CN117159577A - Antitumor pharmaceutical composition for combination therapy - Google Patents

Abstract

The present application relates to an anti-tumor pharmaceutical composition for combination therapy, which contains (a) azivudine or a pharmaceutically acceptable salt, prodrug, hydrate, solvate or isotope-labeled analog thereof and (b) gemcitabine prodrug or its pharmaceutically acceptable salts, hydrates, solvates or isotopically labeled analogs.

Description

Anti-tumor pharmaceutical composition for combination therapy

Technical field

The present application relates to the field of medicinal chemistry, specifically to anti-tumor pharmaceutical compositions for combination therapy.

Background technique

Gemcitabine (compound 1; trade name ) is a potent nucleoside analog that has been shown to treat breast cancer, non-small cell lung cancer, ovarian cancer, and pancreatic cancer, and is widely used to treat a variety of other cancers, including bladder cancer, biliary tract cancer, and colorectal cancer cancer and lymphoma.

Gemcitabine has shown efficacy in several clinical settings, but it also has side effects such as flu-like symptoms, diarrhea, weakness, mouth sores, and shortness of breath. The clinical efficacy of gemcitabine is limited by multiple intrinsic and acquired resistance mechanisms. Resistance at the cellular level depends on three parameters: (i) downregulation of deoxycytidine kinase necessary for activation into phosphorylated groups; (ii) reduction of hENT1, a nucleoside transporter required for uptake in particular by cancer cells. expression, and (iii) upregulation of catalytic enzymes, especially cytidine deaminase, which degrades gemcitabine.

WO2005/012327 describes a series of nucleotide prodrugs of gemcitabine and related nucleoside drug molecules. Among them, gemcitabine-[phenyl(benzoyloxy-L-alanyl)]phosphate (NUC-1031, compound 2) is considered to be the most effective compound. These prodrugs appear to avoid many of the intrinsic and acquired resistance mechanisms that limit the effectiveness of gemcitabine ('Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent(NUC-1031) in Clinical Development'; Slusarczyk et all; J. Med. Chem.; 2014, 57, 1531-1542). Unfortunately, NUC-1031 (Compound 2) is very lipophilic and therefore poorly soluble in water (by calculation: <0.1 mg/mL), which has implications for the development of formulations that deliver the prodrug at doses high enough to achieve effective therapy . It also has implications for the development of efficient manufacturing processes that allow NUC-1031 to be produced in a cost-effective manner.

WO2014/078295 describes a class of gemcitabine prodrugs: Compound 3 (Example 12) and Compound 3a (Example 22), as follows:

WO2020/001475 describes a class of phosphorus-containing prodrugs of gemcitabine: Compound 4 (Example 1) showed significant overall safety improvements (improved therapeutic index (TI)) compared to the parent compound gemcitabine:

Azivudine (compound 5, abbreviated as FNC) is a pyrimidine nucleoside drug with broad-spectrum antiviral activity. It has a dual-target mechanism of action as a nucleoside reverse transcriptase inhibitor and an accessory protein Vif (viral infection factor) inhibitor. Patent ZL200710137548.0 (Example 15) discloses FNC structural information. FNC can selectively enter CD4 cells or CD14 cells in HIV-1 target cells, peripheral blood mononuclear cells, and exert its function to inhibit the replication of RNA viruses such as HIV, HCV, and EV71.

FNC inhibits positive RNA viruses such as HCV, EV, SARS-COV-2, HBV and retroviruses (including HIV) by inhibiting RNA-dependent polymerase. It also inhibits this enzyme (reverse transcriptase) in human retrotransposons, including human endogenous retroviruses (HERVs). Because activation of retrotransposons may be a major contributor to ongoing cancer genome instability and, thus, higher tumor aggressiveness, FNCs have the potential to improve the efficacy of multiple anticancer therapies. In addition, FNC also shows other anti-cancer activities by inhibiting the adhesion, migration, invasion and proliferation of malignant cells. It is also reported to be involved in cell cycle arrest and apoptosis, thus inhibiting cancer progression through different pathways. FNC is a cell cycle non-specific drug that can cause cell cycle arrest in the G1/S or G2/M phase and induce apoptosis.

Azivudine, as a nucleoside drug, has potential anti-tumor activity. It can compete with the nucleotides produced inside cells and be incorporated into DNA and RNA chains, thereby leading to the weakening of nucleotide synthesis in tumor cells and interfering with the growth and division of tumor cells. . Modulating the immune system may be another important anti-tumor mechanism of FNC. Patent ZL200710137548.0 describes that the 2’-fluoro-4’-azido-nucleoside analog FNC has a significant inhibitory effect on a variety of human cancer cells and animal transplanted tumors. Myelosuppression is one of the most important factors leading to the failure of clinical cancer chemotherapy. FNC has no significant effect on the bone marrow hematopoietic function of animals at effective doses, while 5-Fu and DDP have significant inhibitory effects. FNC shows low cytotoxicity, thus implying acceptable side effects of treatment.

The pathogenesis of tumors is complex, and there are many types of drugs used to treat tumors. They generally have shortcomings such as high side effects and easy development of drug resistance in patients. The inhibitory effect of a single drug on tumors can no longer meet strict treatment requirements. In order to maximize the benefits of gemcitabine-based chemotherapy regimens and improve safety and tolerability, drugs with other mechanisms of action need to be combined with gemcitabine or its medicinal salts, prodrugs, etc. to overcome drug resistance and achieve Earlier and deeper disease remission, extending disease remission time and patient survival time. Actively exploring different forms of combination drug regimens is an inevitable direction for the research and development of anti-tumor drugs.

Contents of the invention

In view of the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a dual inhibitor of nucleoside reverse transcriptase and accessory protein Vif (viral infection factor) and gemcitabine prodrug or a pharmaceutically acceptable salt, hydrate, solvate or The use of a combination of isotope-labeled analogs as anti-tumor drugs has the advantage of dual inhibitors of nucleoside reverse transcriptase and accessory protein Vif (viral infection factor) and gemcitabine prodrugs or pharmaceutically acceptable salts, hydrates, and solvates thereof Or the synergistic effect of isotope-labeled analogues, the anti-tumor effect achieved is better than that of a single drug, and it has a significant effect on inhibiting tumor volume.

In one aspect, the invention provides a pharmaceutical composition comprising:

(a) Dual inhibitors of nucleoside reverse transcriptase and accessory protein Vif (viral infection factor) or pharmaceutically acceptable salts, prodrugs, hydrates, solvates or isotope-labeled analogs thereof, and

(b) Gemcitabine prodrug or its pharmaceutically acceptable salt, hydrate, solvate or isotopically labeled analogue.

In some embodiments, the dual inhibitor of nucleoside reverse transcriptase and accessory protein Vif (viral infection factor) or a pharmaceutically acceptable salt, prodrug, hydrate, solvate or isotope-labeled analog thereof is azivudine ( Abbreviated as FNC, compound 5 disclosed in patent ZL200710137548.0 (Example 15)).

In some embodiments, the gemcitabine prodrug, or a pharmaceutically acceptable salt, hydrate, solvate, or isotopically labeled analog thereof, may be selected from any suitable gemcitabine prodrug, or a pharmaceutically acceptable salt thereof. More preferably, the gemcitabine prodrug is selected from: compound 1, compound 2, compound 3, compound 3a, compound 4; most preferably, the gemcitabine prodrug is compound 4:

In another aspect, the pharmaceutical composition of the invention further comprises an immunotherapeutic agent selected from PD-1 blockers or PD-L1 blockers or CTLA-4 blockers.

In some embodiments, the immunotherapeutic agent is selected from the group consisting of PD-1 antibodies or antigen-binding fragments thereof, PD-L1 antibodies or antigen-binding fragments thereof, soluble PD-1 or CTLA-4 antibodies or antigen-binding fragments thereof.

In some embodiments, the pharmaceutical compositions of the present invention may also include additional drugs for preventing and/or treating cancer or tumors (such as small molecules, polypeptides, nucleic acids, antibodies, antibody conjugates).

In some embodiments, drugs used to prevent and/or treat cancer or tumors are selected from antibody conjugates, and representative examples thereof may include, but are not limited to, trastuzumab emtansine, vibutuxin Brentuximab Vedotin, brentuximab vedotin (BV), Disitamab Vedotin, sacituzumab govitecan, delutican-trastuzumab Trastuzumab Deruxtecan or any combination thereof.

In another aspect, the present invention also provides a dual inhibitor of nucleoside reverse transcriptase and accessory protein Vif (viral infection factor) and gemcitabine or its pharmaceutically acceptable salts, prodrugs, hydrates, solvates or isotope-labeled A method of treating a disease in combination with an analogue, the method comprising administering a composition of the invention to a patient in need thereof.

In another aspect, the present invention also provides the use of the above pharmaceutical composition in the preparation of medicaments for treating diseases.

In some embodiments, the disease is selected from cancer or tumors. Representative examples of cancers and tumors include, but are not limited to, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neuroblastoma, rectal cancer , colon cancer, familial adenomatous polyposis carcinoma, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, adenocarcinoma, medullary thyroid carcinoma, papillary Thyroid cancer, kidney cancer, renal parenchymal cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary cancer, melanoma, brain tumors (such as glioblastoma, astrocytoma , meningiomas, medulloblastomas, and peripheral neuroectodermal tumors), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), adult T-cell leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell Lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, muscle tumors, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma.

In some embodiments, the cancer and tumor are selected from the group consisting of intermediate and advanced tumors, relapsed and refractory tumors, tumors that have failed and/or relapsed after chemotherapy drug treatment, failed and/or relapsed tumors after radiotherapy, failed and/or relapsed tumors after targeted drug treatment, and /or tumor recurrence, and immunotherapy failure and/or tumor recurrence.

As the most preferred technical solution of the present invention, the combination drug involved in the present invention is azivudine (compound 5) and gemcitabine prodrug or its pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled analog (compound 4) Combined, they inhibit the activity of many cancer-causing viruses. In addition, as a nucleotide analog, the combination showed cancer cell growth and cancer progression compared with the administration of Compound 5 alone or the administration of gemcitabine prodrug or its pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled analogs alone. A stronger inhibition of active cell proliferation, which penetrates synthetic nucleic acid chains and causes chain termination, thus inducing apoptosis of tumor cells. At the same time, the combination of drugs has high biological safety, has stronger anti-tumor activity than single drugs, overcomes the resistance of single drugs, and provides new strategies and ideas for the treatment of tumors.

Description of drawings

Figure 1 shows the pharmacodynamic effect of FNC (compound 5) combined with compound 4 on the HCT-116 model.

Detailed ways

definition

As used herein, the term "combination" refers to a mode of administration whereby at least one dose of azivudine and at least one dose of gemcitabine or a pharmaceutically acceptable salt, prodrug or Hydrates, solvates or isotopically labeled analogues and/or at least one dose of a PD-1 blocker or a PD-L1 blocker or a CTLA-4 blocker, two or three of which exhibit pharmacological properties learning function. The time period may be within one administration cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week or within 24 hours, more preferably within 12 hours. Azivudine and gemcitabine or its pharmaceutically acceptable salts, prodrugs, hydrates, solvates or isotopically labeled analogs and/or PD-1 blockers or PD-L1 blockers or CTLA- 4 blockers. Such term includes treatments in which azivudine and gemcitabine or a pharmaceutically acceptable salt, prodrug, hydrate, solvate or isotopically labeled analog thereof and/or PD are administered by the same route of administration or by different routes of administration -1 blockers or PD-L1 blockers or CTLA-4 blockers. The combined administration mode of the present invention is selected from simultaneous administration, independently formulated and co-administered, or independently formulated and administered sequentially.

As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, and sulfuric acid, and salts with organic carboxylic and sulfonic acids such as acetic acid, trifluoroacetic acid, propionic acid, maleic acid, and sulfuric acid. Salts of malic acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid and naphthalenedisulfonic acid; or salts with conventional bases, Such as alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), derived from ammonia and organic amines (such as diethylamine, triethylamine, ethyldiisopropylamine, triethylamine, Lucaine, dibenzylamine, N-methylmorpholine, dihydroabiethylamine, methylpiperidine, L-arginine, creatine, choline, L-lysine, ethylenediamine , N,N-dibenzylethylenediamine (benzathine), ethanolamine, meglumine and tromethamine) ammonium salts.

As used herein, the term "prodrug" refers to a compound that undergoes biotransformation before exhibiting its pharmacological effect. Prodrugs can be prepared using well-known methods, such as those described in Burgers Medicinal Chemistry and Drug Development (1995) 172-178, 949-982 (Manfred E. Wolff).

As used herein, the term "solvate" refers to a complex formed by coordination with a solvent molecule. When the solvent molecule is water, the solvate is a hydrate.

Compounds as described herein may be isotopically labeled, ie, one or more atoms therein are replaced by atoms with different atomic masses or mass numbers. Such isotopically labeled compounds are considered to be within the scope of the present invention. Examples of isotopes that may be incorporated into the compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as, but not limited to, ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, respectively. ^13N , ^15N , ^15O , ^17O , ^18O , ^31P , ^32P , ^35S , ^18F , ^36Cl , ^123I and ^125I .

Example

The invention is illustrated below by the following non-limiting examples.

Example 1. Determination of cell proliferation inhibitory activity of compound combination using CTG (CELLTITER-GLO) luminescence method

Test principle: Promega's CellTiter-Glo ^TM luciferase kit is used. This method uses luciferase as the detection substance. During the luminescence process, luciferase requires the participation of ATP, the respiration of metabolically active cells and other Life activity processes can produce ATP. Add an equal volume of CellTiter-Glo ^TM reagent to the cell culture medium and measure the luminescence value. The light signal is proportional to the amount of ATP in the system. ATP is positively correlated with the number of viable cells, so it is used to detect the inhibition of cell proliferation activity by compounds. In order to detect whether compounds with different mechanisms of action have additive or synergistic effects when combined, this combination experiment was designed.

Test method: Prepare complete culture medium according to different cell requirements, resuscitate the cells (ATCC), select cell lines with good growth status for about two generations, collect and count cells in the logarithmic growth phase, resuspend the cells to an appropriate concentration, and divide the cell suspension into Inoculate in a 384-well plate, add 50 μL of cell suspension to each well, the seed plate density is 600 cells/well, and place the culture plate in a 37°C, 5% _CO2 incubator overnight. The next day, the compound to be tested was prepared into a storage solution with DMSO, and added according to the predetermined arrangement (9*9 matrix) using HPD 300e. After centrifugation at 1000 rpm for 1 minute, it was incubated in a 37°C, 5% carbon dioxide incubator for 72 hours. Add 25 μL/well of CellTiter Glo to end the reaction, incubate at room temperature in the dark for 30 minutes, shake gently and then detect in Envision. Read the luminescence value and calculate the inhibition rate according to the formula cell growth inhibition rate % = (1-As/Ac) × 100. Among them, As is the sample (cells + CTG + test compound), and Ac is the normal growth cell control (cells + CTG + DMSO). Enter the inhibition rate Inh% (Y) corresponding to each concentration (X) in EXCEL, and use the XLfit plug-in to fit the built-in four-parameter formula Y=Bottom+(Top-Bottom)/(1+(IC50/X)*HillSlope) The half inhibitory concentration IC50 value of each compound was calculated. SynergyFinder is used to calculate the synergy score (Bliss) value, where less than -10 indicates that the interaction between the two drugs is antagonistic, between -10 and 10 indicates that the interaction between the two drugs is additive, and greater than 10 Indicates that the interaction between the two drugs is synergistic.

The results showed that the combined use of azivudine (FNC) and gemcitabine showed synergistic effects on multiple test cell lines.

Example 2. Efficacy evaluation of FNC combined with gemcitabine prodrug (compound 4) on HCT-116 human colon cancer model

Experimental principle: First, establish an experimental animal model of tumor. Based on the successful establishment of the animal model of tumor, perform anti-tumor treatment on the animal experimental samples. Finally, make correct judgments on the effectiveness, safety and action characteristics of the drug by observing the treatment effect. evaluate.

experimental method:

Human colon cancer HCT116 cells were cultured in a monolayer in vitro. The culture conditions were McCoy's 5a medium plus 10% fetal calf serum and 1% penicillin/streptomycin/amphotericin B, and cultured in a 37°C, 5% CO2 _incubator . Passage was performed twice a week with routine digestion treatment with trypsin-EDTA. When the cell saturation is 80%-90% and the number reaches the requirement, collect the cells. 0.1 mL (5 × 10 ⁶ cells) of HCT116 cells were inoculated subcutaneously into the right back of each mouse (BALB/c nude mouse, female, 6-8 weeks old) near the upper limb. The average tumor volume reached approximately ~125 mm. Dosing in groups begins at ³ o'clock. Tumor volume was measured twice a week, and at the end of the experiment, the tumor growth inhibition rate was calculated (TGI (%) = [1-(average tumor volume at the end of administration for a certain treatment group - average tumor volume at the beginning of administration for this treatment group)/ (Average tumor volume at the end of treatment in the solvent control group - Average tumor volume at the beginning of treatment in the solvent control group)] × 100%).

The experimental grouping and dosing schedule are as follows:

1. Vehicle control group

2.FNC, 1mg/kg, PO (oral), QD (administered once a day)

3. Compound 4, 30 mg/kg, PO (oral), QD (administered once daily)

4.FNC+Compound 4, 1mg/kg+30mg/kg, PO (oral), QD (administered once a day)

As shown in Figure 1, on the 12th day after administration, the tumor volume in the vehicle control group was 787mm ³ , the tumor volume in the FNC group was 257mm ³ , and the TGI was 80.0%, which significantly inhibited tumor growth; the tumor volume in the compound 4 group was 307 mm ³ , TGI was 72.5%, significantly inhibiting tumor growth; the tumor volume of FNC combined with compound 4 group was 169 mm ³ , TGI was 93.2%, showing significant synergistic tumor inhibition effect.

The results showed that the combination of azivudine (FNC) and gemcitabine prodrug (compound 4) showed better inhibitory effect on cancer cells than single drugs.

Claims (8)

1. A pharmaceutical composition, said pharmaceutical composition comprising: (a) Azivudine or its pharmaceutically acceptable salts, prodrugs, hydrates, solvates or isotopically labeled analogs, as well as (b) Gemcitabine prodrug or its pharmaceutically acceptable salt, hydrate, solvate or isotopically labeled analogue. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises an immunotherapeutic agent selected from the group consisting of PD-1 blockers or PD-L1 blockers or CTLA-4 blockers. Breaking agent. 3. The pharmaceutical composition according to claim 1, wherein the gemcitabine prodrug is selected from: 4. The pharmaceutical composition according to claim 3, wherein the gemcitabine prodrug is: 5. The pharmaceutical composition according to any one of claims 1-4, wherein the pharmaceutical composition further comprises additional drugs for preventing and/or treating cancer or tumors (such as small molecules, polypeptides, nucleic acids, Antibodies, antibody-drug conjugates). 6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of medicaments for the treatment of cancer and tumors. 7. Use according to claim 6, wherein the cancers and tumors include: skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, Neurocytoma, rectal cancer, familial adenomatous polyposis carcinoma, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, adenocarcinoma, medullary thyroid cancer , papillary thyroid cancer, renal cancer, renal parenchymal cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary cancer, melanoma, brain tumors such as glioblastoma, astrocytoma Cytomas, meningiomas, medulloblastomas and peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, leukemia, acute lymphoblastic leukemia (ALL), chronic lymphoid Leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), adult T-cell leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small Cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, Sarcomas, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma. 8. Use according to claim 7, wherein the cancer and tumor are selected from the group consisting of colon cancer, small cell lung cancer, pancreatic cancer and gastric cancer.