Application of monocarboxylic acid transporter 1 inhibitor in preparation of medicine for preventing, relieving and / or treating pancreatic cancer

Abstract

The invention relates to application of a monocarboxylic acid transporter 1 inhibitor in preparation of a medicine for preventing, relieving and / or treating pancreatic cancer. By using the MCT1 inhibitor, the lactic acid level in the pancreatic stellate cells PSCs is effectively reduced, so that the lactic acid of Vps34 is inhibited, and the activation process of autophagy-dependent PSCs mediated by the lactic acid is blocked. By inhibiting the activation of the PSCs, the MCT1 inhibitor further reduces the secretion of downstream chemokines CXCL9 and CXCL10. The CXCL9 and the CXCL10 are key factors for inducing CD8 + T cells to up-regulate PD-1 expression through CXCR3 and STAT3 signal channels, so that the MCT1 inhibitor substantially cuts off an induction signal causing CD8 + T cell depletion on the upstream, further, the in-vivo growth and in-situ tumorigenesis ability of pancreatic cancer can be inhibited, and the purpose of preventing, relieving and / or treating pancreatic cancer is achieved.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the use of a monocarboxylic acid transporter 1 inhibitor in the preparation of a drug for the prevention, relief and / or treatment of pancreatic cancer. Background Technology

[0002] Pancreatic cancer (PC) is a highly malignant tumor of the digestive system, often referred to as the "king of cancers." Although cancer immunotherapy has shown significant efficacy in various solid tumors, pancreatic cancer, due to its highly immunosuppressive tumor microenvironment (TME), responds poorly to existing immune checkpoint blockade (ICB) therapies, including PD-1 (programmed death-1) / PD-L1 (programmed cell death ligand 1) inhibitors and CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) inhibitors, resulting in limited treatment efficacy.

[0003] Pancreatic stellate cells (PSCs), as the main source of pancreatic cancer-associated fibroblasts (CAFs), play a crucial role in maintaining the normal physiological structure of the pancreas. However, activated pancreatic stellate cells (PSCs) can mediate the formation of an immunosuppressive microenvironment in pancreatic cancer through matrix reprogramming, recruitment of myeloid-derived suppressor cells (MDSCs), and induction of M2 macrophage polarization. Therefore, identifying novel targets and related strategies that can regulate the activation of pancreatic stellate cells (PSCs) is of significant clinical importance for improving the efficacy of immunotherapy for pancreatic cancer. Summary of the Invention

[0004] This application provides the use of a monocarboxylic acid transporter 1 inhibitor in the preparation of a drug for the prevention, relief and / or treatment of pancreatic cancer. It can inhibit the activity of pancreatic stellate cells (PSCs), thereby inhibiting the in vivo growth and in situ tumorigenesis of pancreatic cancer, achieving the purpose of preventing, alleviating and / or treating pancreatic cancer.

[0005] In a first aspect, embodiments of this application provide the use of a monocarboxylic acid transporter 1 inhibitor in the preparation of a medicament for the prevention, relief, and / or treatment of pancreatic cancer.

[0006] In conjunction with the first aspect, in one embodiment, the monocarboxylic acid transporter 1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof: .

[0007] In conjunction with the first aspect, in one embodiment, the monocarboxylic acid transporter 1 inhibitor reduces lactate levels in pancreatic stellate cells and inhibits lactation of Vps34, a key regulator of autophagy.

[0008] Secondly, embodiments of this application provide a medicament for the prevention, relief, and / or treatment of pancreatic cancer, the medicament comprising an active substance, the active substance comprising a monocarboxylic acid transporter 1 inhibitor.

[0009] In conjunction with the second aspect, in one embodiment, the monocarboxylic acid transporter 1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof: .

[0010] In conjunction with the second aspect, in one embodiment, the monocarboxylic acid transporter 1 inhibitor reduces lactate levels in pancreatic stellate cells and inhibits lactation of Vps34, a key regulator of autophagy.

[0011] In conjunction with the second aspect, in one embodiment, the drug further includes a pharmaceutically acceptable carrier.

[0012] In conjunction with the second aspect, in one embodiment, the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable solid or liquid excipients.

[0013] In conjunction with the second aspect, in one embodiment, the drug further includes a buffer solution selected from acetate, citrate, borate, or phosphate.

[0014] In conjunction with the second aspect, in one embodiment, the dosage form of the drug is a tablet, capsule, powder, suppository, injection, or nasal spray.

[0015] The beneficial effects of the technical solution provided in this application include: The tumor microenvironment (TME) of pancreatic cancer contains a large amount of lactate produced by tumor cell glycolysis. MCT1 inhibitors specifically block the MCT1 transporter protein on the surface of pancreatic stellate cell (PSC) membranes, thereby cutting off the pathway for lactate to enter PSCs. The applicant's research found that lactate entering PSCs leads to lysine lactation modification of the key autophagy regulator Vps34 (specifically at K356 and K781). The use of MCT1 inhibitors effectively reduced lactate levels within PSCs, thereby inhibiting Vps34 lactation and blocking the lactation-mediated autophagy-dependent PSC activation process. Activated pancreatic stellate cells (PSCs) are a major source of immunosuppressive signals in the TME. By inhibiting PSC activation, MCT1 inhibitors further reduced the secretion of downstream chemokines CXCL9 and CXCL10. Since CXCL9 and CXCL10 are key factors that induce the upregulation of PD-1 expression in CD8+ T cells through the CXCR3 and STAT3 signaling pathways, MCT1 inhibitors essentially cut off the inducing signals that lead to CD8+ T cell exhaustion upstream, thereby inhibiting the in vivo growth and in situ tumorigenesis of pancreatic cancer, achieving the purpose of preventing, alleviating and / or treating pancreatic cancer. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a fluorescence staining pattern provided in Example 1 of this application; Figure 2 This is a statistical chart of mouse tumor weight provided in Example 2 of this application; Figure 3 This is a statistical chart of mouse weight provided in Example 2 of this application. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0019] Metabolic abnormalities are a key characteristic of many malignant tumors, including pancreatic cancer. Pancreatic cancer cells undergo metabolic reprogramming, manifesting as aerobic glycolysis, leading to excessive accumulation of lactate in the tumor microenvironment. The applicant discovered that lactate participates in the tumor microenvironment (TME) remodeling of various solid tumors, such as activating fibroblast CAFs by reducing the NAD+ / NADH ratio in prostate cancer, or promoting macrophage M2 polarization in pancreatic cancer. Furthermore, lactate can increase PD-1 expression in Treg cells by promoting NFAT1 nuclear translocation. Although lactate participates in immune reprogramming within the TME, whether it mediates the interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), and the specific molecular mechanisms involved, remains unknown, limiting the progress in developing effective antitumor drugs targeting this metabolic pathway.

[0020] The applicant's research found that lactate promotes the progression and deterioration of pancreatic cancer by upregulating histone H3K18 or non-histone lactylation. In the related research of this application, pancreatic cancer cells, as the main source of lactate in the TME (tumor endothelial cell line), were found to enter PSCs via the lactate transporter MCT1 (monocarboxylic acid transporter 1). Lactate entering PSCs induces lysine lactylation modification of the key autophagy regulator Vps34 at K356 and K781 sites, thereby activating PSCs through an autophagy-dependent mechanism. Activated PSCs then secrete the chemokines CXCL9 and CXCL10, which activate the CXCR3 and STAT3 signaling pathways and upregulate PD-1 expression in CD8+ T cells, ultimately leading to immune escape.

[0021] The applicant's research found that by specifically inhibiting the lactate transporter MCT1 with a monocarboxylic acid transporter 1 inhibitor, lactate entry into pancreatic cysts (PSCs) and its mediated Vps34 lactation and PSC activation can be blocked. This inhibitor can suppress the in vivo growth and in situ tumorigenesis of pancreatic cancer, and may become a potential candidate for targeted therapy. This application has potential clinical value for finding new therapeutic targets for pancreatic cancer and improving the treatment outcomes for pancreatic cancer patients.

[0022] In view of this, embodiments of this application provide the use of a monocarboxylic acid transporter 1 inhibitor in the preparation of a medicament for the prevention, relief and / or treatment of pancreatic cancer.

[0023] In the technical solution of this application, the core function of the specific inhibitor of monocarboxylic acid transporter 1 (MCT1) is not to directly kill tumor cells, but to inhibit the formation of the highly immunosuppressive tumor microenvironment (TME) from the source by blocking the metabolic signal transduction of pancreatic stellate cells (PSCs). The specific mechanism is as follows: (1) The tumor microenvironment (TME) of pancreatic cancer contains a large amount of lactic acid produced by glycolysis of tumor cells. MCT1 inhibitors specifically block the MCT1 transporter protein on the surface of pancreatic stellate cells (PSCs), thereby cutting off the channel for lactic acid to enter pancreatic stellate cells (PSCs).

[0024] (2) The applicant's research found that lactate entering pancreatic stellate cells (PSCs) leads to lysine lactation modification of Vps34, a key regulator of autophagy (specifically at sites K356 and K781). The use of MCT1 inhibitors effectively reduced lactate levels in pancreatic stellate cells (PSCs), thereby inhibiting Vps34 lactation and blocking the lactation-mediated autophagy-dependent PSC activation process.

[0025] (3) Activated pancreatic stellate cells (PSCs) are the main source of immunosuppressive signals in the tumor microenvironment (TME). By inhibiting PSC activation, MCT1 inhibitors further reduce the secretion of downstream chemokines CXCL9 and CXCL10. Since CXCL9 and CXCL10 are key factors that induce CD8+ T cells to upregulate PD-1 expression through the CXCR3 and STAT3 signaling pathways, MCT1 inhibitors essentially cut off the induction signals that lead to CD8+ T cell exhaustion upstream, thereby inhibiting the in vivo growth and in situ tumorigenesis of pancreatic cancer, achieving the purpose of preventing, alleviating and / or treating pancreatic cancer.

[0026] As an example, the monocarboxylic acid transporter 1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof: .

[0027] The compound shown in formula (I) above is also known as AZD3965, and its CAS number is 1448671-31-5.

[0028] Furthermore, embodiments of this application also provide a medicament for the prevention, relief, and / or treatment of pancreatic cancer, the medicament comprising an active substance, the active substance comprising a monocarboxylic acid transporter 1 inhibitor.

[0029] The monocarboxylic acid transporter 1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof: .

[0030] Furthermore, the drug also includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from pharmaceutically acceptable solid or liquid excipients.

[0031] A wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0032] Furthermore, the drug also includes a buffer solution selected from acetate, citrate, borate, or phosphate.

[0033] Furthermore, the dosage form of the drug is a tablet, capsule, powder, suppository, injection, or nasal spray.

[0034] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0035] To form capsules, the monocarboxylic acid transporter 1 inhibitor provided in this application can be mixed with a diluent and a flow aid, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, the monocarboxylic acid transporter 1 inhibitor provided in this application, can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and flow aids used to prepare tablets of the monocarboxylic acid transporter 1 inhibitor provided in this application can also be used to prepare capsules of the monocarboxylic acid transporter 1 inhibitor provided in this application.

[0036] To formulate the monocarboxylic acid transporter 1 inhibitor provided in this application into an injectable preparation, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, cosolvents, pH adjusters, and osmotic pressure adjusters can be added. Solubilizers or cosolvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure adjusters can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder for injection, mannitol, glucose, etc., can also be added as a supporting agent. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, or other additives can be added to the pharmaceutical preparation. To achieve the intended therapeutic effect, the drug of this application can be administered using any known method of administration.

[0037] Example 1: Inhibitory effect of AZD3965 on pancreatic stellate cell activation The pancreatic stellate cells used were extracted from the pancreatic tissue of SPF-grade C57 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.).

[0038] Resuscitation and culture of primary pancreatic stellate cells (PSCs): The pancreas was isolated from mice using aseptic surgical techniques and cut into 1 mm pieces. 3 The tissue fragments were collected and then digested for 40 minutes in a digestion solution containing collagenase IV (5 mg / ml, Sigma-Aldrich, catalog number 12352204). The pancreatic tissue was then aspirated by pipette and filtered through a 70µm cell sieve. After centrifugation (1000 rpm, 3 minutes), the supernatant was cultured in DMEM / F-12 medium containing 20% ​​fetal bovine serum (FBS). The medium was changed continuously for three days until all suspended cells were removed, leaving only adherent cells.

[0039] After removing the cryovials of primary PSCs from the liquid nitrogen tank, quickly place them in a 37°C water bath and agitate to rapidly thaw them. To avoid the DMSO added during cell cryopreservation affecting cell growth, transfer the thawed cell solution to centrifuge tubes pre-filled with fresh complete culture medium, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Transfer the cells to culture flasks, add DMEM / F12 medium containing 10% fetal bovine serum, mix well, and incubate at 37°C in a 5% CO2 incubator according to standard procedures. Use the cells for subsequent experiments when they are in good growth condition (no obvious tentacles, similar cell morphology, and natural extension) and have reached 80% confluence.

[0040] Experimental steps: Cell inoculation and spreader preparation: Primary PSCs in the logarithmic growth phase were digested with trypsin and dispersed in culture medium to prepare a single-cell suspension. Sterile cell spreaders were pre-placed in 24-well plates, and the cell suspension was inoculated into each well at a density of 2 × 10⁶ cells / well. 4 Add 500 μL of culture medium to each cell and incubate at 37°C in an incubator containing 5% CO2 and saturated humidity for 24 hours to allow the cells to fully adhere and spread.

[0041] Grouping and Drug Action: After cells adhered for 24 hours, the old culture medium was removed from each well using a pipette. According to the experimental design, the experimental groups were given fresh culture medium containing different concentrations of lactate or specific inhibitors, while the control group received an equal volume of fresh complete culture medium. Cells were then cultured in an incubator for another 24 hours to induce or inhibit cell activation. Details are as follows: Lactic acid group: Fresh culture medium containing 15 mmol / L lactic acid was added.

[0042] Inhibitor group: Fresh culture medium containing 5 mmol / L of MCT1 inhibitor was added.

[0043] Lactate + Inhibitor Group: Fresh culture medium containing 15 mmol / L lactate and 5 mmol / L MCT1 inhibitor was added.

[0044] Control group: Add an equal amount of fresh complete culture medium.

[0045] Fixation and blocking: Discard the culture medium in the wells and wash the cell slides three times with PBS. Add 4 wt% paraformaldehyde (Shanghai Beyotime Biotechnology Co., Ltd.) to each well and fix for 30 min at 4°C. Discard the fixative, wash with PBS, add Triton X-100 immunostaining permeabilization buffer (Shanghai Beyotime Biotechnology Co., Ltd.), and incubate at room temperature for an appropriate time. Then discard the permeabilization buffer, wash with PBS, and add 1 wt% BSA solution to block for 60 min at room temperature to prevent nonspecific binding.

[0046] Immunofluorescence staining and detection: Discard the blocking solution, add α-SMA primary antibody working solution (14395-1-AP, Wuhan Sanying Biotechnology Co., Ltd.) diluted 1:200 (v / v) to each well, and incubate overnight in a humidified chamber at 4°C. The next day, remove the slides and wash three times with TBST buffer, 5 min each time. Then add Cy3-labeled fluorescent secondary antibody diluted 1:1000 (v / v) and incubate at room temperature in the dark for 60 min. Finally, counterstain the cell nuclei with DAPI staining solution. After washing with PBS, remove the slides, mount them, and observe and acquire images using an Olympus fluorescence microscope. The intensity of α-SMA red fluorescence represents the activation level of PSCs.

[0047] according to Figure 1It can be seen that MCT1 inhibitors have a significant inhibitory effect on lactate-induced pancreatic stellate cell activation.

[0048] Example 2: Treatment and toxic side effects of AZD3965 on mice with orthotopic pancreatic cancer.

[0049] This experiment used SPF-grade C57 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), which were 4-6 weeks old, male, and weighed 18-20g. All animal experimental procedures complied with the guidelines for laboratory animal care and use.

[0050] The pancreatic cancer cell line KPC (purchased from Cyagen Biotech) was injected into the pancreas of mice via in situ injection. Since in situ tumor formation was performed, this group was designated as the cancer group. The other group was not implanted with tumors and was designated as the normal group because in situ tumor formation was not performed. The normal group was only observed for drug toxicity and side effects.

[0051] The MCT1 inhibitor was prepared into a solution with a concentration of 1 mg / mL using physiological saline, filtered through a 0.22 μm microporous membrane, and stored under sterile conditions for later use.

[0052] The cancer group and the normal group were further subdivided into four groups in the same manner: C57 mice were randomly divided into four groups: control group, inhibitor group 1, inhibitor group 2, and inhibitor group 3, and administered the drug via intraperitoneal injection every two days. The control group was injected with saline; inhibitor groups 1, 2, and 3 were injected with 100 mg / kg of MCT1 inhibitor.

[0053] For the four subgroups of cancer, the survival status of mice was observed and the survival rate was recorded every day. On the 40th day, the mice were euthanized by cervical dislocation and the weight of the tumor was measured.

[0054] Mouse survival rates are shown in Table 1 below, and tumor weight is as follows: Figure 2 As shown.

[0055] Table 1

[0056] As shown in Table 1 above, compared with the control group, MCT1 inhibitors improved the survival rate of mice with pancreatic tumors. The survival rate of mice in inhibitor groups 1, 2, and 3 was above 90% after 40 days of treatment, especially in inhibitor group 1, where the survival rate was 100%. Figure 2 As shown, the final tumor weight in inhibitor groups 1, 2, and 3 was significantly lower than that in the control group. In conclusion, the results indicate that MCT1 inhibitors have a significant inhibitory effect on tumor growth.

[0057] For the four subgroups further subdivided into the normal group, see [link to relevant documentation]. Figure 3 As shown, the body weight of mice in each group was measured on day 40 of the experiment, according to... Figure 3 The mouse weight statistics shown in the figure indicate that, compared with the control group, there was no significant difference in weight among the mice in the inhibitor 1, inhibitor 2 and inhibitor 3 groups treated with the MCT1 inhibitor, suggesting that this MCT1 inhibitor has few toxic side effects and strong safety.

[0058] Therefore, MCT1 inhibitors can significantly inhibit the activation of pancreatic stellate cells. In vivo animal experiments have shown that MCT1 inhibitors have few toxic side effects, strong safety, and can significantly inhibit the progression of pancreatic cancer.

[0059] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. The use of a monocarboxylic acid transporter 1 inhibitor in the preparation of a medicament for the prevention, relief and / or treatment of pancreatic cancer, wherein the monocarboxylic acid transporter 1 inhibitor reduces lactate levels in pancreatic stellate cells and inhibits lactation of Vps34, a key regulator of autophagy.

2. The use of the monocarboxylic acid transporter 1 inhibitor as described in claim 1 in the preparation of a medicament for the prevention, alleviation, and / or treatment of pancreatic cancer, characterized in that: The monocarboxylic acid transporter 1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof.

3. A drug for the prevention, relief, and / or treatment of pancreatic cancer, characterized in that: The drug comprises an active substance, which includes a monocarboxylic acid transporter 1 inhibitor that reduces lactate levels in pancreatic stellate cells and inhibits lactation of Vps34, a key regulator of autophagy.

4. The medicament for preventing, alleviating, and / or treating pancreatic cancer as described in claim 3, characterized in that: The monocarboxylic acid transporter 1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof.

5. The medicament for preventing, alleviating, and / or treating pancreatic cancer as described in claim 3, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

6. The medicament for preventing, alleviating, and / or treating pancreatic cancer as described in claim 5, characterized in that: Pharmaceutically acceptable carriers are selected from pharmaceutically acceptable solid or liquid excipients.

7. The medicament for preventing, alleviating, and / or treating pancreatic cancer as described in claim 5, characterized in that: The drug also includes a buffer solution selected from acetate, citrate, borate or phosphate.

8. The medicament for preventing, alleviating, and / or treating pancreatic cancer as described in claim 3, characterized in that: The dosage form of the drug is tablet, capsule, powder, suppository, injection, or nasal spray.

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