Pharmaceutical composition, preparation thereof and use thereof

Abstract

Provided are an anticancer drug containing azvudine and the use thereof. The combination of azvudine with one or more VEGF / VEGFR inhibitors and / or one or more immunotherapeutic PD-1 / PD-L1 inhibitors achieves more effective anti-cancer therapeutic effects, and can be used for treating cancer, including rectal cancer, colon cancer, squamous cell lung carcinoma, gastric cancer, adenocarcinoma of the esophagogastric junction, esophageal cancer, renal cancer, liver cancer, cholangiocarcinoma, etc.

Description

A pharmaceutical composition, its formulation and its use Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to a pharmaceutical composition, formulation comprising azvudine or a pharmaceutically acceptable salt thereof, and its use in the preparation of a medicament for treating cancer. Background Technology

[0002] Azvudine (FNC) is a broad-spectrum RNA virus inhibitor. As a synthetic nucleoside analog of viral RNA-dependent RNA polymerase (RdRp), it is metabolized intracellularly into an antiviral 5'-triphosphate metabolite (azvudine triphosphate). It inhibits positive RNA viruses, such as HCV, EV, SARS-CoV-2, HBV, and retroviruses (including HIV), by inhibiting RNA-dependent RNA polymerase. In July 2021, azvudine tablets were approved for marketing in my country for the treatment of adult HIV-1 infected patients with high viral loads. In July 2022, azvudine was approved for the treatment of COVID-19 infection.

[0003] Patent document CN201010506595.X discloses the use of azvudine for the treatment of tumors, such as colon cancer, liver cancer, stomach cancer, esophageal cancer, lung cancer, breast cancer, cervical cancer, leukemia, and lymphoma. It was found that azvudine has a significant inhibitory effect on various human cancer cells and transplanted tumors in animals.

[0004] VEGF inhibitors are a class of drugs that inhibit the activity of vascular endothelial growth factor (VEGF). They are primarily used to inhibit tumor angiogenesis, thereby blocking the tumor's nutrient supply and achieving the goal of treating tumors. Common VEGF inhibitors include bevacizumab, ranibizumab, aflibercept, and conbercept. These drugs are commonly used to treat various types of cancer, such as non-small cell lung cancer (NSCLC), colon cancer, and renal cell carcinoma. In the treatment of NSCLC, VEGF inhibitors such as bevacizumab are often used in combination with chemotherapy drugs to improve treatment efficacy.

[0005] VEGFR inhibitors are a class of drugs used to block the vascular endothelial growth factor receptor (VEGFR) signaling pathway. They primarily limit tumor growth and spread by inhibiting tumor angiogenesis. VEGFR inhibitors reduce angiogenesis and achieve their anti-tumor effect by blocking the tyrosine kinase activity of VEGFR.

[0006] PD-1 inhibitors, also known as programmed cell death protein 1 (PD-1) inhibitors, are an important class of immunotherapeutic drugs. PD-1 is an immunosuppressive molecule that inhibits T cell activity by binding to programmed cell death ligand 1 (PD-L1), thereby preventing autoimmune responses. PD-1 inhibitors work by blocking the binding of PD-1 to its ligand PD-L1, thus relieving this inhibitory effect, activating T cells, and enhancing the immune system's ability to kill tumor cells. Currently, PD-1 inhibitors are widely used in the treatment of various cancers, and ongoing research explores their combination with other drugs to improve treatment efficacy and reduce drug resistance.

[0007] The pathogenesis of tumors is complex, and there are numerous drugs available for treating them, but many suffer from drawbacks such as significant side effects and the tendency for patients to develop drug resistance. The inhibitory effect of single-agent chemotherapy is no longer sufficient to meet stringent treatment requirements. To maximize the efficacy, safety, and tolerability of single-agent-based chemotherapy regimens, achieve earlier and deeper disease remission, and prolong remission time and patient survival, actively exploring different forms of combination therapy is an inevitable direction for anti-tumor drug development. Summary of the Invention

[0008] Azvudine (FNC) has been approved for marketing as a treatment for HIV and COVID-19, and its safety has been guaranteed. Through extensive research and screening, the inventors discovered that combining azvudine with one or more VEGF / VEGFR inhibitors and / or one or more immunotherapy PD-1 / PD-L1 inhibitors can achieve more effective anti-cancer treatment results.

[0009] In view of this, the present invention provides an anticancer drug containing azvudine, which can be used to treat cancers including rectal cancer, colon cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastric cancer, esophagogastric junction cancer, bile duct cancer, small cell lung cancer, etc., especially cancers in the above-mentioned cancers with overexpression of VEGF / VEGFR and PD-1 / PD-L1.

[0010] In one aspect, the present invention provides a pharmaceutical composition comprising the following active ingredients:

[0011] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0012] (b) VEGF / VEGFR inhibitors.

[0013] Optionally, the VEGF / VEGFR inhibitor is selected from one or more of fruquintinib or a pharmaceutically acceptable salt thereof, anlotinib or a pharmaceutically acceptable salt thereof;

[0014] Optionally, the VEGF / VEGFR inhibitor is axitinib or a pharmaceutically acceptable salt thereof;

[0015] Optionally, the VEGF / VEGFR inhibitor is selected from one or more of fruquintinib or a pharmaceutically acceptable salt thereof, anlotinib or a pharmaceutically acceptable salt thereof, and axitinib or a pharmaceutically acceptable salt thereof.

[0016] Optionally, the pharmaceutically acceptable salt of the anlotinib is anlotinib hydrochloride.

[0017] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to the VEGF / VEGFR inhibitor is 0.045:1 to 3.21:1, preferably 1.37:1 to 3.21:1, preferably 1.83:1 to 2.75:1, preferably 0.045:1 to 1.37:1, and preferably 0.59:1 to 1.37:1.

[0018] Optionally, the active ingredient of the pharmaceutical composition comprises the following components:

[0019] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0020] (b) VEGF / VEGFR inhibitors.

[0021] In a preferred embodiment of the present invention, the VEGF / VEGFR inhibitor is anlotinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to anlotinib or a pharmaceutically acceptable salt thereof is 0.045:1 to 1.37:1, preferably 0.59:1;

[0022] Alternatively, the VEGF / VEGFR inhibitor is fruquintinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to fruquintinib or a pharmaceutically acceptable salt thereof is 1.37:1 to 3.21:1, preferably 1.37:1, 1.83:1, 2.29:1, 2.75:1, or 3.21:1;

[0023] Alternatively, the VEGF / VEGFR inhibitor is axitinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to axitinib or a pharmaceutically acceptable salt thereof is 0.045:1 to 1.37:1, preferably 0.045:1.

[0024] In a preferred embodiment of the present invention, the pharmaceutical composition further includes: (c) a PD-1 / PD-L1 inhibitor.

[0025] Optionally, the PD-1 / PD-L1 inhibitor is selected from one or more of penpulimab and tislelizumab.

[0026] Optionally, the PD-1 / PD-L1 inhibitor is a mouse PD-1 antibody, and the mouse PD-1 antibody clone number is one or more of RMP1-14, 29F.1A12, and J43, preferably RMP1-14;

[0027] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to a VEGF / VEGFR inhibitor is 0.045:1 to 3.21:1, preferably 1.37:1 to 3.21:1, preferably 1.83:1 to 2.75:1, preferably 0.045:1 to 1.37:1, and preferably 0.59:1 to 1.37:1.

[0028] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to a PD-1 / PD-L1 inhibitor is 7.86:1 to 52.41:1, preferably 7.86:1 to 18.34:1, more preferably 10.48:1 to 18.34:1, and even more preferably 13.10:1 to 18.34:1.

[0029] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the active ingredient of the pharmaceutical composition comprises the following components:

[0030] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0031] (b) VEGF / VEGFR inhibitors;

[0032] (c) PD-1 / PD-L1 inhibitors.

[0033] In a preferred embodiment of the present invention, the pharmaceutically acceptable salt of azvudine comprises salts formed by azvudine with the following acids: hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, mandelic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, ascorbic acid palmitic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, phthalic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, or leucine.

[0034] In this article, the term "stereoisomer" refers to compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and hindered isomers.

[0035] The term "isotope-labeled compound" refers to a compound whose structure differs only in the presence of one or more isotope-enriched atoms. Examples of isotopes used in the azvine of this invention can include isotopes of hydrogen, such as... 2 H(D) and 3 H(T), isotopes of carbon, such as 11 C 13 C and 14 C, isotopes of fluorine, such as 18 F, such as 13 N and 15 N, an isotope of oxygen, such as 15 O、 17 O and 18 O.

[0036] In a preferred embodiment of the present invention, a pharmaceutical composition is provided, wherein the active ingredient of the pharmaceutical composition comprises the following components: (a) azvudine or a pharmaceutically acceptable salt thereof, and (b) fruquintinib or a pharmaceutically acceptable salt thereof, and (c) penpulimab;

[0037] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to fruquintinib or its pharmaceutically acceptable salt is 1.37:1 to 3.21:1, preferably 1.37:1, 1.83:1, 2.29:1, 2.75:1, or 3.21:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to penpulimab is 10.48:1 to 18.34:1, preferably 10.84:1, 13.10:1, 15.72:1, or 18.34:1.

[0038] Optionally, the active ingredient of the pharmaceutical composition comprises the following components:

[0039] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0040] (b) fruquintinib or a pharmaceutically acceptable salt thereof, and

[0041] (c) Tislelizumab;

[0042] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to fruquintinib or its pharmaceutically acceptable salt is 1.37:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to tislelizumab is 7.86:1.

[0043] In a preferred embodiment of the present invention, a pharmaceutical composition is provided, wherein the active ingredient of the pharmaceutical composition comprises the following components:

[0044] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0045] (b) Mouse PD-1 antibody, clone number RMP1-14, and

[0046] (c) Axitinib or a pharmaceutically acceptable salt thereof;

[0047] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to RMP1-14 is 52.41:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to axitinib or its pharmaceutically acceptable salt is 0.045:1.

[0048] In another aspect, the present invention provides a pharmaceutical formulation in which the active ingredients comprise (a) azvudine or a pharmaceutically acceptable salt thereof, and (b) a VEGF / VEGFR inhibitor, wherein the two active ingredients (a) and (b) are either mixed together in the same formulation or separated from each other in two formulations.

[0049] Optionally, the VEGF / VEGFR inhibitor is selected from one or more of fruquintinib or a pharmaceutically acceptable salt thereof, anlotinib or a pharmaceutically acceptable salt thereof, and axitinib or a pharmaceutically acceptable salt thereof;

[0050] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to a VEGF / VEGFR inhibitor is 0.045:1 to 3.21:1, preferably 1.37:1 to 3.21:1, preferably 1.83:1 to 2.75:1, preferably 0.045:1 to 1.37:1, and preferably 0.59:1 to 1.37:1.

[0051] Optionally, the active ingredient of the pharmaceutical preparation consists of: (a) azvudine or a pharmaceutically acceptable salt thereof, and (b) a VEGF / VEGFR inhibitor;

[0052] Optionally, the VEGF / VEGFR inhibitor is anlotinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to anlotinib or a pharmaceutically acceptable salt thereof is 0.045:1 to 1.37:1, preferably 0.59:1;

[0053] Alternatively, the VEGF / VEGFR inhibitor is fruquintinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to fruquintinib or a pharmaceutically acceptable salt thereof is 1.37:1 to 3.21:1, preferably 1.37:1, 1.83:1, 2.29:1, 2.75:1, or 3.21:1;

[0054] Alternatively, the VEGF / VEGFR inhibitor is axitinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to axitinib or a pharmaceutically acceptable salt thereof is 0.045:1 to 1.37:1, preferably 0.045:1.

[0055] Optionally, the pharmaceutical formulation further includes (c) a PD-1 / PD-L1 inhibitor, wherein the three active ingredients (a), (b), and (c) are mixed together in the same formulation, or any two of the active ingredients are mixed so that the three active ingredients are present in two formulations, or the three active ingredients are separated from each other in three formulations;

[0056] Optionally, the PD-1 / PD-L1 inhibitor is selected from one or more of penpulimab and tislelizumab;

[0057] Optionally, the PD-1 / PD-L1 inhibitor is a mouse PD-1 antibody, and the mouse PD-1 antibody clone number is one or more of RMP1-14, 29F.1A12, and J43, preferably RMP1-14;

[0058] Optionally, in the pharmaceutical formulation, the molar ratio of azvudine or its pharmaceutically acceptable salt to the VEGF / VEGFR inhibitor is 0.045:1 to 3.21:1, preferably 1.37:1 to 3.21:1, preferably 1.83:1 to 2.75:1, preferably 0.045:1 to 1.37:1, preferably 0.59:1 to 1.37:1; optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to the PD-1 / PD-L1 inhibitor is 7.86:1 to 52.41:1, preferably 7.86:1 to 18.34:1, preferably 10.48:1 to 18.34:1, more preferably 13.10:1 to 18.34:1;

[0059] Optionally, the active ingredient of the pharmaceutical preparation comprises the following components:

[0060] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0061] (b) VEGF / VEGFR inhibitors, and

[0062] (c) PD-1 / PD-L1 inhibitors.

[0063] Optionally, the active ingredient of the pharmaceutical preparation comprises the following components:

[0064] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0065] (b) fruquintinib or a pharmaceutically acceptable salt thereof, and

[0066] (c) Penprilmab;

[0067] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to fruquintinib or its pharmaceutically acceptable salt is 1.37:1 to 3.21:1, preferably 1.37:1, 1.83:1, 2.29:1, 2.75:1, or 3.21:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to penpulimab is 10.48:1 to 18.34:1, preferably 10.84:1, 13.10:1, 15.72:1, or 18.34:1.

[0068] Optionally, the active ingredient of the pharmaceutical preparation comprises the following components:

[0069] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0070] (b) fruquintinib or a pharmaceutically acceptable salt thereof, and

[0071] (c) Tislelizumab;

[0072] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to fruquintinib or its pharmaceutically acceptable salt is 1.37:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to tislelizumab is 7.86:1.

[0073] Optionally, the active ingredient of the pharmaceutical preparation comprises the following components:

[0074] (a) Azvudine or a pharmaceutically acceptable salt thereof, and

[0075] (b) Mouse PD-1 antibody, clone number RMP1-14, and

[0076] (c) Axitinib or a pharmaceutically acceptable salt thereof;

[0077] Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to RMP1-14 is 52.41:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to axitinib or its pharmaceutically acceptable salt is 0.045:1.

[0078] In another aspect, the present invention provides the use of the above-described pharmaceutical composition or pharmaceutical preparation in the preparation of a medicament for treating cancer in humans or animals;

[0079] In a preferred embodiment, the cancer includes rectal cancer, colon cancer, squamous cell carcinoma of the lung, gastric cancer, adenocarcinoma of the esophagogastric junction, esophageal cancer, kidney cancer, liver cancer, and bile duct cancer; optionally, the colon cancer is transverse colon cancer, ascending colon cancer, descending colon cancer, or sigmoid colon cancer.

[0080] In another aspect, the present invention also provides a method for treating cancer in humans or animals, comprising administering the aforementioned commercially available pharmaceutical composition or pharmaceutical preparation to a subject in need (e.g., a human animal). In a preferred embodiment, the cancer includes rectal cancer, colon cancer, squamous cell carcinoma of the lung, gastric cancer, adenocarcinoma of the esophagogastric junction, esophageal cancer, renal cancer, liver cancer, and bile duct cancer; optionally, the colon cancer is transverse colon cancer, ascending colon cancer, descending colon cancer, or sigmoid colon cancer.

[0081] Specifically, when used to treat human cancer, the molar ratio of azvudine or its pharmaceutically acceptable salt to a VEGF / VEGFR inhibitor is 1.37:1 to 3.21:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to a PD-1 / PD-L1 inhibitor is 7.86:1 to 18.34:1; when used to treat cancer in animals, the molar ratio of azvudine or its pharmaceutically acceptable salt to a VEGF / VEGFR inhibitor is 0.045:1 to 1.37:1, preferably 0.045:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to a PD-1 / PD-L1 inhibitor is 52.41:1.

[0082] All of the VEGF / VEGFR inhibitors and PD-1 / PD-L1 inhibitors mentioned above are commercially available, and their pharmaceutically acceptable salts can be prepared by conventional methods.

[0083] In this invention, the active ingredients azvudine or a pharmaceutically acceptable salt thereof, fruquintinib or a pharmaceutically acceptable salt thereof, anlotinib or a pharmaceutically acceptable salt thereof, axitinib or a pharmaceutically acceptable salt thereof may be their isotope markers, stereoisomers, and various physical forms, including crystals, amorphous substances, solvates such as hydrates, etc.

[0084] This invention combines azvudine with one or more VEGF / VEGFR inhibitors and / or one or more immunotherapy PD-1 / PD-L1 inhibitors to achieve a more effective anti-cancer treatment effect, and can be used to treat cancers including rectal cancer, colon cancer, squamous cell carcinoma of the lung, gastric cancer, adenocarcinoma of the esophagogastric junction, esophageal cancer, kidney cancer, liver cancer, bile duct cancer, etc. Detailed Implementation

[0085] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0086] Trial Example 1: Clinical trials validated the effectiveness of azvudine in combination with one or more of the following: VEGF / VEGFR inhibitors and / or immunotherapy PD-1 / PDL1 inhibitors, for anti-tumor purposes.

[0087] This trial is a single-arm, single-center, open-label clinical study, divided into dose escalation and dose expansion phases.

[0088] Dosage escalation phase: A combination of accelerated titration and Bayesian optimal interval (BOIN) design was used. The FNC was pre-set with 6 dose groups: 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, and 8 mg, orally, once daily, for escalation. The 3 mg group was the accelerated titration dose group, and the subsequent dose groups adopted the BOIN design.

[0089] During dose escalation, with SMC approval, the sponsor may expand to an appropriate dose group based on safety, efficacy, and external data obtained during dose escalation. The number of patients in the expanded dose group shall not exceed 12, and safety monitoring shall be conducted during the dose expansion period. Patient data from the expanded dose group shall not be used for dose increase / decrease decisions or order-preserving regression calculations during the dose escalation period.

[0090] Dose expansion phase: The safety and efficacy of FNC will be further evaluated. After completing the FNC dose escalation and determining the RP2D, 1-2 dose levels will be selected to conduct a dose expansion study in 1-3 solid tumor populations (including but not limited to HCC, colorectal cancer, lung cancer, etc.), involving approximately 30-120 subjects.

[0091] 1. Selection criteria for included cases:

[0092] (1) Age 18-80 years old (inclusive);

[0093] (2) Patients with histological or cytologically confirmed advanced solid tumors (see Table 1 below for specific tumor type selection) who have failed or cannot tolerate standard treatment; patients with colorectal cancer and rectal cancer who are mismatch repair-proficient (pMMR) or microsatellite stable (MSS) status are enrolled.

[0094] (3) ECOG Physical Condition (PS) score 0-1;

[0095] (4) At least one measurable lesion must be present at baseline (meeting RECIST 1.1 criteria).

[0096] 2. Before enrollment and during the middle of the study, researchers may perform tumor tissue biopsies to assess the tumor microenvironment based on the patient's condition, in order to guide the medication for enrollment.

[0097] 3. Tumor assessments were conducted according to the RECIST 1.1 criteria (Standards for Evaluation of Treatment Efficacy in Solid Tumors) during the study period, with assessments performed every two treatment cycles. The specific criteria for evaluating treatment efficacy in solid tumors are as follows:

[0098] Target lesion assessment:

[0099] Complete remission (CR): All target lesions disappear and the short axis value of any pathological lymph node (whether or not it is a target lesion) must be <10 mm.

[0100] Partial remission (PR): The sum of the radii of all target lesions is reduced by at least 30%, with the sum of the critical radii as a reference.

[0101] Stable disease (SD): The minimum total radius of the target lesion recorded after the start of treatment is used as a reference. The degree of reduction does not meet the criteria for partial remission (PR), and the degree of increase does not meet the criteria for disease progression (PD).

[0102] Disease progression (PD): Using the minimum sum of the studied (target lesion radii) as a reference (including cases where the minimum value equals the critical value), the sum of the radii of all target lesions must increase by at least 20%. In addition, the absolute value of the increase in the sum of radii must be at least 5 mm (the appearance of one or more new lesions can also be considered as disease progression).

[0103] Non-target lesion assessment:

[0104] Complete remission (CR): All non-target lesions disappeared, and tumor markers returned to normal levels. All lymph nodes were of non-pathological size (short diameter <10mm).

[0105] Non-complete remission / non-progression (Non-CR / Non-PD): The presence of one or more non-target lesions and / or persistent tumor marker levels above normal.

[0106] Disease progression (PD): Clear progression of existing non-target lesions. Note: The appearance of one or more new lesions is also considered disease progression.

[0107] 4. The dosing regimens and antitumor efficacy results for the subjects are shown in Table 1. Table 1

[0108]

[0109]

[0110]

[0111]

[0112] Clinical trial results show that:

[0113] FNC, in combination with penaplimab and fruquintinib, was still effective in subjects who had failed multiple lines of therapy for advanced pMMR colorectal cancer. Most subjects had stable disease, and the best overall response (BOR) was partial remission (PR).

[0114] The combination of FNC with the VEGF / VEGFR inhibitor anlotinib also achieved a more effective anti-cancer treatment effect.

[0115] Experiment 2 evaluated the efficacy of azvudine alone, or in combination with mouse PD-1 antibody and / or axitinib, in a subcutaneous BALB / c mouse model inoculated with the CT26.WT mouse colon cancer cell line.

[0116] Experimental Objective

[0117] To evaluate the antitumor activity and safety of azvudine alone, or in combination with mouse PD-1 antibody and / or axitinib, in a mouse subcutaneous model of BALB / c colon cancer cell line CT26.WT.

[0118] Experimental materials

[0119] 1. Testing compounds

[0120] Name: ZSSW2102001 (FNC, Azvudine); Batch No.: 04; Appearance: Powder; Purity and Weight: 99.0%, 56.2 mg; Salt Coefficient: 1; Storage Conditions: 4℃; Manufacturer: Henan Zhenshi Biotechnology Co., Ltd.

[0121] Name: Anti-mouse PD-1 antibody (hereinafter abbreviated as Anti-PD-1, clone number RMP1-14); Batch number: 924625A1; Catalog number: BE0146; Concentration: 9.66mg / mL; Storage conditions: 2-8℃; Manufacturer: BioXCell.

[0122] Name: Axitinib; Batch Number: L2103397; Product Number: A129732; Appearance: Flocculent solid; Storage Conditions: -20℃; Manufacturer: Aladdin Biochemical Technology Co., Ltd.

[0123] 2. Compound solvent (5% DMSO+5% Solutol+90% Saline)

[0124] Name: DMSO; Batch Number: F0217F; Storage Conditions: RT; Supplier: Dalian Meilun.

[0125] Name: Solutol HS15; Part Number: P7014234; Lot Number: C21PC5505A; Storage Conditions: RT; Supplier: Sigma-Aldrich.

[0126] Name: Physiological saline; Batch number: 20230809; Storage conditions: RT; Supplier: Jiangsu Hengfengqiang Biotechnology.

[0127] 3. Laboratory animals

[0128] The animals required for this experiment are listed in Table 2 below. Table 2: Animal Information for this Experiment

[0129]

[0130] The animal husbandry conditions are as follows:

[0131] All experimental animals were housed in individually ventilated enclosures with constant temperature and humidity, and were allowed to acclimatize to the environment for 3-7 days prior. The enclosure temperature was 20-26℃, humidity 40-70%, with 10-20 air changes per hour. A day-night cycle was implemented, with light from 7:00 AM to 7:00 PM and darkness from 7:00 PM to 7:00 AM the following day. Animals were continuously provided with cobalt-60 radioactively sterilized complete pelleted rat feed, with unlimited access. Tap water (sterilized by autoclaving) was provided via a continuous water bottle supply. The enclosures were made of polysulfone, autoclaved, and measured 325mm × 210mm × 180mm. The bedding was autoclaved corn cobs. Each enclosure contained 3-5 animals. Cage tags indicated the IACUC approval number, experiment number, experiment start time, project leader, researchers, animal origin, group, and animal number. Animals were also marked with ear tags.

[0132] Experimental methods

[0133] 1. Cell Culture

[0134] CT26.WT cells were purchased from ATCC (Cat#CRL-2638) and cultured in 1640 medium containing 10% fetal bovine serum. CT26.WT cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice. The cells were confirmed by a third party to have the correct STR and be mycoplasma-negative.

[0135] 2. Random grouping

[0136] Before administration, all animals were weighed and tumor volume was measured using calipers. Since tumor volume affects treatment effectiveness, a randomized controlled trial was conducted, grouping mice according to their tumor volume to ensure similar tumor volumes across different groups.

[0137] 3. Cell seeding and drug administration

[0138] The experimental mice were subcutaneously inoculated with 5×10⁶ mice on their right back. 5 CT26.WT cells were resuspended in PBS (0.10 ml / cell), and tumor growth was observed regularly. Tumors were allowed to grow to an average volume of 50–60 mm². 3 The mice were randomly grouped and given medication based on tumor size and body weight.

[0139] At the time of administration, each mouse should weigh more than 18g, and the average weight of mice in each group should be controlled between 19-20g. Detailed administration methods, dosages, and routes are shown in Table 3. The day of cell inoculation is defined as day 0. Table 3 Note: QD, once a day; BIW, twice a week; BID, twice a day; PO (oral enema); IP (intraperitoneal injection).

[0140] 4. Drug preparation

[0141] The preparation methods for each group of drugs are shown in Table 4 below. Table 4

[0142]

[0143] 5. Animal observation and data collection

[0144] Following tumor cell inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss, and any abnormalities observed in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. After drug administration began, mouse weight and tumor size were measured three times per week. Tumor size was calculated using the formula: Tumor volume (mm²) = ... 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 ).

[0145] 6. Data statistics and analysis

[0146] All experimental results are expressed as MEAN (arithmetic mean) ± SEM (mean standard error). Statistical analysis between different groups was performed to select the optimal point of drug treatment (usually after the last dose). Independent samples t-tests were used to compare the relative tumor volume and tumor weight between the treatment group and the control group to determine if there were any significant differences.

[0147] All data were analyzed using SPSS 28.0. p < 0.05 was considered statistically significant.

[0148] 7. Experimental Results

[0149] 7.1 Test sample tolerance results

[0150] The test products FNC 1 mg / kg QD×4 weeks, anti-PD-1 10 mg / kg BIW×4 weeks, and Axitinib 30 mg / kg BID×4 weeks were all well tolerated by the tumor-bearing animals during the administration of the test products alone, and there was no weight loss.

[0151] Axitinib 30 mg / kg BID × 4 weeks combined with anti-PD-1 10 mg / kg BIW × 4 weeks was well tolerated in tumor-bearing animals during the administration process, and there was no weight loss.

[0152] During the combined administration of the test drug FNC 1 mg / kg QD×4 weeks with Axitinib 30 mg / kg BID×4 weeks, and the combined administration of the test drug FNC 1 mg / kg QD×4 weeks + Axitinib 30 mg / kg BID×4 weeks + Anti-PD-1 10 mg / kg BIW×4 weeks, tumor-bearing animals tolerated the drugs well with mild weight loss. The lowest mean weight change rates were -2.39% (PG-D29) and -3.41% (PG-D6), respectively.

[0153] The average weight change rate for each group is shown in Figure 1.

[0154] 7.2 Tumor volume analysis results

[0155] Since the euthanasia time points varied among the animals in this experiment, the average tumor volume at the time when all animals survived and the drug administration time was longest (PG-D17) was selected for statistical analysis. Specific statistical results are shown in Table 4 and Figure 2.

[0156] At PG-D17, the mean tumor volume in the Vehicle group was 3092.81 mm. 3 The average tumor volume in Groups 2 to 7 was 615.75 mm. 3 2833.89mm 3 1962.11mm 3 1972.38mm 3 497.98mm 3 and 381.26mm 3The corresponding tumor growth inhibition rates were 82%, 9%, 37%, 37%, 86%, and 89%, respectively. Statistical analysis showed that all test groups, except for the Anti-PD-1 10 mg / kg monotherapy group, significantly inhibited tumor growth. The TGI% of the FNC+Axitinib+Anti-PD-1 1+30+10 mg / kg triple therapy group was 61% higher than that of the Axitinib+Anti-PD-1 2-drug therapy group. By the end of the observation period, four patients achieved complete remission (CR) in the triple therapy group, while no CR was observed in the Axitinib+Anti-PD-1 2-drug therapy group. In summary, the triple therapy group showed superior efficacy compared to the Axitinib+Anti-PD-1 2-drug therapy group. The TGI values ​​and statistics for each group are shown in Table 5. Table 5: Statistical analysis of the mean tumor volume at PG-D17 in each treatment group.

[0157] Note: a, vs Vehicle group; Note: TGI (%) Tumor inhibition rate TGI (%) = [1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the beginning of treatment in solvent control group)] × 100%;

[0158] 7.3 Analysis of the lifespan of animals in each group

[0159] In this experiment, the total tumor volume of individual animals was defined as exceeding 3000 mm. 3 As a humanitarian endpoint, animals were euthanized individually; PG-D43 was the experimental endpoint. Median survival and survival extension rates for each treatment group are shown in Table 6, and survival curve statistics are shown in Figure 3. Notably, the survival curve of the FNC+Axitinib+Anti-PD-1 (Group 7) triple therapy group was overridden by the survival curve of the FNC+Axitinib (Group 6) dual therapy group. Table 6: Median Survival Statistics of Animals in Different Groups

[0160] Note: a, vs. Vehicle group;

[0161] In conclusion,

[0162] 1) The triple therapy of FNC with Anti-PD-1 and Axitinib was more effective than the double therapy of Anti-PD-1 + Axitinib;

[0163] 2) The TGI of the triple therapy group reached 89%, and 4 complete remissions were observed on day 43. The tumor continued to shrink after drug withdrawal, and the median survival time was greater than 43 days.

[0164] 3) The TGI of the Anti-PD-1 + Axitinib double therapy group reached 37%, and the median survival time was 22 days;

[0165] 4) The TGI of the FNC+Axitinib dual-drug group reached 86%, and the median survival time was greater than 43 days, which was superior to the Axitinib monotherapy group (TGI=37%, median survival time was 21 days).

Claims

1. A pharmaceutical composition, characterized in that, It contains the following active ingredients: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) VEGF / VEGFR inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that, The VEGF / VEGFR inhibitor is selected from one or more of fruquintinib or a pharmaceutically acceptable salt thereof, and anlotinib or a pharmaceutically acceptable salt thereof; Optionally, the VEGF / VEGFR inhibitor is selected from one or more of fruquintinib or a pharmaceutically acceptable salt thereof, anlotinib or a pharmaceutically acceptable salt thereof, and axitinib or a pharmaceutically acceptable salt thereof; Optionally, the pharmaceutically acceptable salt of the anlotinib is anlotinib hydrochloride.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The molar ratio of azvudine or its pharmaceutically acceptable salt to the VEGF / VEGFR inhibitor is 0.045:1 to 3.21:1, preferably 1.37:1 to 3.21:1, preferably 1.83:1 to 2.75:1, preferably 0.045:1 to 1.37:1, and preferably 0.59:1 to 1.37:

1.

4. The pharmaceutical composition according to any one of claims 1-3, characterized in that, Its active ingredients consist of the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) VEGF / VEGFR inhibitors.

5. The pharmaceutical composition according to any one of claims 1-4, characterized in that, The VEGF / VEGFR inhibitor is anlotinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to anlotinib or a pharmaceutically acceptable salt thereof is 0.045:1 to 1.37:1, preferably 0.59:1; Alternatively, the VEGF / VEGFR inhibitor is fruquintinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to fruquintinib or a pharmaceutically acceptable salt thereof is 1.37:1 to 3.21:1, preferably 1.37:1, 1.83:1, 2.29:1, 2.75:1, or 3.21:1; Alternatively, the VEGF / VEGFR inhibitor is axitinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to axitinib or a pharmaceutically acceptable salt thereof is 0.045:1 to 1.37:1, preferably 0.045:

1.

6. The pharmaceutical composition according to any one of claims 1-3 and 5, characterized in that, The pharmaceutical composition further includes: (c) a PD-1 / PD-L1 inhibitor; Optionally, the PD-1 / PD-L1 inhibitor is selected from one or more of penpulimab and tislelizumab; Optionally, the PD-1 / PD-L1 inhibitor is a mouse PD-1 antibody, and the mouse PD-1 antibody clone number is one or more of RMP1-14, 29F.1A12, and J43, preferably RMP1-14; Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to a VEGF / VEGFR inhibitor is 0.045:1 to 3.21:1, preferably 1.37:1 to 3.21:1, preferably 1.83:1 to 2.75:1, preferably 0.045:1 to 1.37:1, and preferably 0.59:1 to 1.37:

1. Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to a PD-1 / PD-L1 inhibitor is 7.86:1 to 52.41:1, preferably 7.86:1 to 18.34:1, more preferably 10.48:1 to 18.34:1, and even more preferably 13.10:1 to 18.34:

1.

7. The pharmaceutical composition according to claim 6, characterized in that, The active ingredient of the pharmaceutical composition comprises the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) VEGF / VEGFR inhibitors, and (c) PD-1 / PD-L1 inhibitors.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that, The active ingredient of the pharmaceutical composition comprises the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) fruquintinib or a pharmaceutically acceptable salt thereof, and (c) Penprilmab; Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to fruquintinib or its pharmaceutically acceptable salt is 1.37:1 to 3.21:1, preferably 1.37:1, 1.83:1, 2.29:1, 2.75:1, or 3.21:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to pendiflubenzumab is 10.48:1 to 18.34:1, preferably 10.84:1, 13.10:1, 15.72:1, or 18.34:

1. Optionally, the active ingredient of the pharmaceutical composition comprises the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) fruquintinib or a pharmaceutically acceptable salt thereof, and (c) Tislelizumab; Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to fruquintinib or its pharmaceutically acceptable salt is 1.37:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to tislelizumab is 7.86:

1.

9. The pharmaceutical composition according to claim 1 or 6, characterized in that, The active ingredient of the pharmaceutical composition comprises the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) Mouse PD-1 antibody, clone number RMP1-14, and (c) Axitinib or a pharmaceutically acceptable salt thereof; Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to RMP1-14 is 52.41:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to axitinib or its pharmaceutically acceptable salt is 0.045:

1.

10. The pharmaceutical composition according to any one of claims 1-9, characterized in that, Pharmaceutically acceptable salts of azvudine include salts formed by azvudine with the following acids: hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, mandelic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, ascorbic acid palmitic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, phthalic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, or leucine.

11. A pharmaceutical preparation, characterized in that, Its active ingredients include (a) azvudine or a pharmaceutically acceptable salt thereof, and (b) a VEGF / VEGFR inhibitor, wherein the two active ingredients (a) and (b) are either mixed together in the same formulation or separated from each other in two formulations; Optionally, the VEGF / VEGFR inhibitor is selected from one or more of fruquintinib or a pharmaceutically acceptable salt thereof, anlotinib or a pharmaceutically acceptable salt thereof, and axitinib or a pharmaceutically acceptable salt thereof; Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to a VEGF / VEGFR inhibitor is 0.045:1 to 3.21:1, preferably 1.37:1 to 3.21:1, preferably 1.83:1 to 2.75:1, preferably 0.045:1 to 1.37:1, and preferably 0.59:1 to 1.37:

1. Optionally, the active ingredient of the pharmaceutical preparation consists of: (a) azvudine or a pharmaceutically acceptable salt thereof, and (b) a VEGF / VEGFR inhibitor; Optionally, the VEGF / VEGFR inhibitor is anlotinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to anlotinib or a pharmaceutically acceptable salt thereof is 0.045:1 to 1.37:1, preferably 0.59:1; Alternatively, the VEGF / VEGFR inhibitor is fruquintinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to fruquintinib or a pharmaceutically acceptable salt thereof is 1.37:1 to 3.21:1, preferably 1.37:1, 1.83:1, 2.29:1, 2.75:1, or 3.21:1; Alternatively, the VEGF / VEGFR inhibitor is axitinib or a pharmaceutically acceptable salt thereof, and the molar ratio of azvudine or a pharmaceutically acceptable salt thereof to axitinib or a pharmaceutically acceptable salt thereof is 0.045:1 to 1.37:1, preferably 0.045:

1.

12. The pharmaceutical preparation according to claim 11, characterized in that, The pharmaceutical formulation further includes (c) a PD-1 / PD-L1 inhibitor, wherein the three active ingredients (a), (b), and (c) are mixed together in the same formulation, or any two of the active ingredients are mixed so that the three active ingredients are present in two formulations, or the three active ingredients are separated from each other in three formulations. Optionally, the PD-1 / PD-L1 inhibitor is selected from one or more of penpulimab and tislelizumab; Optionally, the PD-1 / PD-L1 inhibitor is a mouse PD-1 antibody, and the mouse PD-1 antibody clone number is one or more of RMP1-14, 29F.1A12, and J43, preferably RMP1-14; Optionally, in the pharmaceutical formulation, the molar ratio of azvudine or its pharmaceutically acceptable salt to the VEGF / VEGFR inhibitor is 0.045:1 to 3.21:1, preferably 1.37:1 to 3.21:1, preferably 1.83:1 to 2.75:1, preferably 0.045:1 to 1.37:1, preferably 0.59:1 to 1.37:1; optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to the PD-1 / PD-L1 inhibitor is 7.86:1 to 52.41:1, preferably 7.86:1 to 18.34:1, preferably 10.48:1 to 18.34:1, more preferably 13.10:1 to 18.34:1; Optionally, the active ingredient of the pharmaceutical preparation comprises the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) VEGF / VEGFR inhibitors, and (c) PD-1 / PD-L1 inhibitors. Optionally, the active ingredient of the pharmaceutical preparation comprises the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) fruquintinib or a pharmaceutically acceptable salt thereof, and (c) Penprilmab; Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to fruquintinib or its pharmaceutically acceptable salt is 1.37:1 to 3.21:1, preferably 1.37:1, 1.83:1, 2.29:1, 2.75:1, or 3.21:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to pendiflubenzumab is 10.48:1 to 18.34:1, preferably 10.84:1, 13.10:1, 15.72:1, or 18.34:

1. Optionally, the active ingredient of the pharmaceutical preparation comprises the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) fruquintinib or a pharmaceutically acceptable salt thereof, and (c) Tislelizumab; Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to fruquintinib or its pharmaceutically acceptable salt is 1.37:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to tislelizumab is 7.86:

1. Optionally, the active ingredient of the pharmaceutical preparation comprises the following components: (a) Azvudine or a pharmaceutically acceptable salt thereof, and (b) Mouse PD-1 antibody, clone number RMP1-14, and (c) Axitinib or a pharmaceutically acceptable salt thereof; Optionally, the molar ratio of azvudine or its pharmaceutically acceptable salt to RMP1-14 is 52.41:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to axitinib or its pharmaceutically acceptable salt is 0.045:

1.

13. The use of the pharmaceutical composition according to any one of claims 1-10, or the pharmaceutical preparation according to claim 11 or 12, in the preparation of a medicament for treating cancer in humans or animals; in, When used to treat human cancer, the molar ratio of azvudine or its pharmaceutically acceptable salt to a VEGF / VEGFR inhibitor is 1.37:1 to 3.21:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to a PD-1 / PD-L1 inhibitor is 7.86:1 to 18.34:1; when used to treat cancer in animals, the molar ratio of azvudine or its pharmaceutically acceptable salt to a VEGF / VEGFR inhibitor is 0.045:1 to 1.37:1, preferably 0.045:1, and the molar ratio of azvudine or its pharmaceutically acceptable salt to a PD-1 / PD-L1 inhibitor is 52.41:

1. Optionally, the cancers include rectal cancer, colon cancer, squamous cell carcinoma of the lung, gastric cancer, adenocarcinoma of the esophagogastric junction, esophageal cancer, kidney cancer, liver cancer, and bile duct cancer; Optionally, the colon cancer is transverse colon cancer, ascending colon cancer, descending colon cancer, or sigmoid colon cancer.

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