Pharmaceutical composition containing carboxamide triazole or pharmaceutically acceptable salt thereof and application of pharmaceutical composition in preparation of medicine for preventing or treating fibrosis-related diseases
By combining carboxytriazole orotate with PD-1 antibody, the problems of insufficient penetration of chemotherapy drugs and limited immune response in solid tumors were solved, thereby reducing tumor collagen deposition and improving the efficacy of chemotherapy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-03-10
AI Technical Summary
Current treatment strategies suffer from insufficient penetration of chemotherapy drugs and limited immune response in solid tumors, especially due to the formation of a dense matrix barrier caused by collagen deposition from cancer-associated fibroblasts, which restricts the penetration of chemotherapy drugs and immune cells, leading to decreased efficacy.
The combination of carboxytriazole orotate (CTO) and immune checkpoint inhibitors (such as PD-1 antibodies) can synergistically reduce tumor collagen deposition, promote the penetration of chemotherapy drugs, and enhance anti-tumor efficacy by targeting matrix remodeling and immune regulation.
It significantly reduces tumor collagen deposition, improves the penetration of chemotherapy drugs in tumor tissue, enhances the effect of chemotherapy, and further enhances the anti-tumor effect by combining with chemotherapy drugs such as gemcitabine.
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Figure CN121622908A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition comprising carboxytriazole or a pharmaceutically acceptable salt thereof, and its use in the preparation of medicaments for the prevention or treatment of fibrosis-related diseases. Background Technology
[0002] Malignant tumors are a major disease that seriously threatens national health, not only leading to a decline in patients' quality of life but also imposing a heavy burden on families and society. A thorough understanding of the mechanisms of tumor development and progression, and the development of highly effective, low-toxicity, and readily accessible anti-tumor drugs, are urgent needs for safeguarding public health.
[0003] In recent years, treatment strategies targeting the tumor microenvironment (TME) have emerged as a new direction for overcoming tumor drug resistance. The core of these strategies lies in disrupting the biological support system upon which tumor cells depend for survival. The therapeutic efficacy of solid tumors has long been limited by the barrier effect of the TME. In stromal-rich tumors, such as pancreatic ductal adenocarcinoma, the extensive fibrosis and collagen deposition significantly limit the effective penetration and anti-tumor effects of chemotherapy drugs. Traditional treatment strategies largely focus on the tumor cells themselves, while tumor immunotherapy, represented by immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) and cell therapies (such as CAR-T), although hailed as the "third revolution" in cancer treatment, still faces the following challenges: Limitations of efficacy: The immunosuppressive microenvironment in solid tumors, especially cancer-associated fibroblasts (CAFs), leads to insufficient response rates in most patients; Safety risks: PD-1 monoclonal antibodies may induce immune-related hepatitis / pneumonia, and CAR-T therapy carries the risk of cytokine release syndrome and neurotoxicity; Metabolic interference: Immunosuppressive metabolites in the microenvironment (such as kynurenine produced by the IDO1 pathway) can directly inhibit T cell function, and similar mechanisms are also widely present in the process of resistance to non-immunotargeted therapies.
[0004] The abundant collagen deposits generated by coronary artery follicles (CAFs) form a dense matrix barrier, significantly limiting the penetration of chemotherapeutic drugs and immune cells. Furthermore, collagen cross-linking and remodeling processes further enhance the physical strength of this barrier, leading to decreased drug efficacy and low immunotherapy response rates. Current treatment methods neglect the limitations imposed by matrix structure on drug efficacy; therefore, directly increasing chemotherapeutic drug penetration and efficacy by altering the matrix structure is a more direct treatment approach.
[0005] Carboxytriazole orotate (CTO) is an oxidative phosphorylation (OXPHOS) inhibitor that has shown significant potential in regulating tumor metabolism and stromal cell status. Meanwhile, immune checkpoint inhibitors (such as PD-1 antibodies) can activate anti-tumor immune responses, but their efficacy in solid tumors still faces the bottleneck of microenvironment suppression.
[0006] The inventors have discovered that, in addition to regulating tumor metabolism, the mechanism of action of CTO on tumor matrix structure has not been fully utilized. They found that CTO can not only directly inhibit collagen synthesis and cross-linking in fibroblasts in vitro, but also significantly reduce intratumoral collagen deposition in vivo, and produce a synergistic effect when used in combination with PD-1 antibodies, enhancing the penetration of chemotherapy drugs and anti-tumor efficacy. Summary of the Invention
[0007] This invention aims to address the bottlenecks of insufficient drug penetration and limited immune response in solid tumors through a combined strategy of targeting matrix remodeling and immune regulation, and to further expand the therapeutic potential of CTO in fibrotic diseases. Based on this, this invention provides a pharmaceutical composition comprising carboxytriazole or a pharmaceutically acceptable salt thereof, and its use in the preparation of medicaments for the prevention or treatment of fibrosis-related diseases.
[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of fibrosis-related diseases, said pharmaceutical composition comprising carboxytriazole or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.
[0009] Alternatively, in the above pharmaceutical composition, the pharmaceutically acceptable salt of carboxytriazole is selected from one or more of the following: hydrochloride, sulfate, orotate, or acetate, and the immune checkpoint inhibitor is selected from one or more of the following: PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor.
[0010] Alternatively, in the above pharmaceutical composition, the pharmaceutically acceptable salt of carboxytriazole (CAI) is carboxytriazole orotate (CTO), and the PD-1 inhibitor is a PD-1 antibody, namely a programmed death protein-1 antibody (aPD-1).
[0011] Alternatively, in the above pharmaceutical composition, the fibrosis-related disease is a solid tumor with a high degree of fibrosis, and the carboxytriazole or a pharmaceutically acceptable salt thereof is used in combination with the immune checkpoint inhibitor to synergistically reduce collagen deposition in tumor tissue.
[0012] Alternatively, the pharmaceutical composition may further comprise a chemotherapeutic drug, wherein the carboxytriazole or a pharmaceutically acceptable salt thereof is used in combination with the immune checkpoint inhibitor to reduce the collagen content in tumor tissue, thereby promoting matrix normalization, facilitating the penetration of chemotherapeutic drugs, and improving antitumor efficacy by inhibiting ECM generation and cross-linking maturation.
[0013] Alternatively, in the above pharmaceutical composition, the matrix normalization is manifested as downregulation of the gene expression of Col1a1, Col3a1, Lox, Loxl2 and Mmp9.
[0014] Alternatively, in the above pharmaceutical composition, the chemotherapeutic agent is selected from one or more of the following: gemcitabine, paclitaxel, docetaxel, oxaliplatin, or 5-FU.
[0015] Alternatively, the dosage used in the above pharmaceutical composition will depend on the specific disease being treated and other factors, including age, weight, health status, severity of symptoms, route of administration, frequency of treatment, and whether other medications are being used concurrently during treatment. The dosage of the active compound can be readily determined using conventional methods known to those skilled in the art.
[0016] For example, in mouse models, the CTO dose was 30–120 mg / kg and the aPD-1 antibody dose was 100–250 µg / mouse. The synergistic effect was assessed using the HSA model, with ΔHSA ≥ 0.2.
[0017] In a second aspect, the present invention provides the use of the pharmaceutical composition described in the first aspect above in the preparation of a medicament for the prevention or treatment of fibrosis-related diseases.
[0018] Alternatively, in the above-described uses, the fibrosis-related disease is a solid tumor with a high degree of fibrosis.
[0019] Alternatively, in the above-described uses, the solid tumor is selected from one or more of the following: pancreatic cancer, colorectal cancer, breast cancer, or lung adenocarcinoma.
[0020] Alternatively, in the above-described uses, the fibrosis-related disease is pancreatic fibrosis, liver fibrosis, or pulmonary fibrosis.
[0021] In the course of research, the present invention provides the following technical solutions: 1. Antifibrotic effect of CTO as a single drug: CTO can inhibit ECM synthesis, cross-linking maturation, and CAF phenotype in pancreatic stellate cells (PSCs, which form pCAF after induction by tumor conditioned medium) in vitro, significantly reduce the expression of key genes such as Col1a1, Col3a1, Lox, Loxl2, and Itga11, and confirm that the COL1A1 protein level is significantly downregulated in Western blot analysis, thus possessing the function of reducing collagen deposition.
[0022] 2. Antitumor effect of combined CTO and PD-1 antibody: In an animal model of pancreatic cancer, CTO combined with PD-1 antibody significantly reduced intratumoral collagen deposition (Masson staining), with a ΔHSA value significantly greater than 0 (p<0.001), indicating a synergistic effect. This synergistic effect stems from the combined action of the two in inducing tumor stroma normalization, including synergistic inhibition of abnormal collagen production (Col1a1, Col3a1), cross-linking (Lox, Loxl2), and degradation (Mmp9). Further combination with the chemotherapeutic drug gemcitabine (GEM) significantly increased intratumoral drug concentration and reduced tumor burden, suggesting that it can significantly enhance the efficacy of chemotherapeutic drugs.
[0023] 3. Scope of application: This invention is applicable to various collagen-rich solid tumors, including pancreatic cancer, colorectal cancer, breast cancer, and lung adenocarcinoma, and is also applicable to non-tumor fibrotic diseases such as liver fibrosis, pulmonary fibrosis, and pancreatic fibrosis. Attached Figure Description
[0024] Figure 1 : The expression of genes related to collagen production and matrix crosslinking decreased in pancreatic stellate fibroblasts (PSCs) after CTO treatment following tumor cell induction (qPCR results). Figure 2 Col1a1 protein expression was significantly decreased after CTO treatment in PSC. Figure 3 Immunohistochemical staining (IHC) results of collagen deposition levels in tumor tissues of different treatment groups in a subcutaneous pancreatic cancer model showed that the CTO combined with aPD-1 antibody group had the lowest collagen expression. Figure 4 Comparison of tumor suppression effects among different treatment groups (con, CTO+aPD-1, GEM, CTO+aPD-1+GEM) in a KPC orthotopic pancreatic cancer model; Western Blot results of Collagen I expression in tumor tissue (con, CTO+aPD-1 group); Figure 5 The CTO+aPD-1+GEM group significantly increased the intratumoral concentration of GEM compared to the GEM group; Figure 6Comparison of tumor suppression effects among different treatment groups (three drugs used in combination) in a KPC orthotopic pancreatic cancer model. Detailed Implementation
[0025] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments and comparative examples. The purpose of this description is to provide a detailed understanding of the invention, not to limit its scope. All other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this invention. Unless otherwise specified, the experimental reagents, raw materials, and instruments designed in the embodiments and comparative examples of this invention are all commonly used reagents, raw materials, and instruments.
[0026] Tumor cells, drugs, and experimental animals used in the embodiments of this invention: The Pan02 mouse pancreatic cancer cell line was obtained from the National Biomedical Experimental Cell Resource Bank; the KPC mouse pancreatic cancer cell line was donated by a collaborative research group and underwent STR and Sanger sequencing; and the PSC cells were obtained from Yimo Biotechnology Co., Ltd. Mycoplasma testing was performed monthly.
[0027] The HSA model is a statistical model used to quantitatively analyze drug interactions. In this model, when the ΔHSA value is greater than 0 (ΔHSA>0) and statistically significant (p<0.05), it usually indicates that the combination of the two drugs produces a synergistic effect, rather than a simple additive effect.
[0028] Male C57 mice aged 6-8 weeks were purchased from the Laboratory Animal Center of Chinese Academy of Medical Sciences and Peking Union Medical College.
[0029] Carboxytriazole orotate (CTO), purchased from MCE.
[0030] PD-1 antibody (aPD-1), purchased from InVivoMab TM .
[0031] Collagen1a1 antibody, purchased from Selleck.
[0032] Gemcitabine-5'-triphosphorylation standard (GTCB) was purchased from Biorbyt.
[0033] Western blotting materials were purchased from Yisheng Biotechnology Co., Ltd.
[0034] Example 1: Experimental methods: Pan02 PSC cells were cultured in 0.4 µm transwell plates for 48 h, and then the cultured PSC cells were treated with CTO for 48 h. RNA was collected, reverse transcribed, and then cDNA was used for qPCR detection. Figure 1 Proteins were collected, and Col1a1 expression levels were determined by Western blotting. Figure 2 ).
[0035] The results show: The expression levels of matrix deposition-related genes in the CTO-treated group were significantly reduced (p<0.05). Figure 1 Western blotting further confirmed that CTO can downregulate Col1a1 protein expression. Figure 2 These results demonstrate that CTO can normalize the matrix, directly inhibit ECM generation and cross-linking maturation, and induce fibroblasts to transform into a matrix-normalized phenotype.
[0036] Example 2: Experimental methods: Pan02 cell line was used at 1x10 6 Cells per mouse were subcutaneously inoculated into c57 mice. Mice were treated with CAI (90 mg / kg) and aPD-1 (10 mg / kg) alone or in combination. After the experiment was terminated, subcutaneous tumors were harvested and collagen Masson staining was performed.
[0037] The results show: Masson staining results showed that the collagen deposition level in the combined group was significantly lower than that in the single-drug group. Figure 3 The HSA model calculated ΔHSA = 0.2228, with a 95% CI of [0.1209, 0.3272] and p < 0.001, suggesting that CTO and aPD-1 have a synergistic effect.
[0038] Example 3: Experimental methods: Establishment of KPC in situ tumor model (C57 mice): Mice were randomly grouped and tagged, cultured for one week, and weighed before the experiment; surgical instruments were soaked in alcohol 2 hours in advance; anesthesia: each mouse was anesthetized by intraperitoneal injection of approximately 0.25 mL of sodium pentobarbital solution; hair removal: hair was removed from the left abdomen below the ribs using a hair removal device; exposure of the surgical site: the skin and abdominal cavity at the hair removal site were cut open with scissors, making an incision of approximately 1 cm, and the pancreas was pulled out through the spleen; inoculation: 1 x 10^6 KPC cells were inoculated into the tail of the pancreas using an insulin needle. 6Individuals / animal; Suturing: The membrane and skin were sutured using a surgical needle with a suture length of 19 mm. After recovery, the animals were observed and fed according to standard feeding practices. Groups were formed based on marking: CON (PEG), CTO+aPD-1, GEM, and GEM+CTO+aPD-1. CTO was administered 90 mg / kg once daily, aPD-1 200 µg / animal every three days, and gemcitabine 80 mg / kg once weekly. At the end of the experiment, tumors were harvested, weighed, and protein samples were prepared for Western blotting to verify intratumoral collagen content.
[0039] HPLC assay for intratumoral GEM concentration: 1. Chromatographic conditions: Phenomenex chromatography was used, with 5-bromouracil as the internal standard. The mobile phase was acetonitrile-0.1% trifluoroacetic acid solution (3:97 V / V) at a flow rate of 1.0 mL / min. -1 The detection wavelength was 268 nm and the column temperature was 45°C. After protein precipitation with methanol-acetonitrile (1:9 V / V), the tissue sample was dried under nitrogen in a 55°C constant temperature water bath, dissolved in the mobile phase, and then injected at a volume of 50 μL.
[0040] 2. Preparation of standard products: Accurately weigh 10 mg of GCTB reference standard and place it in a 10 mL volumetric flask. Dissolve and dilute with ultrapure water to obtain a mass concentration of 1 g·L⁻¹. -1 The 1000 mg / L reference stock solution was stored at 4°C for later use. This stock solution was used as the stock solution to prepare a series of reference solutions of different concentrations. Blank mouse tissue homogenate was added.
[0041] Separately, accurately weigh an appropriate amount of 5-bromouracil reference standard, dissolve and dilute it with ultrapure water to prepare an internal standard solution with a mass concentration of 4 mg·L⁻¹, and store it at 4°C for later use.
[0042] 3. Tumor sample processing: Approximately 0.2 g of organ tissue samples were added to each sample with 1 mL of physiological saline. Samples in group C included three GEM, cto+aPD-1, and GEM+cto+aPD-1 groups. The samples were thoroughly homogenized using an electric homogenizer. For the standard curve group, corresponding control solutions of 1, 5, 10, 25, 50, and 100 mg / L were added. The homogenates were vortexed for 1 min and centrifuged (4 °C, 5500 rpm). -1 5 min; take 0.2 mL of tissue supernatant; add 0.1 mL of internal standard solution; add 1 mL of precipitation solvent methanol-acetonitrile (1:9 V / V); vortex mix for 2 min; centrifuge (4°C, 5500 r·min) -15 min. Collect the supernatant; dry it under nitrogen in a 5°C constant temperature water bath; dissolve the residue in 125 μL of mobile phase; vortex mix for 2 min and centrifuge (4°C, 5500 r·min). -1 5 min; take the supernatant and inject it for analysis.
[0043] Experimental results: The experimental results are shown in Figure 5 R = peak area of activated form of gemcitabine / peak area of internal standard.
[0044] In the KPC orthotopic pancreatic cancer model, the tumor volume decreased significantly in the CTO + aPD-1 + GEM triple therapy group, and the intratumoral GEM concentration increased as detected by HPLC.
[0045] The results show that: CTO+aPD-1 can significantly reduce tumor collagen deposition ( Figure 4 ), increasing the intratumoral concentration of the drug ( Figure 5 The combination therapy of three drugs improves drug penetration through ECM remodeling. Figure 3 and Figure 6 This improves the efficacy of chemotherapy.
[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. A pharmaceutical composition for preventing or treating a fibrosis-related disease, characterized by: The pharmaceutical composition comprises a carboxyamidotriazole or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.
2. The pharmaceutical composition of claim 1, wherein: The pharmaceutically acceptable salt of carboxyamidotriazole is selected from one or more of hydrochloride, sulfate, orotate or acetate, and the immune checkpoint inhibitor is selected from one or more of a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor.
3. The pharmaceutical composition of claim 2, wherein: The pharmaceutically acceptable salt of carboxyamidotriazole is carboxyamidotriazole orotate, and the PD-1 inhibitor is a PD-1 antibody.
4. The pharmaceutical composition of claim 3, wherein: The fibrosis-related disease is a solid tumor with high degree of fibrosis, and the carboxyamidotriazole or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor are used in combination to synergistically reduce collagen deposition in tumor tissue.
5. The pharmaceutical composition of claim 4, wherein: The pharmaceutical composition further comprises a chemotherapeutic drug, and the carboxyamidotriazole or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor are used in combination to reduce collagen content in tumor tissue, inhibit ECM generation and cross-linking maturation, promote stromal normalization, promote penetration of the chemotherapeutic drug, and improve anti-tumor efficacy.
6. The pharmaceutical composition of claim 5, wherein: The chemotherapeutic drug is selected from one or more of gemcitabine, paclitaxel, docetaxel, oxaliplatin or 5-FU.
7. Use of the pharmaceutical composition of any one of claims 1 to 6 in the preparation of a medicament for preventing or treating a fibrosis-related disease.
8. Use according to claim 7, characterized in that: The fibrosis-related disease is a solid tumor with high degree of fibrosis.
9. Use according to claim 8, characterized in that: The solid tumor is selected from one or more of pancreatic cancer, colorectal cancer, breast cancer or lung adenocarcinoma.
10. Use according to claim 7, characterized in that: The fibrosis-related disease is pancreatic fibrosis, liver fibrosis or lung fibrosis.