Use of combination of piperidine alkene compounds in preparation of drug for treating cancer

Abstract

The present invention relates to use of a combination of piperidine alkene compounds in the preparation of a drug for treating cancer.

Description

Use of piperidine derivatives in combination therapy in the preparation of drugs for treating cancer. Technical Field

[0001] This application belongs to the pharmaceutical field, specifically relating to the use of a combination of piperidine compounds in the preparation of a drug for treating cancer. Background Technology

[0002] Prostate cancer is one of the most common malignant tumors of the male genitourinary system. According to the 2020 edition of the World Cancer Statistics report published by the International Agency for Research on Cancer (IARC) of the World Health Organization, there were 1.41 million new cases of prostate cancer globally in 2020, accounting for 7.3% of all malignant tumors, ranking third in incidence after breast and lung cancer; there were 375,000 deaths from prostate cancer, accounting for 3.8% of all malignant tumors, ranking eighth in mortality. Data from GLOBOCAN 2020 shows that China accounts for 8.2% of global prostate cancer cases and 13.6% of global prostate cancer deaths.

[0003] The survival time of prostate cancer patients is closely related to the stage of malignancy at clinical diagnosis. Almost all uncured prostate cancers eventually develop into metastatic prostate cancer, including metastatic castration-sensitive prostate cancer (mCSPC). Androgen deprivation therapy is the main systemic basic treatment for patients with advanced metastatic prostate cancer. The use of new endocrine therapy drugs and paclitaxel-based drugs has significantly prolonged the survival of mCSPC patients compared to the past. However, mCSPC patients may progress to metastatic castration-resistant prostate cancer (mCRPC) within 12 to 30 months. Therefore, there is still a great unmet need for treatment in the field of mCSPC.

[0004] The efficacy of polyadenosine diphosphate ribose polymerase (PARP) inhibitors in homologous recombination repair (HRR) gene-mutant prostate cancer has been demonstrated in multiple clinical studies and has been approved for the treatment of mCRPC patients carrying HRR gene mutations. DNA damage repair pathways and androgen receptor signaling pathways can influence each other, and inhibiting both pathways has a potential synergistic mechanism. This synergistic mechanism has been demonstrated in multiple studies in the field of mCRPC. The PROpel study showed that the PAPR1 / 2 inhibitor olaparib combined with abiraterone provided a median survival of 42 months in the entire population as first-line treatment for mCRPC, exceeding the control group by 7.4 months. This combination therapy is currently approved in Europe and the United States. Furthermore, in the field of mCSPC, several registrational phase III studies of PARP inhibitors combined with novel endocrine therapies are actively underway.

[0005] Because selective PARP1 inhibitors can avoid a variety of serious side effects caused by inhibiting PARP2 enzymes, the technical solution of this invention aims to study a combination therapy regimen of PARP1 inhibitors that has fewer side effects while retaining its good anti-tumor efficacy. Summary of the Invention

[0006] This invention provides a combination formulation or pharmaceutical composition comprising:

[0007] (1) The first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof.

[0008] (2) The second active ingredient comprises one or more of the following drugs: (a) androgen receptor antagonists; (b) AR inhibitors; (c) CYP17 inhibitors; (d) B7H3 inhibitors; (e) tyrosine kinase inhibitors; (f) natural antitumor drugs and immunomodulators, other cytotoxic drugs; (g) DNA topoisomerase I inhibitors;

[0009] The inhibitor drugs mentioned above can be any form containing a specific drug, such as salts, crystal forms, complexes, solvates, hydrates, active metabolites, or any other form containing its active molecule.

[0010] The pharmaceutical combination formulation or pharmaceutical composition of the present invention includes, wherein the androgen receptor antagonist is enzalutamide; preferably N-desmethylenzalutamide; the AR inhibitor is revelutamide; the CYP17 inhibitor is abiraterone; the B7H3 inhibitor is an antibody-drug conjugate; the tyrosine kinase inhibitor is apatinib or bevacizumab; the natural antitumor drug and immunomodulator, and other cytotoxic drugs are albumin-bound paclitaxel or docetaxel; and the DNA topoisomerase I inhibitor is topotecan, irinotecan and its metabolites, SN38 or its liposomes.

[0011] The B7H3 inhibitor described in this invention is an antibody-drug conjugate; the structure of the antibody-drug conjugate is shown in formula (I):

[0012] in:

[0013] n is 1 to 10, preferably 2 to 8, more preferably 3 to 8, and n is a decimal or an integer;

[0014] Pc is an anti-B7H3 antibody or its antigen-binding fragment.

[0015] The anti-B7H3 antibody or its antigen-binding fragment described in this invention comprises: heavy chains HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO:01, 02, and 03, respectively, and light chains LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO:04, 05, and 06, respectively.

[0016] In this invention, the amino acid sequences of the CDRs listed above are all as shown in the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways. Although the scope of protection claimed in this invention is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0017] The CDR sequences mentioned above are shown in the table below:

[0018] Table 1. CDR sequences of each heavy chain and light chain Note: The CDR sequence is derived from the rules defined by Kabat.

[0019] The anti-B7H3 antibody or its antigen-binding fragment described in this invention is selected from humanized antibodies or their fragments.

[0020] In some alternative embodiments, the anti-B7H3 antibody or its antigen-binding fragment described in this application is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments.

[0021] In some alternative embodiments, the anti-B7H3 antibody or its antigen-binding fragment described in this application comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotypes, preferably comprising a heavy chain constant region of IgG1 or IgG4 isotypes.

[0022] In some alternative embodiments, the anti-B7H3 antibody or its antigen-binding fragment contains a constant region of the light chain of κ or λ.

[0023] The heavy chain variable region sequence of the anti-B7H3 antibody or its antigen-binding fragment described in this invention is as shown in SEQ ID NO: 07 or a variant thereof, and the light chain variable region sequence is as shown in SEQ ID NO: 08 or a variant thereof.

[0024] The sequences of the aforementioned anti-B7H3 antibody or its antigen-binding fragment heavy and light chain variable regions are shown below:

[0025] Heavy chain variable region sequence

[0026] Light chain variable region sequence Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italics in the sequence represent the FR sequence, and the underlined sequences represent the CDR sequence. The CDR sequence is derived from the Kabat definition rules.

[0027] Furthermore, the heavy chain sequence of the anti-B7H3 antibody or its antigen-binding fragment is preferably the sequence shown in SEQ ID NO:09 or a variant thereof, and the light chain sequence is the sequence shown in SEQ ID NO:10 or a variant thereof.

[0028] The sequences of the heavy and light chains of the aforementioned anti-B7H3 antibody or its antigen-binding fragment are shown below:

[0029] Heavy chain (IgG1) amino acid sequence: (SEQ ID NO:09)

[0030] Light chain (λ) amino acid sequence: (SEQ ID NO:10)

[0031] The pharmaceutical combination formulation or pharmaceutical composition of the present invention, wherein the first active ingredient is a pharmaceutically acceptable salt thereof, wherein the salt is selected from hydroxyethyl sulfonate, hydrochloride, sulfate, 1,5-naphthalenedisulfonate, methanesulfonate, hydrobromide, ethanesulfonate, phosphate, benzenesulfonate, oxalate, maleate, adipate, hydrochloride, citrate, malonate, L-malate, pamoate, p-toluenesulfonate or fumarate; preferably hydrochloride, sulfate, methanesulfonate, hydrobromide or p-toluenesulfonate.

[0032] The pharmaceutical combination formulation or composition of the present invention, wherein the content range of the first active ingredient or the second active ingredient is independently from 0.01% to 99.99%; preferably 0.05% to 99.95%; preferably 1% to 99%; preferably 1.5% to 98.5%; preferably 1% to 90%; preferably 2% to 98%; preferably 3% to 97%; preferably 4% to 96%; more preferably 1% to 80%; 1% to 70%; 1% to 60%; 1% to 50%; 1% to 40%; 1% to 30%. 1% to 20%, 1% to 10%, 5% to 10%, 5% to 20%, 5% to 30%, 5% to 40%, 5% to 50%, 5% to 60%, 5% to 70%, 5% to 80%, 5% to 85%, 5% to 90%, 5% to 95%, 5% to 99%, 10% to 99%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 40%, 10% to 20%, 15% to 99%, 15% to 99% 0%, 15% to 80%, 15% to 70%, 15% to 60%, 15% to 50%, 15% to 40%, 15% to 30%, 20% to 99%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 99%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 99%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 99%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 99%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 99%, 70% to 90%, 70% to 80%, 80% to 99%, 80% to 90%, 85% to 95%, 90% to 99%, 90% to 95%, by total weight of active ingredients.

[0033] The pharmaceutical combination formulation or pharmaceutical composition of the present invention, wherein the first active ingredient or the second active ingredient is administered independently via a route selected from gastrointestinal administration, injection administration, respiratory administration or transdermal administration; the gastrointestinal administration is preferably oral administration, sublingual administration or rectal administration; the injection administration is preferably intravenous injection, intramuscular injection or subcutaneous injection; oral administration is preferred.

[0034] In the pharmaceutical combination formulation or pharmaceutical composition of the present invention, the dosage of the first active ingredient is 5-300 mg; preferably 10-150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg.

[0035] The pharmaceutical combination formulation or pharmaceutical composition of the present invention, wherein the administration frequency of the first active ingredient is once daily, twice daily, three times daily, four times daily, every other day, once weekly, twice weekly, three times weekly, every other week, three times every two months, four times every two months, five times every two months, twice a month, or once a month.

[0036] The pharmaceutical combination formulation or composition of the present invention, wherein the oral dose of the second active ingredient is 50-1000 mg; preferably 150-1000 mg; more preferably 50-100 mg, 100-150 mg, 150-200 mg, 200-250 mg, 250-300 mg, 300-350 mg, 350-400 mg, 400-450 mg, 450-500 mg, 500-550 mg, 550-600 mg, 650-700 mg, 750-800 mg, 800-850 mg, 850-900 mg, or 950-1000 mg; the intravenous dose of the second active ingredient is 30-500 mg / m². 2 Preferred concentration: 30–50 mg / m³ 2 50-100 mg / m 2 100-150 mg / m 2 150-200 mg / m 2 200-250 mg / m 2 250~300mg / m 2 300-350 mg / m 2 350~400mg / m 2 400-450 mg / m 2 Or 450-500 mg / m 2 ;

[0037] Specifically, the dosage of enzalutamide is 80–200 mg, preferably 80 mg, 120 mg, 140 mg, or 160 mg;

[0038] The dosage of reverutamide is 80–300 mg, preferably 80 mg, 160 mg, or 240 mg;

[0039] The dosage of abiraterone or its nanocrystalline formulation is 100-1000 mg, preferably 150 mg, 225 mg, 300 mg, 500 mg, 750 mg, or 1000 mg;

[0040] The dosage of the B7H3 antibody-drug conjugate is from 0.1 mg / kg to 12.0 mg / kg, preferably from 1.0 mg / kg to 12.0 mg / kg, and the dosing frequency is once a week, once every two weeks, once every three weeks, or once every four weeks.

[0041] The dosage of apatinib is 200–900 mg, preferably 250 mg, 375 mg, 500 mg, 750 mg, or 850 mg;

[0042] The dosage of bevacizumab is 1.0 mg / kg to 100 mg / kg, preferably 1.0 mg / kg to 40 mg / kg, more preferably 1.0 mg / kg to 30 mg / kg, more preferably 10-25 mg / kg, more preferably 15-20 mg / kg, and more preferably 15 mg / kg. The dosing frequency is once a week, once every two weeks, once every three weeks, or once every four weeks.

[0043] The dosage of albumin-bound paclitaxel is 150–300 mg / m². 2 Preferred concentration: 180 mg / m 2 220mg / m 2 260mg / m 2 280mg / m 2 ;

[0044] The dosage of docetaxel is 40–200 mg / m². 2 Preferred concentration: 40–50 mg / m³ 2 50-65 mg / m 2 70-80 mg / m 2 80-85 mg / m 2 ;

[0045] The dosage of topotecan, irinotecan and its metabolites, SN38 or its liposomes is 1–400 mg / m². 2 Preferred concentration: 30–400 mg / m³ 2 Preferred concentration: 1–10 mg / m³ 2 1.2~8mg / m 2 1.4~7mg / m 230-40 mg / m 2 40-50 mg / m 2 50-60 mg / m 2 330mg / m 2 350mg / m 2 380mg / m², 1.2mg / m² 2 1.25 mg / m 2 1.4 mg / m 2 1.6 mg / m 2 1.8 mg / m 2 2.0 mg / m 2 .

[0046] In the pharmaceutical combination formulation or pharmaceutical composition of the present invention, the intravenous dose of the second active ingredient is 0.1 mg / kg to 100 mg / kg; preferably 1-30 mg / kg; more preferably 5-25 mg / kg; and even more preferably 6-12 mg / kg or 10-15 mg / kg.

[0047] The pharmaceutical combination formulation or composition of the present invention contains bevacizumab at a dosage of 1.0 mg / kg to 100 mg / kg, preferably 1.0 mg / kg to 40 mg / kg, more preferably 1.0 mg / kg to 30 mg / kg, and even more preferably 1.0 mg / kg, 1.2 mg / kg, 1.4 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2.0 mg / kg, 2.2 mg / kg, 2.4 mg / kg, 2.6 mg / kg, 2.8 mg / kg, 3.0 mg / kg, 3.2 mg / kg, 3.4 mg / kg, 3.6 mg / kg, 3.8 mg / kg, 4.0 mg / kg, etc. 4.2mg / kg, 4.4mg / kg, 4.6mg / kg, 4.8mg / kg, 5.0mg / kg, 5.2mg / kg, 5.4mg / kg, 5.6mg / kg, 5.8mg / kg, 6.0mg / kg, 6.2mg / kg, 6.4mg / kg, 6.6mg / kg, 6.8mg / k g, 7.0mg / kg, 7.2mg / kg, 7.4mg / kg, 7.6mg / kg, 7.8mg / kg, 8.0mg / kg, 8.2mg / kg, 8.4mg / kg, 8.6mg / kg, 8.8mg / kg, 9.0mg / kg, 9.2mg / kg, 9.4mg / kg, 9.6mg / kg, 9.8mg / kg, 10.0mg / kg, 10.2mg / kg, 10.4mg / kg, 10.6mg / kg, 10.8mg / kg, 11.0mg / kg, 11.2mg / kg, 11.4mg / kg, 11.6mg / kg, 11.8mg / kg, 12.0mg / kg, 12.2mg / kg, 12.4mg / kg, 12.6mg / kg, 12.8mg / kg, 13.0mg / kg, 13.2mg / kg, 13.4mg / kg, 13.6mg / kg, 13.8mg / kg, 14.0mg / kg, 14.2mg / kg, 14.4mg / kg, 14.6mg / kg g / kg, 14.8mg / kg, 15.0mg / kg, 15.2mg / kg, 15.4mg / kg, 15.6mg / kg, 15.8mg / kg, 16.0mg / kg, 16.2mg / kg, 16.4mg / kg, 16.6mg / kg, 16.8mg / kg, 17.0mg / kg , 17.2mg / kg, 17.4mg / kg, 17.6mg / kg, 17.8mg / kg, 18.0mg / kg, 18.2mg / kg, 18.4mg / kg, 18.6mg / kg, 18.8mg / kg, 19.0mg / kg, 19.2mg / kg, 19.4mg / kg, 19.6mg / kg, 19.8mg / kg, 20.0mg / kg, 20.2mg / kg, 20.4mg / kg, 20.6mg / kg, 20.8mg / kg, 30.0mg / kg. .

[0048] The present invention relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the administration frequency of the second active ingredient is once daily, twice daily, three times daily, four times daily, every other day, once weekly, five times every three weeks, twice weekly, three times weekly, every other week, three times every two months, four times every two months, five times every two months, twice a month, or once a month; the oral administration frequency is preferably once daily or five times every three weeks, and the intravenous administration frequency is preferably once every two weeks, once every three weeks, or five times every three weeks;

[0049] Specifically, enzalutamide is administered once daily; revelutamide is administered once daily; abiraterone is administered once daily; B7H3 antibody-drug conjugates are administered once every week, once every two weeks, once every three weeks, or once every four weeks; bevacizumab is administered once every week, once every two weeks, or once every three weeks, preferably once every three weeks; apatinib is administered once daily; albumin-bound paclitaxel or docetaxel is administered once every three weeks; topotecan, irinotecan and its metabolites, SN38 or its liposomes are administered once every three weeks, once every two weeks, or five times every three weeks.

[0050] The pharmaceutical combination formulation or pharmaceutical composition of the present invention may further include one or more pharmaceutically acceptable carriers, excipients, and diluents.

[0051] The pharmaceutical combination formulation or pharmaceutical composition of the present invention, wherein the first active ingredient and the second active ingredient may be administered simultaneously, in parallel, sequentially, continuously, alternately or separately.

[0052] The treatment cycle of the drug combination formulation or drug composition described in this invention is every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, or every 6 weeks.

[0053] This invention relates to the use of the aforementioned pharmaceutical combination formulation or pharmaceutical composition in the preparation of medicaments for the treatment or prevention of cancer.

[0054] The present invention also relates to a method for treating cancer, which may employ the pharmaceutical combination formulations or pharmaceutical compositions of the present invention as described above.

[0055] The cancers described in this invention are selected from at least one of the following: head and neck cancer, lung cancer, stomach cancer, liver cancer, kidney cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, bladder cancer, esophageal cancer, salivary gland cancer, skin cancer, pharyngeal cancer, laryngeal cancer, gallbladder cancer, bile duct cancer, thyroid cancer, uterine cancer, vulvar cancer, penile cancer, testicular cancer, urothelial carcinoma, urethral cancer, colon cancer, rectal cancer, colorectal cancer, esophagogastric junction adenocarcinoma, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, fallopian tube cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tumor, nerve sheath tumor, mesothelioma, Paget's disease, and sarcoma; preferably prostate cancer, human head and neck squamous cell carcinoma (including human tongue squamous cell carcinoma and human pharyngeal squamous cell carcinoma), small cell lung cancer, colorectal cancer, breast cancer, ovarian cancer, fallopian tube cancer, stomach cancer, or peritoneal cancer; the prostate cancer is advanced prostate cancer, preferably metastatic. The cancer is selected from the following: metastatic hormone-sensitive prostate cancer; more preferably, metastatic prostate cancer with homologous recombination repair gene mutation; more preferably, metastatic castration-sensitive prostate cancer with homologous recombination repair gene mutation and metastatic castration-sensitive prostate cancer with homologous recombination repair gene mutation or unknown mutation status; further preferably, metastatic castration-resistant prostate cancer that has progressed after at least one first-line treatment; wherein the homologous recombination repair gene mutation is selected from one or more germline mutations or somatic mutations of ATM, ATR, BRCA1, BRCA2, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, MLH1, MRE11, NBN, PALB2, RAD51B, RAD51C, RAD51D or RAD54L.

[0056] The breast cancer mentioned is advanced breast cancer, and more specifically, advanced HER2- breast cancer with germline BRCA1 / 2 or PALB2 mutations that has progressed after at least one first-line treatment, or advanced triple-negative breast cancer that has progressed after at least one first-line treatment.

[0057] The ovarian cancer mentioned is advanced ovarian cancer, preferably platinum-sensitive recurrent advanced ovarian cancer, newly diagnosed advanced ovarian cancer, or HRR-mutant advanced ovarian cancer that has failed standard treatment; or preferably first-line HRD-positive ovarian cancer.

[0058] The gastric cancer mentioned is advanced gastric cancer, preferably advanced gastric cancer that has progressed after at least two lines of treatment;

[0059] The peritoneal cancer is preferably primary peritoneal cancer.

[0060] This invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof.

[0061] The second active ingredient is enzalutamide, revelutamide, or abiraterone;

[0062] The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumor is selected from prostate cancer, preferably advanced prostate cancer, and more preferably metastatic castration-sensitive prostate cancer.

[0063] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is enzalutamide, revivalamide or abiraterone;

[0064] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg.

[0065] The second active ingredient is enzalutamide, and its dosage is 80-200 mg, preferably 80 mg, 120 mg, 140 mg, or 160 mg; more preferably 160 mg.

[0066] Alternatively, the second active ingredient may be revelutamide, with a dosage of 80–300 mg, preferably 80 mg, 160 mg, or 240 mg; more preferably 240 mg.

[0067] Alternatively, the second active ingredient may be abiraterone or its nanocrystalline formulation, with an administration dose of 100–1000 mg, preferably 150 mg, 225 mg, 300 mg, 500 mg, 750 mg, or 1000 mg; more preferably 300 mg.

[0068] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, wherein the solid tumor is selected from prostate cancer, preferably advanced prostate cancer, and more preferably metastatic castration-sensitive prostate cancer.

[0069] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is enzalutamide, revivalamide or abiraterone;

[0070] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg; and the dosing frequency is once daily.

[0071] The second active ingredient is enzalutamide, and the dosage is 80-200 mg; preferably 80 mg, 120 mg, 140 mg, or 160 mg; more preferably 160 mg; the dosage is once daily.

[0072] Alternatively, the second active ingredient may be revelutamide, with a dosage of 80–300 mg; preferably 80 mg, 160 mg, or 240 mg; more preferably 240 mg; and administered once daily.

[0073] Alternatively, the second active ingredient may be abiraterone or its nanocrystalline formulation, with a dosage of 100–1000 mg; preferably 150 mg, 225 mg, 300 mg, 500 mg, 750 mg, or 1000 mg; more preferably 300 mg; and administered once daily.

[0074] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, wherein the solid tumor is selected from prostate cancer, preferably advanced prostate cancer, and more preferably metastatic castration-sensitive prostate cancer.

[0075] This invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof.

[0076] The second active ingredient is a B7H3 inhibitor, which is an antibody-drug conjugate. The structure of the antibody-drug conjugate is shown in formula (I) above, wherein Pc is an anti-B7H3 antibody or its antigen-binding fragment, wherein the heavy chain variable region sequence of the anti-B7H3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 07 or a variant thereof, the light chain variable region sequence is as shown in SEQ ID NO: 08 or a variant thereof, the heavy chain sequence of the anti-B7H3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 09 or a variant thereof, and the light chain sequence is as shown in SEQ ID NO: 10 or a variant thereof.

[0077] The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumors are selected from head and neck squamous cell carcinoma, small cell lung cancer, prostate cancer and colorectal cancer, preferably in the treatment of advanced head and neck squamous cell carcinoma, advanced small cell lung cancer, advanced colorectal cancer, advanced prostate cancer or other solid tumors, and more preferably in the treatment of metastatic castration-resistant prostate cancer that has progressed after at least one first-line treatment.

[0078] This invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof.

[0079] The second active ingredient is a B7H3 inhibitor, which is an antibody-drug conjugate. The structure of the antibody-drug conjugate is shown in formula (I) above, wherein Pc is an anti-B7H3 antibody or its antigen-binding fragment, wherein the heavy chain variable region sequence of the anti-B7H3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 07 or a variant thereof, the light chain variable region sequence is as shown in SEQ ID NO: 08 or a variant thereof, the heavy chain sequence of the anti-B7H3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 09 or a variant thereof, and the light chain sequence is as shown in SEQ ID NO: 10 or a variant thereof.

[0080] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg.

[0081] The dosage of the second active ingredient is from 0.1 mg / kg to 12.0 mg / kg; preferably from 1.0 mg / kg to 12.0 mg / kg; more preferably 6 mg / kg;

[0082] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, the solid tumors being selected from head and neck squamous cell carcinoma, small cell lung cancer, prostate cancer, and colorectal cancer, more preferably in the treatment of advanced head and neck squamous cell carcinoma, advanced small cell lung cancer, advanced colorectal cancer, advanced prostate cancer, or other solid tumors, and more preferably in the treatment of metastatic castration-resistant prostate cancer that has progressed after at least first-line treatment.

[0083] This invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof.

[0084] The second active ingredient is a B7H3 inhibitor, which is an antibody-drug conjugate. The structure of the antibody-drug conjugate is shown in formula (I) above, wherein Pc is an anti-B7H3 antibody or its antigen-binding fragment, wherein the heavy chain variable region sequence of the anti-B7H3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 07 or a variant thereof, the light chain variable region sequence is as shown in SEQ ID NO: 08 or a variant thereof, the heavy chain sequence of the anti-B7H3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 09 or a variant thereof, and the light chain sequence is as shown in SEQ ID NO: 10 or a variant thereof.

[0085] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg; and the dosing frequency is once daily.

[0086] The dosage of the second active ingredient is from 0.1 mg / kg to 12.0 mg / kg; preferably from 1.0 mg / kg to 12.0 mg / kg; more preferably 6 mg / kg; and the dosing frequency is once every three weeks.

[0087] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, the solid tumors being selected from head and neck squamous cell carcinoma, small cell lung cancer, prostate cancer, and colorectal cancer, more preferably in the treatment of advanced head and neck squamous cell carcinoma, advanced small cell lung cancer, advanced colorectal cancer, advanced prostate cancer, or other solid tumors, and more preferably in the treatment of metastatic castration-resistant prostate cancer that has progressed after at least first-line treatment.

[0088] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is apatinib;

[0089] The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumor is selected from breast cancer or ovarian cancer, preferably in the treatment of advanced breast cancer or ovarian cancer or other solid tumors, and more preferably in the treatment of advanced HER2- breast cancer with germline BRCA1 / 2 or PALB2 mutations that has progressed after at least first-line treatment.

[0090] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is apatinib;

[0091] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg.

[0092] The dosage of the second active ingredient is 200–900 mg, preferably 250 mg, 375 mg, 500 mg, 750 mg or 850 mg; preferably 500 mg.

[0093] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, the solid tumors being selected from breast cancer or ovarian cancer, more preferably in the treatment of advanced breast cancer or ovarian cancer or other solid tumors, and more preferably in the treatment of advanced HER2- breast cancer with germline BRCA1 / 2 or PALB2 mutations that has progressed after at least first-line treatment.

[0094] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is apatinib;

[0095] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg; and the dosing frequency is once daily.

[0096] The dosage of the second active ingredient is 200–900 mg, preferably 250 mg, 375 mg, 500 mg, 750 mg or 850 mg; preferably 500 mg; the frequency of administration is once daily;

[0097] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, the solid tumors being selected from breast cancer or ovarian cancer, more preferably in the treatment of advanced breast cancer or ovarian cancer or other solid tumors, and more preferably in the treatment of advanced HER2- breast cancer with germline BRCA1 / 2 or PALB2 mutations that has progressed after at least first-line treatment.

[0098] This invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof.

[0099] The second active ingredient is albumin-bound paclitaxel or docetaxel;

[0100] The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumors are selected from breast cancer, gastric cancer or ovarian cancer, preferably patients with advanced breast cancer, advanced gastric cancer or advanced ovarian cancer or other advanced solid tumors, more preferably patients with HRR-mutant advanced ovarian cancer who have failed standard treatment or advanced gastric cancer that has progressed after at least two lines of treatment.

[0101] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is albumin-bound paclitaxel or docetaxel;

[0102] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg.

[0103] The second active ingredient is albumin-bound paclitaxel, with a dosage of 150–300 mg / m². 2 Preferred concentration: 180 mg / m³ 2 220mg / m 2 260mg / m 2 Or 280mg / m 2 More preferably 260 mg / m 2 ;

[0104] Alternatively, the second active ingredient may be docetaxel, with a dosage of 40–200 mg / m². 2 Preferred concentration: 40–50 mg / m³ 2 50-65 mg / m 270-80 mg / m 2 80-85 mg / m 2 More preferably 75 mg / m 2 ;

[0105] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, the solid tumors being selected from breast cancer, gastric cancer, or ovarian cancer, preferably in patients with advanced breast cancer, advanced gastric cancer, or advanced ovarian cancer or other advanced solid tumors, and more preferably in the treatment of HRR-mutant advanced ovarian cancer that has failed standard treatment or advanced gastric cancer that has progressed after at least two lines of treatment.

[0106] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is albumin-bound paclitaxel or docetaxel;

[0107] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg; and the dosing frequency is once daily.

[0108] The second active ingredient is albumin-bound paclitaxel, with a dosage of 150–300 mg / m². 2 Preferred concentration: 180 mg / m³ 2 220mg / m 2 260mg / m 2 Or 280mg / m 2 More preferably 260 mg / m 2 The dosing frequency is once every three weeks;

[0109] Alternatively, the second active ingredient may be docetaxel, with a dosage of 40–200 mg / m². 2 Preferred concentration: 40–50 mg / m³ 2 50-65 mg / m 2 70-80 mg / m 2 Or 80-85 mg / m 2 More preferably 75 mg / m 2 The dosing frequency is once every three weeks;

[0110] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, the solid tumors being selected from breast cancer, gastric cancer, or ovarian cancer, preferably in patients with advanced breast cancer, advanced gastric cancer, or advanced ovarian cancer or other advanced solid tumors, and more preferably in the treatment of HRR-mutant advanced ovarian cancer that has failed standard treatment or advanced gastric cancer that has progressed after at least two lines of treatment.

[0111] This invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof.

[0112] The second active ingredient is topotecan, irinotecan and their metabolites, SN38 or its liposomes;

[0113] The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumors are selected from breast cancer, gastric cancer or ovarian cancer, preferably in the treatment of patients with advanced breast cancer, advanced gastric cancer or ovarian cancer or other advanced solid tumors, and more preferably in the treatment of advanced gastric cancer that has progressed after at least two lines of therapy.

[0114] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is topotecan, irinotecan and its metabolites, SN38 or its liposomes;

[0115] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg.

[0116] The second active ingredient is irinotecan liposomes, with a dosage of 30–400 mg / m². 2 Preferred concentration: 30–40 mg / m³ 2 40-50 mg / m 2 50-60 mg / m 2 330mg / m 2 350mg / m 2 Or 380mg / m 2 More preferably 56.5 mg / m 2 ;

[0117] Alternatively, the second active ingredient may be topotecan, with a dosage of 1–400 mg / m².2 Preferred concentration: 1–10 mg / m³ 2 1.2~8mg / m 2 Or 1.4–7 mg / m² 2 More preferably 1.2 mg / m 2 1.25mg / m 2 1.4 mg / m 2 1.6 mg / m 2 1.8 mg / m 2 Or 2.0 mg / m 2 ;

[0118] Preferably, the pharmaceutical composition is used in a drug for treating solid tumors, the solid tumor being selected from breast cancer, gastric cancer, or ovarian cancer, more preferably in a drug for patients with advanced breast cancer, advanced gastric cancer, or ovarian cancer or other advanced solid tumors, and more preferably in a drug for advanced gastric cancer that has progressed after at least two lines of treatment.

[0119] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is topotecan, irinotecan and its metabolites, SN38 or its liposomes;

[0120] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg; and the dosing frequency is once daily.

[0121] The second active ingredient is irinotecan liposomes, with a dosage of 30–400 mg / m². 2 Preferred concentration: 30–40 mg / m³ 2 40-50 mg / m 2 50-60 mg / m 2 330mg / m 2 350mg / m 2 Or 380mg / m 2 More preferably 56.5 mg / m 2 The dosing frequency is once every 2 weeks;

[0122] Alternatively, the second active ingredient may be topotecan, with a dosage of 1–400 mg / m². 2 Preferred concentration: 1–10 mg / m³ 2 1.2~8mg / m 2 Or 1.4–7 mg / m²2 More preferably 1.2 mg / m 2 1.25mg / m 2 1.4 mg / m 2 1.6 mg / m 2 1.8 mg / m 2 Or 2.0 mg / m 2 The dosing frequency is five times every three weeks.

[0123] Preferably, the pharmaceutical composition is used in a drug for treating solid tumors, the solid tumor being selected from breast cancer, gastric cancer, or ovarian cancer, more preferably in a drug for patients with advanced breast cancer, advanced gastric cancer, or ovarian cancer or other advanced solid tumors, and more preferably in a drug for advanced gastric cancer that has progressed after at least two lines of treatment.

[0124] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient of the pharmaceutical combination formulation or pharmaceutical composition is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphth-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof.

[0125] The second active ingredient is bevacizumab;

[0126] The application of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumors are selected from advanced ovarian cancer, fallopian tube cancer or peritoneal cancer, preferably advanced first-line HRD-positive ovarian cancer or primary peritoneal cancer.

[0127] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient of the pharmaceutical combination formulation or pharmaceutical composition is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is bevacizumab;

[0128] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg.

[0129] The dosage of the second active ingredient is from 1.0 mg / kg to 100 mg / kg; preferably from 1.0 mg / kg to 40 mg / kg; more preferably from 1.0 mg / kg to 30 mg / kg; more preferably from 10 to 25 mg / kg; more preferably from 15 to 20 mg / kg; more preferably from 15 mg / kg;

[0130] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, wherein the solid tumors are selected from advanced ovarian cancer, fallopian tube cancer or peritoneal cancer, preferably advanced first-line HRD-positive ovarian cancer or primary peritoneal cancer.

[0131] The present invention also relates to a pharmaceutical combination formulation or pharmaceutical composition, wherein the first active ingredient of the pharmaceutical combination formulation or pharmaceutical composition is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof, and the second active ingredient is bevacizumab;

[0132] The dosage of the first active ingredient is 5–300 mg; preferably 10–150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; more preferably 20 mg, 40 mg or 80 mg; more preferably 40 mg; and the dosing frequency is once daily.

[0133] The dosage of the second active ingredient is from 1.0 mg / kg to 100 mg / kg; preferably from 1.0 mg / kg to 40 mg / kg; more preferably from 1.0 mg / kg to 30 mg / kg; more preferably from 10-25 mg / kg; more preferably from 15-20 mg / kg; more preferably from 15 mg / kg; and the dosing frequency is once every three weeks.

[0134] Preferably, the pharmaceutical composition is used in the treatment of solid tumors, wherein the solid tumors are selected from advanced ovarian cancer, fallopian tube cancer or peritoneal cancer, preferably advanced first-line HRD-positive ovarian cancer or primary peritoneal cancer.

[0135] In this invention, "PARP1 inhibitor" and "first active ingredient" have the same meaning and can be used interchangeably in some cases.

[0136] In this invention, 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide has the following structure: The compound may be used in combination in any form, including but not limited to its stereoisomers, its free bases or various pharmaceutically acceptable salt forms, crystal forms, hydrates, solvates, active metabolites, etc.

[0137] In some embodiments of the invention, the pharmaceutically acceptable salt is p-toluenesulfonate.

[0138] Specifically, 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide can be a free base polymorph A or B, a hydrochloride polymorph A, a sulfate polymorph A, a methanesulfonate polymorph A, a p-toluenesulfonate polymorph A, a p-toluenesulfonate polymorph B, a p-toluenesulfonate polymorph C, a p-toluenesulfonate dihydrate polymorph A, a benzenesulfonate polymorph A, especially the p-toluenesulfonate dihydrate polymorph A. The free base polymorph can be prepared by the method used in the polymorph study section of CN2023113706680, and the salt polymorph can be prepared by the method used in the salt and polymorph study section of Example 1 of PCT / CN2023 / 125513. Enzalutamide as described in this invention may also be present in any form when used in combination, including but not limited to its stereoisomers, its free bases, or various pharmaceutically acceptable salt forms, crystal forms, hydrates, solvates, and active metabolites. Attached Figure Description

[0139] Figure 1-1 Combined index of drug A and drug B inhibiting the proliferation of human prostate cancer cells LNCaP clone FGC.

[0140] Figure 1-2 Combined index of drug A and drug C inhibiting the proliferation of human prostate cancer cells LNCaP clone FGC.

[0141] Figure 2-1 Efficacy of drug A combined with drug B on a subcutaneous xenograft model of human prostate cancer cells LNCaP clone FGC.

[0142] Figure 2-2 Effects of drug A combined with drug B on body weight in mice bearing human prostate cancer cells LNCaP clone FGC.

[0143] Figure 3-1 Efficacy of drug A in combination with drug B or drug C on a subcutaneous xenograft model of human prostate cancer cells LNCaP clone FGC.

[0144] Figure 3-2 Effects of drug A in combination with drug B or drug C on body weight in mice bearing human prostate cancer cells LNCaP clone FGC.

[0145] Figure 4-1 Combined index of drug A and drug D inhibiting the proliferation of human prostate cancer cells LNCaP clone FGC.

[0146] Figure 4-2 Combined index of drug A and drug D inhibiting the proliferation of human prostate cancer cells 22Rv1.

[0147] Figure 4-3 Combined index of drug A and drug D inhibiting the proliferation of human prostate cancer cells DU145.

[0148] Figure 4-4 Combined index of drug A and drug D inhibiting the proliferation of human prostate cancer cells PC3.

[0149] Figure 5-1 Combined index of drug A and drug D inhibiting the proliferation of human head and neck squamous cell carcinoma FaDu cells.

[0150] Figure 5-2 Combined index of drug A and drug D inhibiting the proliferation of human head and neck squamous cell carcinoma CAL-33 cells.

[0151] Figure 5-3 Combined index of drug A and drug D inhibiting the proliferation of human small cell lung cancer NCI-H146 cells.

[0152] Figure 5-4 Combined index of drug A and drug D inhibiting the proliferation of human small cell lung cancer NCI-H82 cells.

[0153] Figure 5-5 Combined index of drug A and drug D inhibiting the proliferation of human colorectal cancer HT-29 cells.

[0154] Figure 6-1 Efficacy of drug A combined with drug D on a subcutaneous xenograft model of human prostate cancer cells LNCaP clone FGC.

[0155] Figure 6-2 Effects of drug A combined with drug D on body weight in mice bearing human prostate cancer cells LNCaP clone FGC.

[0156] Figure 7-1 Combined index of drug A and drug F inhibiting the proliferation of human breast cancer CAL-51 cells.

[0157] Figure 7-2 Combined index of drug A and drug F inhibiting the proliferation of human breast cancer MX-1 cells.

[0158] Figure 8-1 Combined index of drug A and drug G inhibiting the proliferation of human breast cancer CAL-51 cells.

[0159] Figure 8-2 Combined index of drug A and drug G inhibiting the proliferation of human breast cancer MX-1 cells.

[0160] Figure 9-1 Combined index of drug A and drug H inhibiting the proliferation of human breast cancer CAL-51 cells.

[0161] Figure 9-2 Combined index of drug A and drug H inhibiting the proliferation of human breast cancer MX-1 cells.

[0162] Figure 10-1 Efficacy of drug A combined with drug I in a human ovarian cancer cell OVCAR-3 subcutaneous xenograft model. Detailed Implementation

[0163] The present application will be explained in more detail below with reference to the embodiments. The embodiments of the present application are only used to illustrate the technical solutions of the present application and are not intended to limit the substance and scope of the present application.

[0164] Material source:

[0165] Drug A: Prepared using the method described in Example 1 of WO2022223025

[0166] Drug B: Enzalutamide, purchased from MCE, catalog number HY-70002

[0167] Drug C: Abiraterone, purchased from MCE, catalog number HY-70013

[0168] Drug D: Following the manufacturing method described in WO2020063673, an anti-B7H3 antibody-drug conjugate with the structure shown below was prepared using h1702DS (anti-B7H3 antibody) and an eczema analog. The average value calculated by the HIC method was n = 4.1, i.e., FADC-2. The heavy chain sequence of h1702DS is shown in SEQ ID NO:09, and the light chain sequence is shown in SEQ ID NO:10.

[0169] Drug E: Apatinib, purchased from Selleck, catalog number S5248

[0170] Drug F: Paclitaxel, purchased from Selleck, catalog number S1150

[0171] Drug G: Topotecan, purchased from MCE, catalog number HY-13768A

[0172] Drug H: SN38, purchased from MCE, catalog number HY-13704

[0173] Drug I: Bevacizumab for injection, provided by Suzhou Shengdiya Biopharmaceutical Co., Ltd., prepared with physiological saline.

[0174] Bioevaluation methods

[0175] Example 1. Evaluation of the inhibitory effect of drug A in combination with drug B or drug C on the proliferation of human prostate cancer cells.

[0176] 1. Experimental Materials

[0177] 1.1 Cell lines and experimental reagents

[0178] The human prostate cancer cell line LNCaP clone FGC was purchased from ATCC.

[0179] Drug A: Drug was prepared and dissolved using DMSO; Drug B: Drug was prepared and dissolved using DMSO;

[0180] Drug C: The drug is prepared by dissolving it in DMSO;

[0181] The Luminescent Cell Viability Assay was purchased from Promega, product number G7573.

[0182] RPMI 1640 medium was purchased from Gibco, catalog number 11995073;

[0183] RPMI 1640 medium (phenol red-free) was purchased from Gibco, catalog number 11835030;

[0184] FBS was purchased from Gibco, product number 10091148;

[0185] Charcoal Stripped FBS was purchased from Bosheng Biotechnology Co., Ltd., product number BS-0004-500;

[0186] The pancreatic enzyme was purchased from Gibco, product number 25200056;

[0187] PBS was purchased from Gibco, catalog number 10010023.

[0188] 1.2 Instruments: Microplate reader (BioTek Synergy H1); pipettes (Eppendorf & Rainin).

[0189] 2. Experimental methods and results

[0190] 2.1 Evaluation of the inhibitory effect of drug A in combination with drug B or drug C on the growth of human prostate cancer cells.

[0191] LNCaP clone FGC cells in good growth condition were washed twice with PBS and transferred to RPMI 1640 medium (without phenol red) containing Charcoal Stripped FBS for 3 days. Then, the cells were seeded into 96-well plates and allowed to adhere overnight. Different concentrations of the test substance were then administered, and the two test substances were cross-treated at different concentrations.

[0192] Test drug A was initially set at a concentration of 1000 nM, diluted 1:3, for a total of 6 concentration gradients; test drug B was initially set at a concentration of 3000 nM, diluted 1:3, for a total of 6 concentration gradients; and test drug C was initially set at a concentration of 5000 nM, diluted 1:3, for a total of 6 concentration gradients. Specific drug administration protocols are shown in Table 1-1. After 6 days of combined administration of the test drugs to each cell type, the in vitro inhibitory effect of the combined administration on human prostate cancer cells was detected using the CTG method. Three repeated administration experiments were conducted at different time points, with two technical replicates per experiment, resulting in a total of 6 replicates across the three combined administration experiments.

[0193] Table 1-1 Dosage Regimens for Drugs A, B, and C

[0194] Based on the signal value measured by the ELISA reader, the cell growth inhibition rate is calculated using the following formula:

[0195] Growth Inhibition % = [(Maximum Mean - Measured Value) / (Maximum Mean - Minimum Mean)] × 100% (Measured Value: Compound Well Reading; Minimum Value: ZPE Well Reading (Well Reading without Cell Culture Medium); Maximum Value: HPE Well Reading (Cell Reading in DMSO-treated Group).

[0196] The cell growth inhibition rates of the combined action of drug A with novel endocrine therapy drugs (drugs B and C) are shown in Tables 2 and 3. For prostate cancer cells, drug A alone had a long onset of action and could not significantly inhibit prostate cancer cell growth in in vitro pharmacodynamic experiments. In contrast, drugs B and C inhibited prostate cancer cell growth in a concentration-dependent manner across different concentration ranges. The combined action of drug A with drugs B and C, respectively, enhanced the inhibition of cell growth, demonstrating an additive or synergistic effect.

[0197] Table 1-2 Inhibitory effects of combined drug A and drug B on LNCaP clone FGC cell growth

[0198] Table 1-3 Inhibitory effects of combined drug A and drug C on LNCaP clone FGC cell growth.

[0199] 2.2 Evaluation of the combined inhibitory effect of drug A in combination with drug B or drug C on the growth of human prostate cancer cells.

[0200] Based on the experimental results in Experiment 2.1, the cell inhibition rate was converted into cell survival rate, and the combination index of the two drugs was analyzed using Combenefit software.

[0201] Figure 1-1 shows that the combination index of drug A and drug B is >10, indicating that the combined use of drug A and drug B has a synergistic effect. The combination index is between -10 and 10, indicating that the combined use of drug A and drug B has an additive effect. Figure 1-2 shows that the combination index of drug A and drug C is >10, indicating that the combined use of drug A and drug C has a synergistic effect. The combination index is between -10 and 10, indicating that the combined use of drug A and drug C has an additive effect.

[0202] 3. Experimental conclusions:

[0203] In LNCaP clone FGC tumor cells, drug A combined with drug B or drug C has a synergistic effect; at certain drug combination concentrations, it has a strong synergistic effect.

[0204] Example 2. Evaluation of the in vivo inhibitory effect of the combination of drug A and drug B on mouse xenografts of LNCaP clone FGC human prostate cancer cells.

[0205] 1. Experimental Materials

[0206] Drug A: 0.5% HPMC for drug preparation;

[0207] Drug B: Drug preparation uses 10% DMSO + 30% PEG400 + 60% (20% HP-β-CD);

[0208] Human prostate cancer LNCaP clone FGC cells (BRCA2 mutant) were cultured in vitro in a monolayer under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, in a 37°C, 5% CO2 cell culture incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0209] NOD SCID mice, male, weighing 18-22g, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0210] 2. Experimental Methods

[0211] LNCaP clone FGC cells were resuspended in PBS, with a density of 100 × 10⁻⁶ cells / mL. 6 / mL, the resuspended cells were mixed with an equal volume of Matrigel and inoculated subcutaneously into the right back of each mouse, 0.2mL (10×10⁶) per mouse. 6 / animal), until the average tumor volume grows to 100-150mm. 3Time grouping (D0), drug administration started on the day of grouping. The mice were administered the drug by gavage (p.o.), once a day; the administration volume was 10 mL / kg; the solvent group was given the same volume of "solvent"; the specific administration dose and administration schedule are shown in Table 1-1. The tumor volume was measured, the body weight of the mice was weighed, and the data were recorded.

[0212] The experimental index was to investigate the effect of the drug on tumor growth, and the specific index was ΔT / ΔC (%) or tumor growth inhibition rate TGI (%).

[0213] The tumor diameter was measured with a vernier caliper twice a week, and the formula for calculating the tumor volume (V) was:

[0214] V = 1 / 2 × a × b 2 where a and b represent the length and width respectively.

[0215] ΔT / ΔC (%) = (T - T0) / (C - C0) × 100 where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0216] Tumor growth inhibition rate (TGI) (%) = 100 - ΔT / ΔC (%).

[0217] When the tumor regressed, tumor growth inhibition rate (TGI) (%) = 100 - (T - T0) / T0 × 100

[0218] If the tumor shrank compared to the initial volume, i.e., T < T0 or C < C0, it was defined as partial regression (PR) of the tumor; if the tumor disappeared completely, it was defined as complete regression (CR) of the tumor.

[0219] At the end of the experiment, when the experimental endpoint was reached, or when the average tumor volume reached 2000 mm 3 , the animals were anesthetized and sacrificed with CO2, and then the tumors were dissected and photographed.

[0220] The experimental data were analyzed and graphed using GraphPad Prism 9.4.0. Based on the tumor volume data at different time points of each group, Dunnett's multiple comparisons test in Two-way ANOVA was used for statistical analysis to evaluate the differences between groups. The t-test was used to analyze the differences in tumor volume between two groups, and P < 0.05 was defined as statistically significant difference.

[0221] Table 2-1. Administration schedule of Drug A combined with Drug B in the LNCaP clone FGC model

[0222] 3. Experimental results

[0223] The growth-inhibiting effects of drug A combined with drug B on the LNCaP clone FGC model are shown in Table 2-2 and Figure 2-1. The changes in body weight of animals in each group of the LNCaP clone FGC model are shown in Table 2-3 and Figure 2-2.

[0224] Table 2-2. Growth inhibitory effects of drug A combined with drug B on the LNCaP clone FGC model. Note: p-value D28: The value obtained by Dunnett analysis using two-way ANOVA with the solvent group as the control for tumor volume in each animal in different groups. In addition, based on the tumor volume on day 28, using the student t-test, the p-value was 0.0245 for the drug A combined with drug B group and the drug A monotherapy group; and the p-value was 0.0353 for the drug A combined with drug B group and the drug B monotherapy group.

[0225] Table 2-3. Body weight changes in each group of animals in the LNCaP clone FGC model Note: a: Experiment time calculation: Calculation starts from the day of grouping (day0); b: Data is expressed as "mean ± standard error".

[0226] At the end of the experiment, the average tumor volume of drug A, drug B, and the combination of drug A and drug B was 562 mm. 3 780mm 3 and 237mm 3 The tumor inhibition rates were 72.86%, 58.88%, and 93.75%, respectively, all of which were significantly different from the solvent control group (p<0.05). The combination of drug A and drug B showed significant differences compared to their respective single-drug groups (p<0.05). Throughout the experiment, the tumor-bearing mice tolerated the combined dose well, and no significant weight loss occurred.

[0227] 4. Experimental conclusions:

[0228] In a mouse model of human prostate cancer xenograft LNCaP clone FGC, drug A combined with drug B showed stronger antitumor activity compared to treatment with either drug alone, without showing significant toxic side effects.

[0229] Example 3. Evaluation of the in vivo inhibitory effect of drug A in combination with drug B or drug C on mouse xenografts of LNCaP clone FGC human prostate cancer cells.

[0230] 1. Experimental Materials

[0231] Drug A: 0.5% HPMC for drug preparation;

[0232] Drug B: Drug preparation uses 10% DMSO + 30% PEG400 + 60% (20% HP-β-CD);

[0233] Drug C: 0.5% HPMC for drug preparation;

[0234] Human prostate cancer LNCaP clone FGC cells (BRCA2 mutant) were cultured in vitro in a monolayer under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, in a 37°C, 5% CO2 cell culture incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0235] NOD SCID mice, male, weighing 18-29g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0236] 2. Experimental Methods

[0237] LNCaP clone FGC cells were resuspended in PBS, with a density of 1×10⁻⁶ cells / mL. 8 / mL, the resuspended cells were mixed with an equal volume of Matrigel and inoculated subcutaneously into the right back of each mouse, 0.2mL (1×10⁶) per mouse. 7 / animal), until the average tumor volume grows to 100-150mm. 3 Mice were divided into time groups (D0), and drug administration began the day after grouping. Mice were administered the drug via gavage (po), once daily, at a volume of 10 mL / kg. The solvent group received the same volume of "solvent." Specific dosages and administration regimens are shown in Table 3-1. Tumor volume was measured, mouse weight was recorded, and data were recorded.

[0238] The experimental indicators were to examine the effect of the drug on tumor growth, specifically ΔT / ΔC (%) or tumor inhibition rate TGI (%).

[0239] The tumor diameter was measured twice a week using vernier calipers. The tumor volume (V) was calculated using the following formula:

[0240] V = 1 / 2 × a × b 2 Where a and b represent length and width, respectively.

[0241] ΔT / ΔC(%)=(T-T0) / (C-C0)×100 where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the beginning of the experiment.

[0242] Tumor inhibition rate (TGI) (%) = 100 - ΔT / ΔC (%).

[0243] When tumor regression occurs, the tumor growth inhibition rate (TGI) (%) = 100 - (T - T0) / T0 × 100

[0244] If the tumor shrinks compared to the initial volume, i.e., when T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR).

[0245] At the end of the experiment, when the experimental endpoint is reached, or when the average tumor volume reaches 2000 mm 3 , the animals are sacrificed by CO2 anesthesia, and then the tumors are dissected and photographed.

[0246] The experimental data are analyzed and graphed using GraphPad Prism 9.4.0. Based on the tumor volume data at different time points for each group, the Dunnett's multiple comparisons test in Two-way ANOVA is used for statistical analysis to evaluate the differences between groups. The t-test is used to analyze the differences in tumor volume between two groups, and P < 0.05 is defined as statistically significant difference.

[0247] Table 3-1. Administration regimens of drug A combined with drug B or drug C in the LNCaP clone FGC model

[0248] 3. Experimental results

[0249] The growth inhibitory effects of drug A combined with drug B or drug C on the LNCaP clone FGC model are shown in Table 3-2 and Figure 3-1, and the body weight changes of animals in each group in the LNCaP clone FGC model are shown in Table 3-3 and Figure 3-2.

[0250] Table 3-2. Growth inhibitory effects of drug A combined with drug B or drug C on the LNCaP clone FGC model Note: p-value D21: The value obtained by Dunnett analysis using Two-way ANOVA with the solvent group as the control based on the tumor volumes of each animal in different groups. In addition, based on the tumor volume on the 21st day, using the t-test, the p-value for the comparison between the group of drug A combined with drug B and the group of drug A alone is 0.2482; the p-value for the comparison between the group of drug A combined with drug B and the group of drug B alone is 0.0015. The p-value for the comparison between the group of drug A combined with drug C and the group of drug A alone is 0.0475; the p-value for the comparison between the group of drug A combined with drug C and the group of drug C alone is 0.0078.

[0251] Table 3-3. Body weight changes of animals in each group in the LNCaP clone FGC model Note: a: Experimental time calculation: Calculation begins on the day of grouping (D0); b: Data are expressed as "mean ± standard error".

[0252] At the end of the experiment, the average tumor volume of the drug A, drug B, drug C, drug A combined with drug B, and drug A combined with drug C groups was 317 mm. 3 420mm 3 528mm 3 232mm 3 and 174mm 3 The tumor inhibition rates were 75.68%, 61.67%, 46.89%, 87.35%, and 95.29%, respectively, all of which were significantly different from the solvent control group (p<0.0001). The combination of drug A and drug B showed significantly better tumor-inhibiting effects than drug B alone (p=0.0015) and drug A alone (p=0.2482). The combination of drug A and drug C showed significantly better tumor-inhibiting effects than drug A alone and drug C alone (p=0.0475 and p=0.0078, respectively). Throughout the experiment, the tumor-bearing mice tolerated the combined doses well, and no significant weight loss occurred.

[0253] 4. Experimental Conclusions: In the LNCaP clone FGC human prostate cancer xenograft mouse model, drug A combined with drug B showed a stronger antitumor effect compared to treatment with either drug alone. Drug A combined with drug C also showed a stronger antitumor effect compared to treatment with either drug alone, and neither drug combination showed significant toxic side effects.

[0254] Example 4. Evaluation of the inhibitory effect of the combined use of drug A and drug D on the proliferation of human prostate cancer cells.

[0255] 1. Experimental Materials

[0256] 1.1 Cell lines and experimental reagents

[0257] Human prostate cancer cells LNCaP clone FGC were purchased from ATCC, 22Rv1 from Guangzhou Genio, DU 145 from Nanjing Kebai, and PC-3 from the Chinese Academy of Sciences.

[0258] Drug A: The drug was prepared by dissolving in DMSO; Drug D: The drug was prepared by dissolving in PBS.

[0259] The Luminescent Cell Viability Assay was purchased from Promega, catalog number G7573.

[0260] RPMI 1640 medium was purchased from Gibco, catalog number 22400-089;

[0261] Ham's F-12K medium was purchased from Gibco, catalog number 21127-022;

[0262] FBS was purchased from Gibco, product number 10091-148;

[0263] The pancreatic enzyme was purchased from Gibco, catalog number 25200-056;

[0264] PBS was purchased from Gibco, catalog number 10010-023.

[0265] 1.2 Instruments: Microplate reader (BioTek Synergy H1); pipettes (Eppendorf & Rainin).

[0266] 2. Experimental methods and results

[0267] 2.1 Evaluation of the inhibitory effect of the combination of drug A and drug D on the growth of human prostate cancer cells

[0268] LNCaP clone FGC, 22Rv1, DU 145 and PC-3 cells in good growth condition were seeded into 96-well plates and allowed to adhere overnight. Then, different concentrations of the test substance were administered, and the two test substances were cross-treated at different concentrations.

[0269] Test drug A was initially set at a concentration of 100 nM, diluted 1:3, for a total of 6 concentration gradients; test drug D was initially set at a concentration of 1000 nM, diluted 1:3, for a total of 6 concentration gradients. Specific drug administration protocols are shown in Table 4-1. After 6 days of combined administration of the test drugs to each cell type, the in vitro inhibitory effect of the combined administration on human prostate cancer cells was detected using the CTG method. Three repeated administration experiments were conducted at different time points, with two technical replicates per experiment, resulting in a total of 6 replicates across the three combined administration experiments.

[0270] Table 4-1 Dosage Regimens for Drug A and Drug B

[0271] Based on the signal value measured by the ELISA reader, the cell inhibition rate is calculated using the following formula:

[0272] Inhibition % = [(Maximum mean - Measured value) / (Maximum mean - Minimum mean)] × 100% (Measured value: Compound well reading; Minimum value: ZPE well reading (well reading without cell culture medium); Maximum value: HPE well reading (cell reading in DMSO-treated group).

[0273] The cell inhibition rates of the combined action of drug A and drug D are shown in Tables 4-2 to 4-5. Drug D can inhibit the growth of prostate cancer cells in a concentration-dependent manner within different concentration ranges. The combined action of the two drugs can enhance the inhibition of cell growth, and the drug effects have an additive or synergistic effect.

[0274] Table 4-2 Inhibitory effects of combined drug A and drug D on the growth of LNCaP clone FGC cells

[0275] Table 4-3 Inhibitory effects of combined drug A and drug D on 22Rv1 cell growth

[0276] Table 4-4 Inhibitory effects of combined drug A and drug D on DU 145 cell growth

[0277] Table 4-5 Inhibitory effects of combined drug A and drug D on PC-3 cell growth

[0278] 2.2 Evaluation of the combined inhibitory effect of drug A and drug D on the growth of human prostate cancer cells

[0279] Based on the experimental results in Experiment 2.1, the cell inhibition rate was converted into cell survival rate, and the combination index of the two drugs was analyzed using Combenefit software.

[0280] Figures 4-1, 4-2, 4-3, and 4-4 show that in LNCaP clone FGC, 22Rv1, DU145, and PC-3 tumor cells, the combination index of drug A and drug D is >10, suggesting that the combined use of drug A and drug D has a synergistic effect; the combination index is between -10 and 10, suggesting that the combined use of drug A and drug D has an additive effect.

[0281] 3. Experimental conclusion: In LNCaP clone FGC, 22Rv1, DU145 and PC-3 tumor cells, drug A combined with drug D has a synergistic effect.

[0282] Example 5. Evaluation of the inhibitory effect of the combination of drug A and drug D on the proliferation of human head and neck squamous cell carcinoma, small cell lung cancer and colorectal cancer cells.

[0283] 1. Experimental Materials

[0284] 1.1 Cell lines and experimental reagents

[0285] Human head and neck squamous cell carcinoma cells FaDu and CAL-33 were purchased from the Chinese Academy of Sciences and Nanjing Kebai, respectively; human small cell lung cancer cells NCI-H146 and NCI-H82 were purchased from the Chinese Academy of Sciences; and human colorectal cancer cells HT-29 were purchased from Kangyuan Bochuang.

[0286] Drug A: The drug was prepared by dissolving in DMSO; Drug D: The drug was prepared by dissolving in PBS.

[0287] The Luminescent Cell Viability Assay was purchased from Promega, catalog number G7573.

[0288] RPMI 1640 medium was purchased from Gibco, catalog number 22400-089;

[0289] MEM culture medium was purchased from Gibco, catalog number 10370-021;

[0290] DMEM culture medium was purchased from Gibco, catalog number 11995-065;

[0291] FBS was purchased from Gibco, product number 10091-148;

[0292] The pancreatic enzyme was purchased from Gibco, catalog number 25200-056;

[0293] PBS was purchased from Gibco, catalog number 10010-023.

[0294] 1.2 Instruments: Microplate reader (BioTek Synergy H1); pipettes (Eppendorf & Rainin).

[0295] 2. Experimental methods and results

[0296] 2.1 Evaluation of the growth inhibition effect of the combination of drug A and drug D on human solid tumors (head and neck squamous cell carcinoma, small cell lung cancer, and colorectal cancer cells)

[0297] FaDu, CAL-33, NCI-H146, NCI-H82, and HT-29 cells in good growth condition were seeded into 96-well plates and allowed to adhere overnight. Different concentrations of the test substance were then administered, and cross-reaction was performed between different concentrations of the two test substances.

[0298] Test drug A was initially set at a concentration of 10 nM or 100 nM, diluted 1:3, for a total of 6 concentration gradients; test drug B was initially set at a concentration of 1000 nM, diluted 1:3, for a total of 6 concentration gradients. Specific drug administration protocols are shown in Table 5-1. Six days after administration of the test drugs to each cell type, the in vitro inhibitory effect of the combined use of the test drugs on the proliferation of human solid tumors (head and neck squamous cell carcinoma, small cell lung cancer, and colorectal cancer) was detected using the CTG method. Three combined drug administration experiments were repeated at different time points, with two technical replicates per experiment, resulting in a total of 6 replicates across the three combined drug administration experiments.

[0299] Table 5-1 Dosage Regimens for Drugs A and D

[0300] Based on the signal value measured by the ELISA reader, the cell growth inhibition rate is calculated using the following formula:

[0301] Inhibition % = [(Maximum mean - Measured value) / (Maximum mean - Minimum mean)] × 100% (Measured value: Compound well reading; Minimum value: ZPE well reading (well reading without cell culture medium); Maximum value: HPE well reading (cell reading in DMSO-treated group).

[0302] The cell inhibition rates of the combined action of drug A and drug D are shown in Tables 5-2 to 5-6. Drug D can inhibit the growth of prostate cancer cells in a concentration-dependent manner within different concentration ranges. The combined action of the two drugs can enhance the inhibition of cell growth, and the drug effects have an additive or synergistic effect.

[0303] Table 5-2 Inhibitory effects of combined drug A and drug D on FaDu cell growth

[0304] Table 5-3 Inhibitory effects of combined drug A and drug D on CAL-33 cell growth

[0305] Table 5-4 Inhibitory effects of combined drug A and drug D on NCI-H146 cell growth

[0306] Table 5-5 Inhibitory effects of combined drug A and drug D on NCI-H82 cell growth

[0307] Table 5-6 Inhibitory effects of combined drug A and drug D on HT-29 cell growth

[0308] 2.2 Evaluation of the combined inhibitory effect of drug A and drug D on the growth of human solid tumors (head and neck squamous cell carcinoma, small cell lung cancer, and colorectal cancer) cells.

[0309] Based on the experimental results in Experiment 2.1, the cell growth inhibition rate was converted into cell survival rate, and the combination index of the two drugs was analyzed using Combenefit software.

[0310] Figures 5-1, 5-2, 5-3, 5-4, and 5-5 show that the combination index of the two drugs in FaDu, CAL-33, NCI-H146, NCI-H82, and HT-29 cells is >10, suggesting that the combined use of drug A and drug D has a synergistic effect. The combination index is between -10 and 10, suggesting that the combined use of drug A and drug D has an additive effect.

[0311] 3. Experimental conclusions: The combination of drug A and drug D significantly enhanced the growth inhibition of FaDu, CAL-33, NCI-H146, NCI-H82 and HT-29 cells compared with drug A alone, and the combination of drug A and drug D had a synergistic effect.

[0312] Example 6. Evaluation of the in vivo inhibitory effect of the combination of drug A and drug D on mouse xenografts of LNCaP clone FGC human prostate cancer cells.

[0313] 1. Experimental Materials

[0314] Drug A: 0.5% HPMC for drug preparation;

[0315] Drug D: The drug is dissolved in water for injection, and diluted with physiological saline to prepare the animal administration solution;

[0316] Human prostate cancer LNCaP clone FGC cells (BRCA2 mutant) were cultured in vitro in a monolayer under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, in a 37°C, 5% CO2 cell culture incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0317] NOD SCID mice, male, weighing 18-29g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0318] 2. Experimental Methods

[0319] LNCaP clone FGC cells were resuspended in PBS, with a density of 1×10⁻⁶ cells / mL. 8 / mL, the resuspended cells were mixed with an equal volume of Matrigel and inoculated subcutaneously into the right back of each mouse, 0.2mL (1×10⁶) per mouse. 7 / animal), wait until the average tumor volume grows to 100 - 150 mm 3 Group the animals (D0) when the average tumor volume reaches 100 - 150 mm, and start drug administration the next day. Administer the drug to mice by gavage (p.o.) once a day; administer the drug to mice by intravenous injection (i.v.) as a single dose; the administration volume is 10 mL / kg; the solvent group is given the same volume of "solvent"; the specific drug dosage and administration regimen are shown in Table 6-1. Measure the tumor volume, weigh the mice, and record the data.

[0320] The experimental index is to investigate the effect of the drug on tumor growth, and the specific index is ΔT / ΔC (%) or tumor growth inhibition rate TGI (%).

[0321] Measure the tumor diameter with a vernier caliper twice a week. The formula for calculating the tumor volume (V) is:

[0322] V = 1 / 2 × a × b 2 where a and b represent the length and width respectively.

[0323] ΔT / ΔC (%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0324] Tumor growth inhibition rate (TGI) (%) = 100 - ΔT / ΔC (%).

[0325] When the tumor shows regression, tumor growth inhibition rate (TGI) (%) = 100 - (T - T0) / T0 × 100

[0326] If the tumor shrinks compared to the initial volume, i.e., T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR).

[0327] At the end of the experiment, when the experimental endpoint is reached, or when the average tumor volume reaches 2000 mm 3 , anesthetize and sacrifice the animals with CO2, and then dissect to remove the tumor and take pictures.

[0328] Analyze and plot the experimental data using GraphPad Prism 9.4.0. Based on the tumor volume data at different time points for each group, perform statistical analysis using Dunnett's multiple comparisons test in Two-way ANOVA to evaluate the differences between groups. Use t-test to analyze the differences in tumor volume between two groups, and P < 0.05 is defined as statistically significant difference.

[0329] Table 6-1. Administration regimen of drug A combined with drug D in the LNCaP clone FGC model

[0330] 3. Experimental Results

[0331] The growth-inhibiting effects of drug A combined with drug D on the LNCaP clone FGC model are shown in Table 6-2 and Figure 6-1. The changes in body weight of animals in each group of the LNCaP clone FGC model are shown in Table 6-3 and Figure 6-2.

[0332] Table 6-2. Growth inhibitory effects of drug A combined with drug D on the LNCaP clone FGC model. Note: p-value D21: The value obtained by Dunnett analysis using two-way ANOVA with the solvent group as the control for tumor volume in each animal in different groups. In addition, based on the tumor volume on day 21, the p-test was used to compare drug A combined with drug D with drug A alone, and the p-value was 0.0121; the p-value was 0.0010.

[0333] Table 6-3. Weight changes in animals in each group of the LNCaP clone FGC model Note: a: Experimental time calculation: Calculation begins on the day of grouping (D0); b: Data are expressed as "mean ± standard error".

[0334] At the end of the experiment, the average tumor volume of drug A, drug D, and the combination of drug A and drug D was 317 mm. 3 269mm 3 and 133mm 3 The tumor inhibition rates were 75.68%, 82.25%, and 104.49%, respectively, all of which were significantly different from the solvent control group (p<0.0001). The tumor-inhibiting effect of drug A combined with drug D was significantly better than that of drug A alone or drug D alone (p=0.0121 and p=0.0010, respectively). Throughout the experiment, the tumor-bearing mice tolerated the combined dose well, and no significant weight loss occurred.

[0335] 4. Experimental conclusions: In the LNCaP clone FGC human prostate cancer xenograft mouse model, drug A combined with drug D showed stronger anti-tumor effects compared with treatment by either drug alone, and no obvious toxic side effects were observed.

[0336] Example 7. Evaluation of the inhibitory effect of the combined use of drug A and drug E on the proliferation of human breast cancer cells.

[0337] 1. Experimental Materials

[0338] 1.1 Cell lines and experimental reagents

[0339] Human breast cancer cells MX-1 were purchased from Nanjing Kebai.

[0340] Drug A: The drug is prepared by dissolving it in DMSO;

[0341] Drug E: The drug is prepared by dissolving it in DMSO;

[0342] The Luminescent Cell Viability Assay was purchased from Promega, product number G7573.

[0343] DMEM culture medium was purchased from Gibco, catalog number 11995-065;

[0344] FBS was purchased from Gibco, product number 10091-148;

[0345] The pancreatic enzyme was purchased from Gibco, catalog number 25200-056;

[0346] PBS was purchased from Gibco, catalog number 10010-023.

[0347] 1.2 Instruments: Microplate reader (BioTek Synergy H1); pipettes (Eppendorf & Rainin).

[0348] 2. Experimental methods and results

[0349] 2.1 Evaluation of the inhibitory effect of the combination of drug A and drug E on the growth of human breast cancer cells

[0350] MX-1 cells in good growth condition were seeded into 96-well plates and allowed to adhere overnight. Different concentrations of the test substance were then administered, and the two test substances were cross-treated at different concentrations.

[0351] Test drug A was initially set at a concentration of 100 nM, diluted 1:3, for a total of 6 concentration gradients; test drug E was initially set at a concentration of 10 μM, diluted 1:3, for a total of 6 concentration gradients. Specific drug administration protocols are shown in Table 7-1. After 6 days of combined administration of the test drugs to each cell type, the in vitro inhibitory effect of the combined administration of the test drugs on the proliferation of human breast cancer cells was detected using the CTG method.

[0352] Table 7-1 Dosage Regimens for Drugs A and E

[0353] Based on the signal value measured by the ELISA reader, the cell growth inhibition rate is calculated using the following formula:

[0354] Inhibition % = [(Maximum mean - Measured value) / (Maximum mean - Minimum mean)] × 100% (Measured value: Compound well reading; Minimum value: ZPE well reading (well reading without cell culture medium); Maximum value: HPE well reading (cell reading in DMSO-treated group).

[0355] 2. Experimental conclusions: The combination of drug A and drug E can enhance the growth inhibition effect on MX-1 cells compared with the single drug. The combination of drug A and drug E has a synergistic or additive effect.

[0356] Example 8. Evaluation of the in vivo inhibitory effect of the combination of drug A and drug E on human breast cancer MX-1 cell xenografts in mice.

[0357] 1. Experimental Materials

[0358] Drug A: 0.5% HPMC for drug preparation; Drug E: 0.5% HPMC for drug preparation;

[0359] Human breast cancer MX-1 cells were cultured in vitro in a monolayer under the following conditions: DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, in a 37°C, 5% CO2 cell culture incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0360] BALB / c nude mice, female, weighing 18-22g, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0361] 2. Experimental Methods

[0362] MX-1 cells were resuspended in PBS, with a density of 1×10⁻⁶ cells after resuspending. 8 / mL, the resuspended cells were mixed with an equal volume of Matrigel and inoculated subcutaneously into the right back of each mouse, 0.1mL (5×10⁶) per mouse. 6 / animal), until the average tumor volume grows to 100-200 mm 3 Mice were divided into time groups (D0), and drug administration began the day after grouping. Mice were administered the drug via gavage (po), once daily, at a volume of 10 mL / kg. The solvent group received the same volume of "solvent." Specific dosages and administration regimens are shown in Table 8-1. Tumor volume was measured, mouse weight was recorded, and data were recorded.

[0363] The experimental indicators were to examine the effect of the drug on tumor growth, specifically ΔT / ΔC (%) or tumor inhibition rate TGI (%).

[0364] Measure the tumor diameter with a vernier caliper twice a week. The formula for calculating the tumor volume (V) is:

[0365] V = 1 / 2 × a × b 2 where a and b represent the length and width, respectively.

[0366] ΔT / ΔC(%) = (T - T0) / (C - C0) × 100 where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0367] Tumor growth inhibition rate (TGI)(%) = 100 - ΔT / ΔC(%).

[0368] When the tumor shows regression, Tumor growth inhibition rate (TGI)(%) = 100 - (T - T0) / T0 × 100

[0369] If the tumor shrinks compared to the initial volume, i.e., T < T0 or C < C0, it is defined as partial regression (PR) of the tumor; if the tumor completely disappears, it is defined as complete regression (CR) of the tumor.

[0370] [[ID=2G]]At the end of the experiment, when the experimental endpoint is reached, or when the average tumor volume reaches 2000 mm 3 , anesthetize the animals with CO2 and sacrifice them. Then dissect to remove the tumors and take pictures.

[0371] The experimental data were analyzed and graphed using GraphPad Prism 9.4.0. Based on the tumor volume data at different time points for each group, the Dunnett's multiple comparisons test in Two-way ANOVA was used for statistical analysis to evaluate the differences between groups. The t-test was used to analyze the differences in tumor volume between two groups, and P < 0.05 was defined as statistically significant difference.

[0372] Table 8-1. Administration schedule of drug A combined with drug E in the MX-1 model

[0373] 3. Experimental conclusions:

[0374] In the MX-1 human breast cancer xenograft mouse model, the combination of drug A and drug E showed stronger antitumor effects compared to their individual treatments alone, and no obvious toxic side effects were observed.

[0375] Example 9. Evaluate the inhibitory effect of the combination of drug A and drug F on the proliferation of human breast cancer cells.

[0376] 1. Experimental materials

[0377] 1.1 Cell lines and experimental reagents

[0378] Human breast cancer cells CAL-51 and MX-1 were purchased from Nanjing Kebai.

[0379] Drug A: Drug was prepared using DMSO for dissolution; Drug F: Drug was prepared using DMSO for dissolution;

[0380] The Luminescent Cell Viability Assay was purchased from Promega, product number G7573.

[0381] DMEM culture medium was purchased from Gibco, catalog number 11995-065;

[0382] RPMI 1640 medium was purchased from Gibco, catalog number 22400-089;

[0383] FBS was purchased from Gibco, product number 10091-148;

[0384] The pancreatic enzyme was purchased from Gibco, catalog number 25200-056;

[0385] PBS was purchased from Gibco, catalog number 10010-023.

[0386] 1.2 Instruments: Microplate reader (BioTek Synergy H1); pipettes (Eppendorf & Rainin).

[0387] 2. Experimental methods and results

[0388] 2.1 Evaluation of the inhibitory effect of the combination of drug A and drug F on the growth of human breast cancer cells

[0389] CAL-51 and MX-1 cells in good growth condition were seeded into 96-well plates and allowed to adhere overnight. Different concentrations of the test substance were then administered, and the two test substances were cross-treated at different concentrations.

[0390] Test drug A was initially set at a concentration of 100 nM, diluted 1:3, for a total of 6 concentration gradients; test drug F was initially set at a concentration of 30 nM, diluted 1:3, for a total of 6 concentration gradients. Specific drug action protocols are shown in Table 9-1. After 6 days of combined administration of the test drugs to each cell type, the in vitro inhibitory effect of the combined administration of the test drugs on the proliferation of human breast cancer cells was detected using the CTG method.

[0391] Table 9-1 Dosage Regimens for Drugs A and F

[0392] Based on the signal value measured by the ELISA reader, the cell growth inhibition rate is calculated using the following formula:

[0393] Inhibition % = [(Maximum mean - Measured value) / (Maximum mean - Minimum mean)] × 100% (Measured value: Compound well reading; Minimum value: ZPE well reading (well reading without cell culture medium); Maximum value: HPE well reading (cell reading in DMSO-treated group).

[0394] The cell inhibition rates of the combined action of drug A and drug F are shown in Tables 9-2 and 9-3. Both drugs A and F inhibited breast cancer cell growth in a concentration-dependent manner across different concentration ranges. The combined action of the two drugs enhanced the inhibition of cell growth, demonstrating a synergistic effect.

[0395] Table 9-2 Inhibitory effects of combined drug A and drug F on CAL-51 cell growth

[0396] Table 9-3 Inhibitory effects of combined drug A and drug F on MX-1 cell growth

[0397] 2.2 Evaluation of the combined inhibitory effect of drug A and drug F on the growth of human breast cancer cells

[0398] Based on the experimental results in Experiment 2.1, the cell inhibition rate was converted into cell survival rate, and the combination index of the two drugs was analyzed using Combenefit software.

[0399] Figures 7-1 and 7-2 show that the combination index of the two drugs is >10, indicating that the combined use of drug A and drug F has a synergistic effect; the combination index is between -10 and 10, indicating that the combined use of drug A and drug F has an additive effect.

[0400] 3. Experimental conclusions: The combination of drug A and drug F can enhance the growth inhibition effect on CAL-51 and MX-1 cells compared with the single drug, and the combination of drug A and drug F has a synergistic effect.

[0401] Example 10. Evaluation of the inhibitory effect of the combined use of drug A and drug G on the proliferation of human breast cancer cells.

[0402] 1. Experimental Materials

[0403] 1.1 Cell lines and experimental reagents

[0404] Human breast cancer cells CAL-51 and MX-1 were purchased from Nanjing Kebai.

[0405] Drug A: The drug is prepared by dissolving it in DMSO;

[0406] Drug G: The drug is prepared by dissolving it in DMSO;

[0407] The Luminescent Cell Viability Assay was purchased from Promega, product number G7573.

[0408] DMEM culture medium was purchased from Gibco, catalog number 11995-065;

[0409] RPMI 1640 medium was purchased from Gibco, catalog number 22400-089;

[0410] FBS was purchased from Gibco, product number 10091-148;

[0411] The pancreatic enzyme was purchased from Gibco, catalog number 25200-056;

[0412] PBS was purchased from Gibco, catalog number 10010-023.

[0413] 1.2 Instruments: Microplate reader (BioTek Synergy H1); pipettes (Eppendorf & Rainin).

[0414] 2. Experimental methods and results

[0415] 2.1 Evaluation of the inhibitory effect of the combination of drug A and drug G on the growth of human breast cancer cells

[0416] CAL-51 and MX-1 cells in good growth condition were seeded into 96-well plates and allowed to adhere overnight. Different concentrations of the test substance were then administered, and the two test substances were cross-treated at different concentrations.

[0417] Test drug A was initially set at a concentration of 100 nM, diluted 1:3, for a total of 6 concentration gradients; test drug G was initially set at a concentration of 100 nM, diluted 1:3, for a total of 6 concentration gradients. Specific drug administration protocols are shown in Table 10-1. After 6 days of combined administration of the test drugs to each cell type, the in vitro inhibitory effect of the combined administration of the test drugs on the proliferation of human breast cancer cells was detected using the CTG method.

[0418] Table 10-1 Dosage Regimens for Drugs A and G

[0419] Based on the signal value measured by the ELISA reader, the cell growth inhibition rate is calculated using the following formula:

[0420] Inhibition % = [(Maximum mean - Measured value) / (Maximum mean - Minimum mean)] × 100% (Measured value: Compound well reading; Minimum value: ZPE well reading (well reading without cell culture medium); Maximum value: HPE well reading (cell reading in DMSO-treated group).

[0421] The cell inhibition rates of the combined action of drug A and drug G are shown in Tables 10-2 and 10-3. Drugs A and G inhibited breast cancer cell growth in a concentration-dependent manner within different concentration ranges. The combined action of the two drugs enhanced the inhibition of cell growth, and their effects were additive or synergistic.

[0422] Table 10-2 Inhibitory effects of combined drug A and drug G on CAL-51 cell growth

[0423] Table 10-3 Inhibitory effects of combined drug A and drug G on MX-1 cell growth

[0424] 2.2 Evaluation of the combined inhibitory effect of drug A and drug G on human breast cancer cell growth

[0425] Based on the experimental results in Experiment 2.1, the cell inhibition rate was converted into cell survival rate, and the combination index of the two drugs was analyzed using Combenefit software.

[0426] Figures 8-1 and 8-2 show that the combination index of the two drugs is >10, indicating that the combined use of drug A and drug G has a synergistic effect; the combination index is between -10 and 10, indicating that the combined use of drug A and drug G has an additive effect.

[0427] 3. Experimental conclusions: The combination of drug A and drug G significantly enhanced the growth inhibition effect on CAL-51 and MX-1 cells compared with the single drug, and the combination of drug A and drug G had a synergistic effect.

[0428] Example 11. Evaluation of the inhibitory effect of the combined use of drug A and drug H on the proliferation of human breast cancer cells.

[0429] 1. Experimental Materials

[0430] 1.1 Cell lines and experimental reagents

[0431] Human breast cancer cells CAL-51 and MX-1 were purchased from Nanjing Kebai.

[0432] Drug A: The drug is prepared by dissolving it in DMSO;

[0433] Drug H: The drug was prepared by dissolving it in DMSO;

[0434] The Luminescent Cell Viability Assay was purchased from Promega, product number G7573.

[0435] DMEM culture medium was purchased from Gibco, catalog number 11995-065;

[0436] RPMI 1640 medium was purchased from Gibco, catalog number 22400-089;

[0437] FBS was purchased from Gibco, product number 10091-148;

[0438] The pancreatic enzyme was purchased from Gibco, catalog number 25200-056;

[0439] PBS was purchased from Gibco, catalog number 10010-023.

[0440] 1.2 Instruments: Microplate reader (BioTek Synergy H1); pipettes (Eppendorf & Rainin).

[0441] 2. Experimental methods and results

[0442] 2.1 Evaluation of the inhibitory effect of the combination of drug A and drug H on the growth of human breast cancer cells

[0443] CAL-51 and MX-1 cells in good growth condition were seeded into 96-well plates and allowed to adhere overnight. Different concentrations of the test substance were then administered, and the two test substances were cross-treated at different concentrations.

[0444] Test drug A was initially set at a concentration of 100 nM, diluted 1:3, for a total of 6 concentration gradients; test drug G was initially set at a concentration of 100 nM, diluted 1:3, for a total of 6 concentration gradients. Specific drug administration protocols are shown in Table 11-1. After 6 days of combined administration of the test drugs to each cell type, the in vitro inhibitory effect of the combined administration of the test drugs on the proliferation of human breast cancer cells was detected using the CTG method.

[0445] Table 11-1 Dosage Regimens for Drugs A and G

[0446] Based on the signal value measured by the ELISA reader, the cell growth inhibition rate is calculated using the following formula:

[0447] Inhibition % = [(Maximum mean - Measured value) / (Maximum mean - Minimum mean)] × 100% (Measured value: Compound well reading; Minimum value: ZPE well reading (well reading without cell culture medium); Maximum value: HPE well reading (cell reading in DMSO-treated group).

[0448] The cell inhibition rates of the combined action of drug A and drug H are shown in Tables 11-2 and 11-3. Drug H can inhibit the growth of breast cancer cells in a concentration-dependent manner within different concentration ranges. The combined action of the two drugs can enhance the inhibition of cell growth, and the drug effects have an additive or synergistic effect.

[0449] Table 11-2 Inhibitory effects of combined drug A and drug H on CAL-51 cell growth

[0450] Table 11-3 Inhibitory effects of combined drug A and drug H on MX-1 cell growth

[0451] 2.2 Evaluation of the combined inhibitory effect of drug A and drug H on the growth of human breast cancer cells

[0452] Based on the experimental results in Experiment 2.1, the cell inhibition rate was converted into cell survival rate, and the combination index of the two drugs was analyzed using Combenefit software.

[0453] Figures 9-1 and 9-2 show that the combination index of the two drugs is >10, indicating that the combined use of drug A and drug H has a synergistic effect; the combination index is between -10 and 10, indicating that the combined use of drug A and drug H has an additive effect.

[0454] 3. Experimental conclusions: The combination of drug A and drug H significantly enhanced the growth inhibition effect on CAL-51 and MX-1 cells compared with the single drug, and the combination of drug A and drug H had a synergistic effect.

[0455] Example 12. Evaluation of the in vivo inhibitory effect of drug A in combination with drug F, drug G, or drug H on human ovarian cancer OVCAR-3 cell xenografts in mice.

[0456] 1. Experimental Materials

[0457] Drug A: 0.5% HPMC for drug preparation; Drug F: Physiological saline for drug preparation;

[0458] Drug G: Prepared with 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline;

[0459] Drug H: Drug preparation uses 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline;

[0460] Human breast cancer OVCAR-3 cells were cultured in vitro in a monolayer using RPMI 1640 medium supplemented with 20% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.01 mg / mL insulin, in a 37°C, 5% CO2 cell culture incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0461] BALB / c nude mice, female, weighing 18 - 22 g, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0462] 2. Experimental methods

[0463] OVCAR-3 cells were resuspended in PBS at a density of 1×10 8 / mL after resuspension. The resuspended cells were mixed with an equal volume of Matrigel and inoculated subcutaneously into the right dorsal side of each mouse at a dose of 0.2 mL (1×10 7 / animal). When the average tumor volume grew to 100 - 200 mm 3 , the mice were grouped (D0), and drug administration started the next day. The mice were administered drugs by gavage (p.o.) once a day; the administration volume was 10 mL / kg; the solvent group was given the same volume of "solvent"; the specific drug administration doses and regimens are shown in Table 12-1. Tumor volume was measured, and the body weights of the mice were weighed, and the data were recorded.

[0464] The experimental index was to investigate the effect of the drug on tumor growth, and the specific index was ΔT / ΔC (%) or tumor growth inhibition rate TGI (%).

[0465] The tumor diameter was measured twice a week with a vernier caliper. The formula for calculating the tumor volume (V) was:

[0466] V = 1 / 2 × a × b 2 where a and b represent the length and width, respectively.

[0467] ΔT / ΔC (%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0468] Tumor growth inhibition rate (TGI) (%) = 100 - ΔT / ΔC (%).

[0469] When the tumor regressed, tumor growth inhibition rate (TGI) (%) = 100 - (T - T0) / T0 × 100

[0470] If the tumor shrank compared to the initial volume, i.e., T < T0 or C < C0, it was defined as partial tumor regression (PR); if the tumor disappeared completely, it was defined as complete tumor regression (CR).

[0471] At the end of the experiment, when the experimental endpoint was reached, or when the average tumor volume reached 2000 mm 3 , the animals were anesthetized with CO2 and sacrificed, and then the tumors were dissected and photographed.

[0472] Experimental data were analyzed and plotted using GraphPad Prism 9.4.0. Based on tumor volume data at different time points for each group, Dunnett's multiple comparisons test in Two-way ANOVA was used for statistical analysis to assess differences between groups. The t-test was used to analyze the differences in tumor volume between the two groups, and P < 0.05 was defined as statistically significant.

[0473] Table 12-1. Dosing regimens of drug A in combination with drug F, drug G, or drug H in the OVCAR-3 model.

[0474] 3. Experimental conclusions: In the OVCAR-3 human ovarian cancer xenograft mouse model, drug A in combination with drug F, drug G, or drug H showed stronger anti-tumor effects compared with each drug alone, and no obvious toxic side effects were observed.

[0475] Example 13. In vivo pharmacodynamic study of drug A as a monotherapy or in combination with other drugs in a mouse subcutaneous xenograft tumor model of human ovarian cancer cell line OVCAR3.

[0476] 1. Test drug

[0477] Drug A: 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide was prepared using the method disclosed in WO2022223025 and is used as Drug A.

[0478] Drug I: Bevacizumab for injection, provided by Suzhou Shengdiya Biopharmaceutical Co., Ltd., prepared with physiological saline.

[0479] 2. Experimental instruments and reagents

[0480] 2.1 Instruments

[0481] CO2 incubator (HERAcell-240i, Thermo Fisher Scientific); precision balance (SECURA225D-1CN, Sartorius Group, Germany); general balance (HZ2002A, Changzhou Tianzhiping Instrument Equipment Co., Ltd.); biosafety cabinet (BSC1300-II-A2, Shandong Xinhua Medical Instrument Co., Ltd.); digital caliper ((0-150)mm / 0.01mm, Mitutoyo, Japan); pipettes (20-200μL; 100-1000μL, Eppendorf).

[0482] 2.2 Reagents

[0483] RPMI 1640 was purchased from Gibco (catalog number 22400-071); FBS was purchased from Gibco (catalog number 10091148); PBS was purchased from Gibco (catalog number 10010023); trypsin was purchased from Gibco (catalog number 25200056); and Matrigel was purchased from Corning (catalog number 354234).

[0484] 3. Experimental Operation and Data Processing

[0485] 3.1 Animals

[0486] NOD SCID mice, 6-8 weeks old, female, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0487] 3.2 Cell Culture and Cell Suspension Preparation

[0488] a. Take an OVCAR3 cell line from the cell bank, revive the cell with RPMI 1640 medium, place the revive cell in a cell culture flask (label the flask wall with cell type, date, culturer's name, etc.) and place it in a CO2 incubator (incubator temperature is 37℃, CO2 concentration is 5%).

[0489] b. Passage the cells weekly, and after passage, continue culturing them in a CO2 incubator. Repeat this process until the cell count meets the requirements for in vivo drug efficacy.

[0490] c. Collect the cultured cells, count them using an automated cell counter, and resuspend the cells in PBS according to the counting results to prepare a cell suspension (density 10 × 10⁻⁶). 7 Add an equal volume of Matrigel to the cell suspension ( / mL), mix well, and place in an ice box for later use.

[0491] 3.3 Cell Seeding

[0492] a. Tag nude mice with disposable universal ear tags for rats and mice before inoculation;

[0493] b. When inoculating, mix the cell suspension well, use a 1mL syringe to draw 0.2-1mL of cell suspension, remove air bubbles, and then place the syringe on an ice pack for later use;

[0494] c. Hold the NOD SCID mouse firmly with your left hand, disinfect the right back of the nude mouse near the right shoulder with a 75% alcohol swab (inoculation site), and begin inoculation after 30 seconds;

[0495] d. The NOD SCID mice were inoculated sequentially (0.1 mL of cell suspension per mouse).

[0496] 3.4 Tumor measurement, grouping, and drug administration in tumor-bearing mice

[0497] a. Based on the tumor growth, measure the tumor and calculate its size on days 20-30 after inoculation;

[0498] Tumor volume calculation: Tumor volume (mm) 3 = Length (mm) × Width (mm) × Width (mm) / 2

[0499] b. Based on the weight and tumor size of the tumor-bearing mice, they were randomly grouped.

[0500] c. Based on the grouping results, the test drug will be administered. The specific dosage and administration regimen are shown in Table 13-1.

[0501] Table 13-1. Dosage and Grouping

[0502] a. Dosage volume: Administer 10 μL / g based on mouse body weight. Discontinue administration if body weight decreases by more than 15%, and resume administration when body weight recovers to within 10%.

[0503] d. After starting the test drug, the tumor was measured and weighed twice a week.

[0504] e. Euthanize the animals after the experiment.

[0505] f. Data were processed using software such as GraphPad Prism. The antitumor efficacy of the compounds was evaluated using TGI (%) and ΔT / ΔC (%). ΔT / ΔC (%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes of the treatment group and the solvent control group at the end of the experiment, respectively, and T0 and C0 are the tumor volumes of the treatment group and the solvent control group at the beginning of the experiment, respectively. Tumor inhibition rate (TGI) (%) = 100 - ΔT / ΔC (%). When tumor regression occurred, the tumor inhibition rate (TGI) (%) = 100 - (T - T0) / T0 × 100

[0506] 4. Experimental results and conclusions: The growth-inhibiting effects of drug A combined with drug I on the OVCAR3 model are shown in Table 13-2 and Figure 10-1.

[0507] Table 13-2. Growth-inhibiting effects of drug A combined with drug I on the OVCAR3 model

[0508] a. Compared with the solvent control group, the tumor volume of each animal in different groups was analyzed using Dunnett's two-way ANOVA. Furthermore, based on the tumor volume on day 21, the t-test was used to compare the drug A combined with drug I group with drug A alone, and the p-value was 0.0074; the p-value was 0.0046.

[0509] In the OVCAR3 ovarian cancer cell line tumor-bearing model, based on D0-D21 data, the average tumor volume of the drug I, drug A, and drug A combined with drug I groups was 479 mm. 3 727mm 3 and 271mm 3 The tumor inhibition rates were 72.18%, 51.86%, and 89.28%, respectively, all of which were significantly different from the solvent control group (p<0.0001). The tumor-inhibiting effect of drug A combined with drug I was significantly better than that of drug I alone (p=0.0046) and drug A alone (p=0.0074). Throughout the experiment, the tumor-bearing mice tolerated the combined dose well, and no significant weight loss occurred.

[0510] Conclusion: As shown above, in the OVCAR3 human ovarian cancer xenograft mouse model, drug A combined with drug I has a stronger anti-tumor effect than either drug alone, without showing obvious toxic side effects and demonstrating good safety.

[0511] Example 14. In vivo pharmacodynamic study of drug A as a monotherapy or in combination with other drugs in a mouse subcutaneous xenograft tumor model of human endometrial cancer cell line RL95-2.

[0512] 1. Test drug

[0513] Drug B: 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide was prepared using the method disclosed in WO2022223025 and used as drug A.

[0514] Drug I: Bevacizumab for injection, provided by Suzhou Shengdiya Biopharmaceutical Co., Ltd., prepared with physiological saline.

[0515] 2. Experimental objective: To evaluate the in vivo efficacy of compound A as a single agent or in combination therapy in a nude mouse subcutaneous xenograft tumor model of human endometrial cancer cell line RL95-2.

[0516] 3. Experimental instruments and reagents

[0517] 3.1 Instruments

[0518] CO2 incubator (HERAcell-240i, Thermo Fisher Scientific)

[0519] Precision balance (SECURA225D-1CN, Sartorius Group, Germany)

[0520] Ordinary balance (HZ2002A, Changzhou Tianzhiping Instrument Equipment Co., Ltd.)

[0521] Biosafety cabinet (BSC1300-II-A2, Shandong Xinhua Medical Instrument Co., Ltd.)

[0522] Digital caliper ((0-150)mm / 0.01mm, Mitutoyo, Japan)

[0523] Pipettes (20-200 μL; 100-1000 μL, Eppendorf)

[0524] 3.2 Reagents

[0525] RPMI 1640 was purchased from Gibco, part number 22400-071.

[0526] FBS was purchased from Gibco, item number 10091148;

[0527] PBS was purchased from Gibco, catalog number 10010023;

[0528] The pancreatic enzyme was purchased from Gibco, catalog number 25200056;

[0529] Matrigel was purchased from Corning, item number 354234.

[0530] 4. Experimental Operation and Data Processing

[0531] 4.1 Animals

[0532] NOD SCID mice, 6-8 weeks old, female, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0533] 4.2 Cell Culture and Cell Suspension Preparation

[0534] a. Take a line of RL95-2 cells from the cell bank, revive the cells with RPMI 1640 medium, place the revive cells in a cell culture flask (label the flask wall with cell type, date, culturer's name, etc.) and place it in a CO2 incubator (incubator temperature is 37℃, CO2 concentration is 5%).

[0535] b. Passage the cells weekly, and after passage, continue culturing them in a CO2 incubator. Repeat this process until the cell count meets the requirements for in vivo drug efficacy.

[0536] c. Collect the cultured cells, count them using an automated cell counter, and resuspend the cells in PBS according to the counting results to prepare a cell suspension (density 10 × 10⁻⁶). 7 Add an equal volume of Matrigel to the cell suspension ( / mL), mix well, and place in an ice box for later use.

[0537] 3.3 Cell Seeding

[0538] a. Tag nude mice with disposable universal ear tags for rats and mice before inoculation;

[0539] b. When inoculating, mix the cell suspension well, use a 1mL syringe to draw 0.2-1mL of cell suspension, remove air bubbles, and then place the syringe on an ice pack for later use;

[0540] c. Hold the NOD SCID mouse firmly with your left hand, disinfect the right back of the nude mouse near the right shoulder with a 75% alcohol swab (inoculation site), and begin inoculation after 30 seconds;

[0541] d. The NOD SCID mice were inoculated sequentially (0.1 mL of cell suspension per mouse).

[0542] 3.4 Tumor measurement, grouping, and drug administration in tumor-bearing mice

[0543] a. Based on the tumor growth, measure the tumor and calculate its size on days 20-30 after inoculation;

[0544] Tumor volume calculation: Tumor volume (mm) 3 = Length (mm) × Width (mm) × Width (mm) / 2

[0545] b. Based on the weight and tumor size of the tumor-bearing mice, they were randomly grouped.

[0546] c. Based on the grouping results, the test drug will be administered. The specific dosage and administration regimen are shown in Table 14-1.

[0547] Table 14-1. Dosage and Grouping

[0548] a. Dosage volume: Administer 10 μL / g based on mouse body weight. Discontinue administration if body weight decreases by more than 15%, and resume administration when body weight recovers to within 10%.

[0549] d. After starting the test drug, the tumor was measured and weighed twice a week.

[0550] e. Euthanize the animals after the experiment.

[0551] f. Data were processed using software such as GraphPad Prism. The antitumor efficacy of the compounds was evaluated using TGI (%) and ΔT / ΔC (%). ΔT / ΔC (%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes of the treatment group and the solvent control group at the end of the experiment, respectively, and T0 and C0 are the tumor volumes of the treatment group and the solvent control group at the beginning of the experiment, respectively. Tumor inhibition rate (TGI) (%) = 100 - ΔT / ΔC (%). When tumor regression occurred, the tumor inhibition rate (TGI) (%) = 100 - (T - T0) / T0 × 100

[0552] 4. Experimental Results and Conclusions

[0553] In a mouse subcutaneous xenograft tumor model of the human endometrial cancer cell line RL95-2, drug A combined with drug I showed stronger antitumor activity compared with each drug alone, without significant toxic side effects, and demonstrated good safety.

[0554] Example 15: Phase I clinical study of the safety, tolerability, pharmacokinetics, and efficacy of drug A combination therapy in subjects with advanced solid tumors.

[0555] 1. Research Objective:

[0556] Main research objectives

[0557] To evaluate the safety and tolerability of drug A in combination therapy in subjects with advanced solid tumors.

[0558] Secondary research objectives

[0559] 1. To evaluate the pharmacokinetic (PK) characteristics of combination therapy with drug A in subjects with advanced solid tumors;

[0560] 2. Evaluate other safety profiles of drug A in combination therapy in subjects with advanced solid tumors;

[0561] 3. Evaluate the efficacy of combination therapy with drug A in subjects with advanced solid tumors.

[0562] 4. Evaluate the pharmacokinetic (PK) characteristics of combination therapy with drug D in patients with advanced solid tumors;

[0563] 5. Evaluate the immunogenicity of drug D in the combination therapy regimen.

[0564] • Exploratory research objectives

[0565] 1. Explore the relationship between exposure and effect;

[0566] 2. Explore biomarkers that predict or influence the efficacy of combination therapy with drug A.

[0567] 2. Dosing regimen:

[0568] The dose-finding phase of this study was structured as follows:

[0569] The dosing regimens for each cohort are as follows:

[0570] • Cohort 1: Drug A combined with novel endocrine therapy

[0571] Drug A: During the escalation phase, administer an initial dose of 40 mg QD. Based on the results of the escalation phase, 1-2 doses will be selected for the extension phase. Subjects will receive the drug once daily orally starting on day 1 (C1D1), with each treatment cycle lasting 21 days. Subjects should fast for 2 hours before and 1 hour after administration of drug A.

[0572] Novel endocrine therapy: Based on the appropriate cohort of enrolled subjects and the investigator's choice, subjects will take enzalutamide 160 mg QD, revelulide 240 mg QD, or abiraterone 300 mg QD orally consecutively. Dosing will begin on C1D1, with each treatment cycle lasting 21 days. Enzalutamide, revelulide, and abiraterone can be administered regardless of food intake. For subjects taking abiraterone, mCRPC patients should concurrently take prednisone or prednisolone 5 mg BID, and mCSPC patients should concurrently take prednisone or prednisolone 5 mg QD.

[0573] All medications were continued until objective disease progression (except for continued treatment after disease progression) or other criteria for discontinuation of treatment were met.

[0574] There is no specific order of administration for Drug A and novel endocrine therapy, as well as prednisone or prednisolone (in the abiraterone group).

[0575] • Cohort 2: Drug A combined with Drug D

[0576] Drug A: During the escalation phase, administer a starting dose of 40 mg QD. Subjects will begin with C1D1, administered orally once daily for 21 days as one treatment cycle. Subjects should fast for 2 hours before and 1 hour after administration of Drug A.

[0577] Drug D: During the escalation period, the initial dose is 6 mg / kg every 3 weeks. Subjects will receive intravenous drug D starting at C1D1 (+3 days), with each treatment cycle lasting 21 days. Starting from C2, the interval between the first intravenous dose and the previous first intravenous dose will be 21 (±3) days.

[0578] Based on the results of the escalation period, the SRC will select one or two combination dosing regimens for expansion based on the obtained safety, tolerability, PK, and efficacy data.

[0579] All medications were continued until objective disease progression (except for continued treatment after disease progression) or other criteria for discontinuation of treatment were met.

[0580] • Cohort 3: Drug A in combination with apatinib

[0581] Drug A: During the escalation phase, administer an initial dose of 40 mg QD. Based on the results of the escalation phase, 1-2 doses will be selected for the extension phase. Subjects will receive the drug once daily orally starting on C1D1, with each treatment cycle lasting 21 days. Subjects should fast for 2 hours before and 1 hour after administration of Drug A.

[0582] Apatinib: Administer at a dose of 500 mg once daily. Start with C1D1 and administer once daily orally for 21 days as one treatment cycle. Apatinib should be taken half an hour after a meal. Each treatment cycle is 21 days.

[0583] All medications were continued until objective disease progression (except for continued treatment after disease progression) or other criteria for discontinuation of treatment were met.

[0584] • Cohorts 4A-1, 4B-1, and 4B-2: Drug A in combination with a paclitaxel-based chemotherapy drug (albumin-bound paclitaxel or docetaxel)

[0585] Drug A: During the escalation phase, administer an initial dose of 40 mg QD. Based on the results of the escalation phase, 1-2 doses will be selected for the extension phase. Subjects will receive the drug once daily orally starting on C1D1, with each treatment cycle lasting 21 days. Subjects should fast for 2 hours before and 1 hour after administration of Drug A.

[0586] Paclitaxel chemotherapy drugs: Subjects will receive albumin-bound paclitaxel 260 mg / m² according to investigator selection. 2 Q3W or docetaxel 75mg / m 2 The Q3W dose will be administered intravenously. Subjects will begin administration on C1D1 (+3 days), with each treatment cycle lasting 21 days. Starting from C2, the interval between the first intravenous dose and the previous first intravenous dose will be 21 (±3) days.

[0587] All medications should be continued until objective disease progression (except for continued treatment after disease progression) or other criteria for discontinuation of treatment are met.

[0588] • Cohorts 4A-2 and 4B-3: Drug A in combination with irinotecan liposomes

[0589] Drug A: During the escalation phase, administer an initial dose of 40 mg QD. Based on the results of the escalation phase, 1-2 doses will be selected for the extension phase. Subjects will receive the drug once daily orally starting on C1D1, with each treatment cycle lasting 14 days. Subjects should fast for 2 hours before and 1 hour after administration of Drug A.

[0590] Irinotecan liposomes: at 56.5 mg / m² 2Q2W intravenous administration. Subjects will begin administration on C1D1 (+3 days), with each treatment cycle lasting 14 days. Starting from C2, the interval between the first intravenous dose and the previous first intravenous dose will be 14 (±3) days.

[0591] All medications should be continued until objective disease progression (except for continued treatment after disease progression) or other criteria for discontinuation of treatment are met.

[0592] • Cohorts 5A and 5B: Drug A in combination with bevacizumab

[0593] Drug A: During the escalation phase, administer an initial dose of 40 mg QD. Based on the results of the escalation phase, 1-2 doses will be selected for the extension phase. Subjects will receive the drug once daily orally starting on day 1 (C1D1), with each treatment cycle lasting 21 days. Subjects should fast for 2 hours before and 1 hour after administration of drug A.

[0594] Bevacizumab: Subjects will receive intravenous administration of 15 mg / kg every 3 weeks. All medications will continue to be administered until objective disease progression (except for continued treatment after disease progression) or other criteria for discontinuation of treatment are met.

[0595] There is no required order of administration for drug A and bevacizumab.

[0596] All oral medications should be swallowed whole with water; do not break or chew them. If a subject misses a dose, they should take it as soon as possible. If a full day's dose is missed, the subject should continue taking the medication at the usual daily dose the following day. If a subject vomits after taking the medication, no additional dose should be taken; the next dose should be taken at the next scheduled time.

[0597] All intravenous medications should be administered after drug A, and intravenous administration should be completed within 6 hours after drug A. The dosage for each cycle should be calculated based on the subject's weight in the 3 days prior to each administration. If the investigator decides to adjust the dosage based on the subject's specific circumstances, it must be agreed upon with the sponsor.

[0598] Based on the obtained safety, efficacy, and pharmacokinetic (PK) data, the SRC may decide to change the dosing frequency (e.g., changing drug A to BID dosing), dosage, or duration of dosing for drugs A and D.

[0599] Study endpoint:

[0600] • Primary endpoint: MTD or MAD with combination therapy of drug A.

[0601] Secondary study endpoints:

[0602] 1. PK characteristics of combination therapy with drug A.

[0603] 2. Safety of combination therapy with drug A.

[0604] 3. Efficacy of combination therapy with drug A (investigator assessment):

[0605] a. Objective response rate (ORR), disease control rate (DCR), and duration of response (DoR) assessed by investigators according to RECIST v1.1 criteria are applicable to subjects with target lesions at baseline (all solid tumors except prostate cancer);

[0606] b. Progression-free survival (PFS) as assessed by investigators according to RECIST v1.1 criteria (for all solid tumors except prostate cancer);

[0607] c. Overall survival (OS);

[0608] d. ORR (ovarian cancer only) assessed by researchers according to RECIST v1.1 and GCIG CA-125 criteria;

[0609] e. ORR, DCR, DoR, and radiographic progression-free survival (rPFS) as assessed by investigators according to RECIST v1.1 (soft tissue) and PCWG3 criteria (bone lesions) (prostate cancer only);

[0610] f. Proportion of subjects with a CA-125 decrease of ≥50% from baseline and CA-125 progression time (ovarian cancer only);

[0611] g. PSA50 response rate and PSA progression time (prostate cancer only).

[0612] 4. PK characteristics of combination drug D therapy in patients with advanced solid tumors.

[0613] 5. Immunogenicity of drug D in combination therapy: proportion of subjects who were positive for anti-B7H3 antibody (ADA).

[0614] • Exploratory research endpoint:

[0615] 1. Explore the relationship between exposure and effect;

[0616] 2. Explore biomarkers that predict or influence the efficacy of combination therapy with drug A.

Claims

1. A pharmaceutical combination formulation or pharmaceutical composition comprising: (1) The first active ingredient is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof. (2) The second active ingredient comprises one or more of the following drugs: (a) androgen receptor antagonists; (b) AR inhibitors; (c) CYP17 inhibitors; (d) B7H3 inhibitors; (e) tyrosine kinase inhibitors; (f) natural antitumor drugs and immunomodulators, other cytotoxic drugs; (g) DNA topoisomerase I inhibitors; The inhibitor drugs mentioned above can be in any form that contains a specific drug.

2. The pharmaceutical combination formulation or pharmaceutical composition of claim 1, wherein the androgen receptor antagonist is enzalutamide; preferably N-desmethylenzalutamide; the AR inhibitor is revelutamide; the CYP17 inhibitor is abiraterone; the B7H3 inhibitor is an antibody-drug conjugate; the tyrosine kinase inhibitor is apatinib or bevacizumab; the natural antitumor drug and immunomodulator, and other cytotoxic drugs are albumin-bound paclitaxel or docetaxel; and the DNA topoisomerase I inhibitor is topotecan, irinotecan and its metabolites, SN38 or its liposomes.

3. The pharmaceutical combination formulation or pharmaceutical composition according to claim 1 or 2, wherein the B7H3 inhibitor is an antibody-drug conjugate; the structure of the antibody-drug conjugate is shown in formula (I): in: n is 1 to 10, preferably 2 to 8, more preferably 3 to 8, and n is a decimal or an integer; Pc is an anti-B7H3 antibody or its antigen-binding fragment.

4. The pharmaceutical combination formulation or pharmaceutical composition according to claim 3, wherein the anti-B7H3 antibody or its antigen-binding fragment comprises: heavy chains HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO:01, 02, and 03, respectively, and light chains LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO:04, 05, and 06, respectively.

5. The pharmaceutical combination formulation or pharmaceutical composition according to claim 3 or 4, wherein the anti-B7H3 antibody or its antigen-binding fragment is selected from humanized antibodies or fragments thereof; preferably, the anti-B7H3 antibody or its antigen-binding fragment comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotypes, and a light chain constant region comprising κ or λ; preferably, the anti-B7H3 antibody or its antigen-binding fragment comprises a heavy chain constant region of IgG1 or IgG4 isotypes.

6. The pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 3-5, wherein the heavy chain variable region sequence of the anti-B7H3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 07 or a variant thereof, and the light chain variable region sequence is as shown in SEQ ID NO: 08 or a variant thereof; preferably, the heavy chain sequence of the anti-B7H3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 09 or a variant thereof, and the light chain sequence is as shown in SEQ ID NO: 10 or a variant thereof.

7. The pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 1-6, characterized in that, The first active ingredient is a pharmaceutically acceptable salt selected from hydroxyethyl sulfonate, hydrochloride, sulfate, 1,5-naphthalenedisulfonate, methanesulfonate, hydrobromide, ethanesulfonate, phosphate, benzenesulfonate, oxalate, maleate, adipate, hydrochloride, citrate, malonate, L-malate, pamoate, p-toluenesulfonate, or fumarate; preferably hydrochloride, sulfate, methanesulfonate, hydrobromide, or p-toluenesulfonate.

8. The pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 1-7, characterized in that, The content range of the first active ingredient or the second active ingredient is independently from 1% to 99%; preferably from 1% to 90%, more preferably from 1% to 80%; 1% to 70%; 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 5% to 10%, 5% to 20%, 5% to 30%, 5% to 40%, 5% to 50%, 5% to 60%, by total weight of active ingredients.

9. The pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 1-8, characterized in that, The administration routes for the first or second active ingredient are selected from gastrointestinal, injection, respiratory, or transdermal administration; the gastrointestinal administration is preferably oral, sublingual, or rectal administration; the injection administration is preferably intravenous, intramuscular, or subcutaneous administration; oral administration is preferred.

10. The pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 1-9, characterized in that, The dosage of the first active ingredient is 5-300 mg; preferably 10-150 mg; more preferably 10 mg, 20 mg, 40 mg, 80 mg or 120 mg; preferably, the frequency of administration of the first active ingredient is once daily, twice daily, three times daily, four times daily, every other day, once weekly, twice weekly, three times weekly, every other week, three times every two months, four times every two months, five times every two months, twice a month or once a month.

11. The pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 1-10, characterized in that, The oral dosage of the second active ingredient is 50–1000 mg; preferably 150–1000 mg; more preferably 50–100 mg, 100–150 mg, 150–200 mg, 200–250 mg, 250–300 mg, 300–350 mg, 350–400 mg, 400–450 mg, 450–500 mg, 500–550 mg, 550–600 mg, 650–700 mg, 750–800 mg, 800–850 mg, 850–900 mg, or 950–1000 mg; the intravenous dosage of the second active ingredient is 30–500 mg / m². 2 Preferred concentration: 30–50 mg / m³ 2 50-100 mg / m 2 100-150 mg / m 2 150-200 mg / m 2 200-250 mg / m 2 250~300mg / m 2 300-350 mg / m 2 350~400mg / m 2 400-450 mg / m 2 Or 450-500 mg / m 2 ; Specifically, the dosage of enzalutamide is 80–200 mg, preferably 80 mg, 120 mg, 140 mg, or 160 mg; The dosage of reverutamide is 80–300 mg, preferably 80 mg, 160 mg, or 240 mg; The dosage of abiraterone or its nanocrystalline formulation is 100-1000 mg, preferably 150 mg, 225 mg, 300 mg, 500 mg, 750 mg, or 1000 mg. The dosage of the B7H3 antibody-drug conjugate is from 0.1 mg / kg to 12.0 mg / kg, preferably from 1.0 mg / kg to 12.0 mg / kg, and the dosing frequency is once a week, once every two weeks, once every three weeks, or once every four weeks. The dosage of apatinib is 200–900 mg, preferably 250 mg, 375 mg, 500 mg, 750 mg, or 850 mg; The dosage of bevacizumab is 1.0 mg / kg to 100 mg / kg, preferably 1.0 mg / kg to 40 mg / kg, more preferably 1.0 mg / kg to 30 mg / kg, more preferably 10-25 mg / kg, more preferably 15-20 mg / kg, and more preferably 15 mg / kg. The dosing frequency is once a week, once every two weeks, once every three weeks, or once every four weeks. The dosage of albumin-bound paclitaxel is 150–300 mg / m². 2 Preferred concentration: 180 mg / m 2 220mg / m 2 260mg / m 2 280mg / m 2 ; The dosage of docetaxel is 40–200 mg / m². 2 Preferred concentration: 40–50 mg / m³ 2 50-65 mg / m 2 70-80 mg / m 2 80-85 mg / m 2 ; The dosage of topotecan, irinotecan and its metabolites, SN38 or its liposomes is 1–400 mg / m². 2 Preferred concentration: 30–400 mg / m³ 2 Preferred concentration: 1–10 mg / m³ 2 1.2~8mg / m 2 1.4~7mg / m 2 30-40 mg / m 2 40-50 mg / m 2 50-60 mg / m 2 330mg / m 2 350mg / m 2 380mg / m 2 1.2 mg / m 2 1.25 mg / m 2 1.4 mg / m 2 1.6 mg / m 2 1.8 mg / m 2 2.0 mg / m 2 ; Preferably, the administration frequency of the second active ingredient is once daily, twice daily, three times daily, four times daily, every other day, once a week, five times every three weeks, twice a week, three times a week, once every other week, three times every two months, four times every two months, five times every two months, twice a month, or once a month; the oral administration frequency is preferably once daily or five times every three weeks, and the intravenous administration frequency is preferably once every two weeks, once every three weeks, or five times every three weeks. Specifically, enzalutamide is administered once daily; revelutamide is administered once daily; abiraterone is administered once daily; B7H3 antibody-drug conjugates are administered once every week, once every two weeks, once every three weeks, or once every four weeks; apatinib is administered once daily; bevacizumab is administered once every week, once every two weeks, or once every three weeks, preferably once every three weeks; albumin-bound paclitaxel or docetaxel is administered once every three weeks; topotecan, irinotecan and its metabolites, SN38 or its liposomes are administered once every three weeks, once every two weeks, or five times every three weeks.

12. The pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 1-11, characterized in that... It may further include one or more pharmaceutically acceptable carriers, excipients, and diluents.

13. The pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 1-12, wherein the first active ingredient and the second active ingredient may be administered simultaneously, in parallel, sequentially, continuously, alternately or separately.

14. Use of the pharmaceutical combination formulation or pharmaceutical composition according to any one of claims 1-12 in the preparation of a medicament for treating or preventing cancer; wherein the cancer is selected from at least one of the following: head and neck cancer, lung cancer, gastric cancer, liver cancer, kidney cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, bladder cancer, esophageal cancer, salivary gland cancer, skin cancer, pharyngeal cancer, laryngeal cancer, gallbladder cancer, bile duct cancer, thyroid cancer, uterine cancer, vulvar cancer, penile cancer, testicular cancer, urothelial carcinoma, urethral cancer, colon cancer, rectal cancer, colorectal cancer, esophagogastric junction adenocarcinoma, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, fallopian tube cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tumor, nerve sheath tumor, mesothelioma, Paget's disease, and sarcoma; The prostate cancer mentioned is advanced prostate cancer, preferably metastatic prostate cancer; more preferably metastatic hormone-sensitive prostate cancer or metastatic castration-resistant prostate cancer; more preferably metastatic prostate cancer with homologous recombination repair gene mutation; more preferably metastatic castration-sensitive prostate cancer with homologous recombination repair gene mutation and metastatic castration-sensitive prostate cancer with homologous recombination repair gene not mutated or with unknown mutation status; and further preferably metastatic castration-resistant prostate cancer that has progressed after at least one first-line treatment. in, The homologous recombination repair gene mutation is selected from one or more germline or somatic mutations of ATM, ATR, BRCA1, BRCA2, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, MLH1, MRE11, NBN, PALB2, RAD51B, RAD51C, RAD51D or RAD54L; The breast cancer mentioned is advanced breast cancer, and more specifically, advanced HER2- breast cancer with germline BRCA1 / 2 or PALB2 mutations that has progressed after at least one first-line treatment, or advanced triple-negative breast cancer that has progressed after at least one first-line treatment. The ovarian cancer mentioned is advanced ovarian cancer, preferably platinum-sensitive recurrent advanced ovarian cancer, newly diagnosed advanced ovarian cancer, or HRR-mutant advanced ovarian cancer that has failed standard treatment; or preferably first-line HRD-positive ovarian cancer. The gastric cancer mentioned is advanced gastric cancer, preferably advanced gastric cancer that has progressed after at least two lines of treatment; The peritoneal cancer is preferably primary peritoneal cancer.

15. The use as described in claim 14, characterized in that, The first active ingredient in the pharmaceutical combination formulation or pharmaceutical composition is 1'-((7-ethyl-6-carbonyl-5,6-dihydro-1,5-diazanaphthyl-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide or a pharmaceutically acceptable salt thereof. The second active ingredient is enzalutamide, revelutamide, or abiraterone; The application of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumor is selected from prostate cancer, preferably advanced prostate cancer, more preferably metastatic castration-sensitive prostate cancer; or The second active ingredient is the B7H3 inhibitor in claims 3-8, which is an antibody-drug conjugate. The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumors are selected from head and neck squamous cell carcinoma, small cell lung cancer, prostate cancer and colorectal cancer, preferably in the treatment of advanced head and neck squamous cell carcinoma, advanced small cell lung cancer, advanced colorectal cancer, advanced prostate cancer or other solid tumors, and more preferably in the treatment of metastatic castration-resistant prostate cancer that has progressed after at least one first-line treatment. or The second active ingredient is apatinib; The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumor is selected from breast cancer or ovarian cancer, preferably advanced breast cancer or ovarian cancer or other solid tumors, more preferably in the treatment of advanced HER2- breast cancer with germline BRCA1 / 2 or PALB2 mutations that has progressed after at least first-line treatment; or The second active ingredient is albumin-bound paclitaxel or docetaxel; The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumor is selected from breast cancer, gastric cancer, or ovarian cancer, preferably in patients with advanced breast cancer, advanced gastric cancer, or advanced ovarian cancer or other advanced solid tumors, more preferably in patients with HRR-mutant advanced ovarian cancer who have failed standard treatment or advanced gastric cancer that has progressed after at least two lines of treatment; or The second active ingredient is topotecan, irinotecan and their metabolites, SN38 or its liposomes; The use of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumor is selected from breast cancer, gastric cancer, or ovarian cancer, preferably in the treatment of patients with advanced breast cancer, advanced gastric cancer, or ovarian cancer or other advanced solid tumors, and more preferably in the treatment of advanced gastric cancer that has progressed after at least two lines of therapy; or The second active ingredient is bevacizumab; The application of this pharmaceutical composition in the treatment of solid tumors, wherein the solid tumors are selected from advanced ovarian cancer, fallopian tube cancer or peritoneal cancer, preferably advanced first-line HRD-positive ovarian cancer or primary peritoneal cancer.

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