the percentage of the affected basal layer in the tumor-adjacent mammary gland and tumor tissue of the mice, and obtained the results shown in Figure 1F and Figure 1H, respectively.
  • the basal layer in the tumor-adjacent mammary gland tissue and tumor tissue of Brca1-MSK mice with BRCA1 mutation is severely missing. This shows that BRCA1 defect-related tumor occurrence has a greater impact on the structural integrity of the mammary gland than sporadic tumor occurrence.
  • the inventors used immunofluorescence staining to study the structural integrity of the mammary glands of 8-month-old and 10-month-old non-mutated and mutated Brca1-MSK mice, all of which were in the stage before or after tumor formation.
  • I-K of Figure 1 the mammary gland structure of non-BRCA1 mutant mice is more complete, while the mammary gland structure of mice after BRCA1 mutation is abnormal.
  • the 10-month-old mice were further experimented with. Representative images of tumor-adjacent mammary tissues of 10-month-old non-mutated mice and control mice (shown in L of Figure 1) were co-stained with CK18 and CK14, and the percentage of the destroyed basal layer was calculated (shown in M of Figure 1).
  • Cancer metastasis is a very complex process, and epithelial-mesenchymal transition (EMT) is acquired by malignant cells before metastasis.
  • EMT epithelial-mesenchymal transition
  • Examination of tumor-adjacent breast tissues of breast cancer patients with vimentin antibodies showed that higher levels of vimentin were detected in BRCA1, and more multi-organ metastases were observed in mutation carriers than in non-mutation carriers (Figure 2, H) in the Brca1-MSK mouse model ( Figure 2, D-G).
  • Krt19, Krt18, Krt5, and Krt14 in mammary epithelial tumor tissues of Brca1-MSK mice continued to increase during the process of differentiation, while markers of mammary epithelial differentiation, including Krt19, Krt18, Krt5, and Krt14, were significantly reduced in mammary tumor tissues of Brca1-MSK mice.
  • the inventors performed a cytokine array screening experiment using the sera of 6-month-old non-Brca1 mutant (WT) and Brca1 mutant (Brca1-MSK) mice, and obtained the results shown in FIG. 1 and FIG. 2 .
  • the real-time fluorescence quantitative PCR method was used to detect the mammary epithelial cell lines of G600 (Brca1-MT) and B477 (Brca1-WT), respectively.
  • the mRNA levels of 8 of them in G600 cells were significantly increased;
  • the mRNA levels of Sele, Sell, St8sia4 and St3gal1 were found to be increased in the mammary tissue of Brca1-MSK mice;
  • the B477 cell line the mRNA levels of St8sia4 and St3gal1 in G600 mammary epithelial cells of Brca1-MT were increased.
  • PSA polysialic acid
  • SLeA Sialyl-Lewis A
  • PSA antibodies to detect the level of polysialic acid (PSA) in mammary epithelial cells of mice and humans with or without BRCA1 defects, and obtained the results shown in Figure 3.
  • Figure 3 B-C is the detection result of mice, specifically, by IF staining, PSA was used to quantify the PSA distribution pattern (Figure 3 B-a), tumor adjacent mammary tissue (Figure 3 B-b) and tumor epithelial cells (Figure 3 B-c) of 10-month-old Brca1-MSK mice.
  • the inventors used a ratiometric fluorescent probe method to detect the pH values of mammary tissues of 8-month-old Brca1-MSK mice and WT mice of the same age, as well as tumors from Brca1-MSK and WT mice, and obtained the color intensity ratios as shown in Figure 3 F.
  • Figures 3 F-G the average pH values of mammary tissues and tumors of Brca1-MSK mice were 6.6 and 6.25, respectively, while the pH value of control mice was 7.4.
  • the inventors conducted tissue microarray analysis on 95 human breast cancer samples and obtained the results shown in Figure 3.
  • conventional IHC or IF staining was performed on breast cancer tissue using BRCA1 antibody and PSA (as shown in H of Figure 3), and PSA in breast cancer patients with high expression of BRCA1, medium expression of BRCA1 and low expression of BRCA1 was quantified (as shown in I of Figure 3).
  • the expression of Brca1 is negatively correlated with the expression of PSA.
  • the inventors proposed a new method for neutralizing the acidic tumor microenvironment, which includes using a sialyltransferase inhibitor to neutralize the acidic tumor microenvironment.
  • Mammary fat blocks were implanted with B477 cells carrying OE-St8sia4 or G600 cells carrying sgSt8sia4, and empty and The white control was used to examine the changes in pH values, and the results were shown in Figure 3.
  • the tumor images in B477 cells without or with OE-St8sia4 are shown in Figure 3 J
  • the tumor slice images using the ratio fluorescence probe are shown in Figure 3 K
  • the interstitial pH is shown in Figure 3 L.
  • the interstitial tissue in the breast tumor tissue with OE-St8sia4 has a larger tumor volume and a lower pH value; conversely, the tumor images in G600 cells without or with sgSt8sia4 are shown in Figure 3 M, the tumor slice images using the ratio fluorescence probe are shown in Figure 3 N, and the interstitial pH is shown in Figure 3 O. As shown in Figure 3 M, the tumor volume is smaller.
  • FIG4C higher St8sia4 expression in human breast cancer patients is associated with poorer survival outcomes in breast cancer patients.
  • St8sia4 was overexpressed in the Brca1-MT545 cell line with low metastatic ability and the EMT6 cell line with moderate metastatic ability, and the integrity of the adjacent mammary glands was examined on days 7, 14, and 26 of tumor growth.
  • CK14 and CK18 antibodies By co-staining with CK14 and CK18 antibodies, in the EMT6 mouse model, destroyed adjacent mammary gland structures were observed on day 14, and more basal and luminal layer damage could be detected on day 26.
  • the sialyltransferase inhibitor 3Fax-P-Neu5Ac (STi) was used, the damaged mammary gland structure could be restored, as shown in Figure 5B.
  • the inventors used two cell lines, 628W and 545, for experiments, wherein the 628W cell line has high metastatic ability and expresses higher levels of St8ia4 and St3gal1; the 545 cell line has low metastasis and has low levels of St8ia4 and St3gal1 expression.
  • St8sia4 and St3gal1 were first overexpressed in 545 cells, and St8sia4 and St3gal1 in 628W cells were knocked out by expression of sgSt8sia4 and St3gal1.
  • the migration state of these cells was studied in vitro, and the research methods were fat pad implantation and tail vein injection in vivo.
  • the transwell migration assay detected much more migration from 545 cells with OE-St8sia4 and St3gal1, indicating that when St8sia4 and St3gal1 are overexpressed, cell morphology may change.
  • EMT6 with OE-St8sia4 acquired EMT characteristics
  • EMT6 and B477 cells with OE-St8sia4 acquired invasive characteristics in 3D culture.
  • PSA polysialic acid
  • Vegfa and VegfIL6 are both transcriptional regulators involved in angiogenesis and tumor cell invasion, and Vegfa signaling can stimulate the production of TGF- ⁇ in A549 cells. Therefore, the inventors used protein immunoblotting to conduct experiments and found that the protein levels of Vegfa and VegfIl6 increased compared with the control group, and the levels of TGF- ⁇ 1 and its downstream proteins pSmad3/total-Smad3 and pStat3/totalStat3 in the precancerous breast (G in FIG. 7 ) and tumor tissue (H in FIG. 7 ) of Brca1-MSK mice were increased.
  • OE-Vegfa or OE-Il6 in G477 cells increased their own expression together with TGF- ⁇ , St8sia4 and St3gal1, whereas KO of Vegfa or Il6 reduced the expression levels of all these cells (as shown in Figure 7 I). Similar results were obtained in EMT6 and MDA-MB-231 cells (as shown in Figure 8 D-E).
  • TGF- ⁇ could induce the expression of St8sia4 and St3gal1 and other sialyltransferases in WT and Brca1-MT cells (as shown in J of FIG7 and F-G of FIG8), and the protein levels of St8sia4 and St3gal1 were reduced after treatment with TGF- ⁇ inhibitor (as shown in K of FIG7).
  • TIME tumor immunosuppressive microenvironment
  • the inventors used antibodies to CD45, CD11b, Ly6G and Ly6C, CD3, CD4 and CD8 to various immune cell populations in breast tumor tissues from Brca1-MSK mice, as well as EMT6 parental cells, OE-St8sia4 cells, and OE-St8sia4/sgSt8sia4 cells.
  • Mass spectrometry flow analysis was performed on the 14th and 26th days after implantation, and the results of A-B in Figure 9 were obtained.
  • PMN-MDSC polymorphonuclear myeloid-derived suppressor cells
  • M-MDSC mononuclear myeloid-derived suppressor cells
  • the inventors further analyzed bulk RNA-seq from bones, blood, spleen, and peritoneum of WT mice, Brca1-MSK mice without tumors, and Brca1-MSK mice carrying primary breast tumors, and then compared the expression patterns obtained in the TCGA database.
  • B cells, T cells, and dendritic cells (DCs) began to decrease, while immune cells from the myeloid cell lineage began to increase in the blood and spleen of Brca1 mutant mice without tumors, and further increased in Brca1 mutant tumor-bearing mice (M in Figure 10).
  • Gene expression of immune cells further illustrates the increase in macrophages and monocytes in bones, blood, and spleen, especially in the peritoneum of Brca1-MSK compared with WT mice (N in Figure 10).
  • the characteristic gene expression pattern in immune cells showed highly expressed immunosuppressive genes, including TGF- ⁇ 1, Smad3, Mmp9, Arg1, Arg2, Il10, Il1 ⁇ and PD-L1 in blood, spleen and peritoneum (O in Figure 10), indicating that Brca1 deficiency leads to a stronger immunosuppressive environment in those organs.
  • the above gene expression analysis was further supported by the increased levels of TGF- ⁇ signaling proteins by Western blotting (P in Figure 10).
  • the acidic tumor microenvironment (ATPME) induced by high sialylation may damage the structure of the mammary bilayer, thereby promoting the entry of malignant cells into blood vessels through the extracellular matrix (ECM).
  • ECM extracellular matrix
  • the inventors next studied whether the neutralization of ATPME could inhibit tumor metastasis.
  • 3Fax-P-Neu5Ac (STi) is a naturally occurring hyperacetylated analog of Neu5Ac that inhibits sialyltransferase function.
  • Stattic an inhibitor that can disrupt TGF- ⁇ signaling induced by pStat3 and pSmad3 activation, because the levels of proteins pStat3 and pSmad3 are both elevated in cells with low Brca1 expression or high Vegfa/Il6 expression.
  • these two drugs were used for monotherapy and combination therapy by intraperitoneal delivery (as shown in A of Figure 12).
  • the pH of tumor tissue was measured in the same group of mice by using a ratiometric fluorescent probe.
  • the ratio of green to red was much larger in the control group (PBS and PEG treatment groups) compared with the drug treatment group (Figure 11G-H) and after treatment with Stattic or STi, the pH of breast tissue recovered from approximately 6.5 to above 6.8 to 7.0 ( Figure 10H).
  • the inventors used STi, Stattic or double treatment with STi and Stattic, and co-stained tumors and tumor-adjacent breasts with CK18/CollagenIV or CK18/CK14 antibodies.
  • the H-J data of Figure 11 show that when treated with a single or combined drug, more collagen bundles are formed in the tumor tissue detected by CK18/Collagen IV staining in the mammary basement membrane compared with the parental control tumor tissue (D of Figure 11), which can prevent tumor cells from escaping from the original position; by double staining CK14 and CK18 antibodies (J of Figure 11), more mammary ducts with complete basal cell layers are observed after treatment with a single drug or two drugs together (I-J of Figure 11), indicating that the acidic conditions in the mammary tissue caused by high sialylation can be specifically neutralized by nanoparticles containing pan-inhibitors of sialyltransferases and inhibitors of TGF- ⁇ signaling molecules.
  • CD3-positive cells were significantly reduced, accompanied by OE-St8sia4 in breast tumor tissues with clear PD-1 positive immune cells for 7 days ( Figure 13 A), while very strong PD-L1-St8sia4 was detected in the parental tumors and OE tumors of the control for 7 days ( Figure 13 B), indicating that ATPME induced by OE-St8sia4 triggered T cell death.
  • the inventors have determined that breast cancer caused by Brca1 deficiency has a sialyltransferase-mediated mechanism that can induce an acidic tumor microenvironment (ATPME) in Brca1 mutations and most Brca1 low-expressing breast cancers, and that high sialylation caused by polysialic acid (PSA) damages the bilayer structure of the mammary epithelium and establishes an acidic tumor microenvironment (ATPME) and tumor immunosuppressive microenvironment (TIME) that contribute to cancer metastasis.
  • ATPME acidic tumor microenvironment
  • TIME tumor immunosuppressive microenvironment
  • the carcinogenic effect of Vegfa/Il6 induced by Brca1 deficiency or insufficiency activates TGF ⁇ -St8sia4 signaling, increases the accumulation of polysialic acid on the mammary epithelial membrane, and forms a malignant niche that promotes cell escape from immune surveillance, ultimately leading to tumor metastasis and resistance to ⁇ PD-1 treatment. Therefore, the inventors proposed to use sialyltransferase inhibitors to prevent or treat breast cancer caused by Brca1 deficiency.
  • sialyltransferase inhibitors By using sialyltransferase inhibitors, the expression of sialyltransferase can be reduced, and the acidic tumor microenvironment formed can be neutralized, thereby achieving the effect of treating or even reversing the basement membrane defects of breast epithelial cells.
  • sialyltransferase inhibitors with drugs PD-1 or Stattic, ATPME can be further neutralized and tumor growth and metastasis can be blocked, greatly reducing the chance of tumor metastasis.
  • the present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment, and relates to the technical field of tumor treatment.
  • the present disclosure provides an application of a sialyltransferase inhibitor in the preparation of a drug for neutralizing an acidic tumor microenvironment.
  • the sialyltransferase inhibitor can neutralize the acidic tumor microenvironment and establish an ecological barrier for tumor metastasis, providing a new approach for the prevention and treatment of tumors, and having excellent industrial applicability.

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Abstract

La présente invention se rapporte au domaine de la technologie du traitement des tumeurs, et concerne plus particulièrement l'utilisation d'un inhibiteur de sialyl-transférase dans la préparation d'un médicament pour neutraliser un microenvironnement tumoral acide. En fournissant l'utilisation de l'inhibiteur de sialyl-transférase dans la préparation du médicament pour neutraliser le microenvironnement tumoral acide, l'inhibiteur de sialyl-transférase peut neutraliser le microenvironnement tumoral acide et établir une barrière écologique contre la métastase tumorale, fournissant ainsi une nouvelle manière de prévenir et de traiter des tumeurs.
PCT/CN2023/128090 2023-05-09 2023-10-31 Utilisation d'un inhibiteur de sialyl-transférase dans la préparation d'un médicament pour neutraliser un microenvironnement tumoral acide Pending WO2024230090A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016071431A1 (fr) * 2014-11-06 2016-05-12 Stichting Katholieke Universiteit Combinaison pour traitement anticancéreux
CN108026138A (zh) * 2015-08-27 2018-05-11 中央研究院 唾液酸转移酶抑制剂及其用途
CN109364252A (zh) * 2018-11-21 2019-02-22 南京大学 抑制ifn-i至arg1诱导通路在制备抗肿瘤药物组合物中的应用
US20210107931A1 (en) * 2018-01-29 2021-04-15 Stichting Katholieke Universiteit New potent sialyltransferase inhibitors
US20210186999A1 (en) * 2017-10-31 2021-06-24 National University Of Ireland Galway Method for treatment of cancer
CN116549646A (zh) * 2023-05-09 2023-08-08 澳门大学 唾液酸转移酶抑制剂在制备中和酸性肿瘤微环境的药物中应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016071431A1 (fr) * 2014-11-06 2016-05-12 Stichting Katholieke Universiteit Combinaison pour traitement anticancéreux
CN108026138A (zh) * 2015-08-27 2018-05-11 中央研究院 唾液酸转移酶抑制剂及其用途
US20210186999A1 (en) * 2017-10-31 2021-06-24 National University Of Ireland Galway Method for treatment of cancer
US20210107931A1 (en) * 2018-01-29 2021-04-15 Stichting Katholieke Universiteit New potent sialyltransferase inhibitors
CN109364252A (zh) * 2018-11-21 2019-02-22 南京大学 抑制ifn-i至arg1诱导通路在制备抗肿瘤药物组合物中的应用
CN116549646A (zh) * 2023-05-09 2023-08-08 澳门大学 唾液酸转移酶抑制剂在制备中和酸性肿瘤微环境的药物中应用

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