Pharmaceutical composition for realizing co-inhibition of tk1 / ts dual targets and use thereof

Abstract

The application provides a drug composition for realizing TK1 / TS double-target co-inhibition and application thereof. The creativity is derived from the key mechanism disclosed by the inventors for the first time, that is, thymidine kinase 1 (TK1) and thymidylate synthase (TS) present co-expression up-regulation in the DNA synthesis period. In-vivo experiments show that the tumor inhibition rate of the drug composition is significantly higher than that of any single drug, synergistic effect is realized, and the drug composition can be used for treating solid tumors or hematological malignancies.

Description

Technical Field

[0001] This invention pertains to the pharmaceutical field, and specifically relates to a novel pharmaceutical composition comprising a TK1 inhibitor and a TS inhibitor, its dosage, ratio, use, and route of administration. In particular, the pharmaceutical composition exhibits a significantly higher tumor inhibition rate than either single agent, and achieves synergistic effects. Background Technology

[0002] In 2024, Gao Shan et al. of Nankai University (hereinafter referred to as the inventors) discovered for the first time in the world through single-cell transcriptome big data mining that cycling cells are a type of stem cells in a state of rapid proliferation and division[1]. The inventors divided cycling cells into two major categories: cycling immune cells (CICs) and cycling non-immune cells (CNICs). Cycling immune cells are further subdivided into cycling myeloid cells (CMCs) and cycling lymphoid cells (CLCs). In particular, cancer cells are a special type of cycling cell. The inventors can easily and accurately identify cycling cells with only 26 marker genes and further reveal a key mechanism common to all cycling cells: by co-expressing several key enzymes that upregulate the ribonucleotide (NTPs) to deoxyribonucleotide (dNTPs) conversion pathway, the dNTP pool is rapidly expanded in a short time to meet the instantaneous surge demand for dNTPs in DNA replication. Because all cell cycles, including tumor cells, are in a state of rapid proliferation and division, their demand for dNTPs is far higher than that of non-cell cycles (including all normal cell types in vivo). Therefore, the surge in dNTP demand has become the "Achilles' heel" of tumor cells. The above findings will be published immediately in the form of an academic paper after the filing of the invention patent application.

[0003] Among the key enzymes upregulated in expression, thymidylate synthase (TS) and thymidine kinase 1 (TK1) control the de novo and salvage pathways of deoxythymidine triphosphate (dTTP), respectively (see instruction manual appendix). Figure 1 Due to the upregulation of TK1 and TS co-expression (see instruction manual appendix) Figure 2Any single enzyme inhibition will be compensated by another pathway, resulting in limited efficacy of single enzyme inhibitors, which may manifest as clinical drug resistance. Only by co-inhibiting TK1 and TS and blocking the de novo and salvage synthesis of dTTP can tumor cells be made to die due to the obstruction of DNA synthesis. Previous studies and current clinical strategies only target single enzyme TS, the fundamental reason being that the key mechanism of co-expression upregulation of TK1 and TS has not yet been understood. Previously, other researchers have systematically studied the main reasons for the resistance of TS inhibitor 5-fluorouracil (5-FU) [2], which, in addition to the well-known DNA mismatch repair defects, also include: (1) altered anabolic metabolism, which limits the production of the main active metabolite 5-fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP); (2) increased expression or activity of TS; and (3) abnormal regulation of programmed cell death. Later studies have pointed to 5-FU resistance as being involved in tumor resistance by various immune cells, primarily tumor-associated macrophages (TAMs), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and cytotoxic T lymphocytes (CTLs). All of these studies deviated from the ribonucleotide-to-deoxyribonucleotide conversion pathway of the target TS. The inventors proposed a dual-target co-inhibition strategy of TK1 / TS, which not only provides a new paradigm for overcoming single-drug resistance but also brings tumor research and treatment back to the ribonucleotide-to-deoxyribonucleotide conversion pathway. Furthermore, TK1 / TS dual-target co-inhibition can achieve dual "anti-tumor-anti-inflammatory" therapy and also possesses potential broad-spectrum anti-inflammatory potential. Summary of the Invention

[0004] As of the date of this patent application, no literature has reported on the mechanism of TK1 and TS co-expression upregulation discovered by the inventors, nor are there any patents involving pharmaceutical compositions containing TK1 inhibitors and TS inhibitors. This invention is based on the inventors' first discovery worldwide, and accordingly, a pharmaceutical composition co-inhibiting TK1 and TS has been prepared. Experimental results show that the tumor inhibition rate of this pharmaceutical composition is significantly higher than that of any single drug, and synergistic effects are achieved. The inventors are the first to discover the mechanism of TK1 and TS co-expression upregulation and achieve dual-target co-inhibition of TK1 / TS, and have provided relevant experimental data to support this discovery.

[0005] It should be noted that this invention does not follow the conventional drug development paradigm—searching for targets and drugs based on known mechanisms and pathways—but is based on a breakthrough discovery (see appendix to the specification). Figure 2 This paper proposes a novel anti-tumor mechanism and an operable pathway. The dual targets are not simply the superposition of two targets, but are based on the inventor's internationally first-disclosed "ribonucleotide → deoxyribonucleotide" conversion pathway (see appendix to the specification). Figure 1The optimal solution is obtained through systematic analysis.

[0006] The inventors, through single-cell transcriptome big data mining, have revealed for the first time that all types of cycle cells (including all types of cancer cells) exhibit an excessive reliance on the co-regulation of TK1 and TS co-expression compared to normal cells (see appendix to the instruction manual). Figure 2 In particular, the compensatory effect of de novo and salvage synthesis of dTTP (see the attached manual). Figure 1 Therefore, co-inhibiting TK1 and TS, and blocking de novo and salvage synthesis of dTTP, can achieve synergistic effects in tumor therapy.

[0007] Given that existing drugs cannot simultaneously block both de novo and salvage synthesis of dTTP, the purpose of this invention is to simultaneously block these two complementary pathways within tumor cells. To this end, this invention provides a novel pharmaceutical composition for treating tumors, comprising: (a) a TK1 inhibitor, said TK1 inhibitor being a compound or antibody capable of reducing thymidine kinase activity; and (b) a TS inhibitor, said TS inhibitor being a compound or antibody capable of reducing thymidine synthase activity. As of the date of this patent application, there are currently no approved or investigational TK1 inhibitors for human or mammalian use; therefore, this invention designs and synthesizes a competitive TK1 inhibitor. Given that the catalytic mechanism of TK1 is well understood—it catalyzes only the phosphorylation of the 5'-hydroxyl group of thymidine (2'-deoxythymidine, dT) to generate deoxythymidine monophosphate (dTMP)—the development of TK1 inhibitors is extremely simple: simply setting a blocking group at the 5'-hydroxyl position of the deoxyribose of thymidine can directly annihilate the phosphorylation site, thereby inhibiting the catalytic activity of TK1; in a preferred embodiment, the inhibition can be achieved in one step by substituting the 5'-hydroxyl group with the R1 group shown in structural formula (I), without the need for additional skeletal modification or the introduction of complex pharmacophores. Wherein, R1 is selected from (1) halogens F, Cl, Br, I; (2) small molecule groups containing carbon or heteroatoms with a molecular weight less than 50: including alkyl, haloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, hydroxyalkyl, cyano, nitro, thioalkoxy, mercapto, and acyloxy; or (3) amino and amide oxygen groups: including unsubstituted amino, mono-C1-C2 alkyl-substituted amino, di-C1-C2 alkyl-substituted amino, and amide oxygen. The competitive inhibitor may be its pharmaceutically acceptable salt, ester, prodrug, isotope label, or stereoisomer.

[0008] (I) The four embodiments disclosed in this invention all provide a novel pharmaceutical composition for treating tumors, comprising: (a) a TK1 inhibitor, wherein the TK1 inhibitor is 2'-deoxythymidine-5'-fluoro (dT5F), corresponding to R1=F in formula (I), and its SMILES is represented as Cc1cn(C2CC(=C(CF)O2)O)c(=O)[nH]c1=O; and (b) a TS inhibitor, wherein the TS inhibitor is commercially available 5-fluorouracil (5-FU), pemetrexed, capecitabine, or floxuridine; and further defining the drug formulation, mass ratio, dosage, administration method, and tumor type treated in the pharmaceutical composition. The following four embodiments are implemented according to the four embodiments.

[0009] Compared with the prior art, the present invention has at least the following beneficial effects: (1) the pharmaceutical composition has a synergistic effect; and (2) compared with the single drug, there is no significant increase in toxic side effects. Attached Figure Description

[0010] Figure 1 This is a diagram of the ribonucleotide to deoxyribonucleotide conversion pathway constructed by the inventors. The diagram includes several key enzymes identified by the inventors (orange background), among which RNR, DCK, DUT, TK1, and TS are significantly upregulated. TS and TK1 control the de novo and salvage synthesis pathways of dTTP, respectively (within the red boxes).

[0011] Figure 2 The figure shows evidence of upregulated co-expression of TK1 and TS in cycle cells (within the black box). This evidence is obtained through bioinformatics analysis of the NCBI GEO database single-cell transcriptome dataset GSE180286. The X-axis of the figure lists the coding genes and... Figure 1 The correspondences between enzymes with different names are as follows: ribonucleotide reductase (RNR) is encoded by genes RRM1, RRM2, and RRM2B; thymidine synthase (TS) is encoded by gene TYMS; and cytidine kinase (CMK) is encoded by gene CDADC1. Among the 20 cell types (numbered 0-19) in the figure, there are two types of cycle cells: one is human t or b cycle lymphocytes (t / b CLC), numbered 10; the other is breast cancer cells (Cancer), numbered 11.

[0012] Figure 3The figures show the tumor volume changes over time for each group in Example 1. Tumor volume data for each group are presented as mean ± standard deviation (μ ± σ). Day 1 is defined as the date the tumor volume reaches 50 cubic millimeters, and the tumor volume of mice was measured in situ on days 5, 8, 11, and 15 before plotting. The control group, TK1 inhibitor monotherapy group, TS inhibitor monotherapy group, and TK1 / TS inhibitor combination group are labeled as Control (black), TK1 (green), TS (blue), and TK1 / TS (red), respectively. Detailed Implementation

[0013] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the experimental or production procedures involved in this invention are all conventional procedures widely used in the field. To more clearly illustrate the purpose, technical solutions, and advantages of this invention, some necessary technical terms are described in detail below.

[0014] In all embodiments, the efficacy of tumor treatment was evaluated using the tumor growth inhibition rate (TGI), calculated as follows: TGI (%) = (mean tumor volume of the control group − mean tumor volume of the experimental group) / mean tumor volume of the control group × 100%, where tumor volume (mm) 3 = Tumor long axis (mm) × Tumor short axis squared (mm) 2 ) / 2, see reference [3] for details. Synergistic effect is quantified by combination index (CI), and the calculation formula is: CI = TGI_obs − TGI_pred, where TGI_obs is the measured tumor inhibition rate of the drug composition; TGI_pred is its expected tumor inhibition rate, which is calculated based on the Bliss independent model, and the calculation formula is: TGI_pred = TGI_TK1 + TGI_TS − (TGI_TK1 × TGI_TS). According to reference [4], CI > 0 is judged as synergistic, and CI < 0 is judged as antagonistic. The specific calculation steps of each index are detailed in Example 1.

[0015] In all embodiments, the significance of differences in tumor inhibition rate and toxicity indicators (body weight, blood routine) among different groups of animals was determined by heteroscedasticity t-test, with a significance level of α = 0.05.

[0016] To more clearly illustrate the purpose, technical solution, and advantages of this invention, the following detailed description is provided in conjunction with embodiments. It should be understood that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. In the embodiments, experiments without specifically specified conditions or parameters are considered to have been performed under conventional conditions or conditions and parameters recommended by the product supplier. Reagents or instruments without specifically specified manufacturers are considered to be commercially available products.

[0017] Example 1. Tumor Suppression Experiment 1 Cells: COLO205 human colorectal cancer cells, 5×10⁶ 6 Cell suspensions were prepared using Hanks' Balanced Salt Solution (HBSS); Animals: 6-week-old BALB / c nude mice, subcutaneously injected with cell suspension; Grouping: control (physiological saline), TK1 inhibitor (thymidine 5-fluoro), TS inhibitor (5-fluorouracil), TK1 / TS inhibitor combination (mass ratio 2:1), with a total of 1 control group and 3 treatment groups, 6 mice in each group; Administration: Each drug was administered at 25 mg / kg, dissolved in physiological saline at pH ≈ 7, and injected intraperitoneally. Administration began when the tumor volume increased to 50 cubic millimeters (counting from day 1), once daily for 14 consecutive days. The patient was euthanized on day 21 or when the tumor volume increased to 2000 cubic millimeters (as required by ethics). Plotting: Measure and plot the tumor volume of mice on days 5, 8, 11, and 15 (see instruction manual). Figure 3 ); Results: On day 15, the efficacy of each group was compared and evaluated. The tumor inhibition rate of TK1 inhibitor was 49.15% (p<0.05), the tumor inhibition rate of TS inhibitor was 60.17% (p<0.05), and the tumor inhibition rate of TK1 / TS inhibitor combination was 81.36% (p<0.05). The synergy index CI=0.8136 - 0.7975 >0, confirming synergy. The toxicity indicators (body weight, blood routine) of TK1 / TS inhibitor combination were not significantly different from those of single drug (P > 0.05). Calculation process: TGI_TK1= (1180-600) / 1180 =0.4915; TGI_TS= (1180-470) / 1180 =0.6017; TGI_obs= (1180-220) / 1180 =0.8136; TGI_pred = 0.4915+0.6017-0.4915*0.6017 = 0.7975; CI = TGI_obs - TGI_pred = 0.8136 - 0.7975 > 0, therefore it is determined to be cooperative.

[0018] Example 2. Tumor Suppression Experiment 2 Cells: NCI-H358 human non-small cell lung cancer cells 5×10 6 One cell suspension was prepared using Hanks' balanced salt solution; Animals: 6-week-old BALB / c nude mice, subcutaneously injected with cell suspension; Grouping: control (physiological saline), TK1 inhibitor (thymidine 5-fluoro), TS inhibitor (pemetrexed), TK1 / TS inhibitor combination (mass ratio 0.5:1), with a total of 1 control group and 3 treatment groups, 6 mice in each group; Dosing (25 mg / kg for each drug), plotting, and statistical analysis were the same as in Example 1; Results: On day 15, the efficacy of each group was compared and evaluated. The tumor inhibition rate of TK1 inhibitor was 48.31% (p<0.05), the tumor inhibition rate of TS inhibitor was 59.8% (p<0.05), and the tumor inhibition rate of TK1 / TS inhibitor combination was 80.09% (p<0.05). The synergy index CI = 0.8009 - 0.7922>0, confirming synergy. The toxicity indicators (body weight, blood routine) of TK1 / TS inhibitor combination were not significantly different from those of single drug (P > 0.05).

[0019] Example 3. Tumor Suppression Experiment 3 Cells: BT549 breast cancer cells 5×10 6 One cell suspension was prepared using Hanks' balanced salt solution; Animals: 6-week-old BALB / c nude mice, subcutaneously injected with cell suspension; Grouping: control (physiological saline), TK1 inhibitor (thymidine 5-fluoro), TS inhibitor (capecitabine), TK1 / TS inhibitor combination (mass ratio 0.5:1), with a total of 1 control group and 3 treatment groups, 6 mice in each group; Dosing (25 mg / kg for each drug), plotting, and statistical analysis were the same as in Example 1; Results: On day 15, the efficacy of each group was compared and evaluated. The tumor inhibition rate of TK1 inhibitor was 47.08% (p<0.05), the tumor inhibition rate of TS inhibitor was 57.2% (p<0.05), and the tumor inhibition rate of TK1 / TS inhibitor combination was 79.01% (p<0.05). The synergy index CI = 0.7901 - 0.7735>0, confirming synergy. The toxicity indicators (body weight, blood routine) of TK1 / TS inhibitor combination were not significantly different from those of single drug (P > 0.05).

[0020] Example 4. Tumor Suppression Experiment 4 Cells: HepG2 liver cancer cells 5×10 6 One cell suspension was prepared using Hanks' balanced salt solution; Animals: 6-week-old BALB / c nude mice, subcutaneously injected with cell suspension; Grouping: control (physiological saline), TK1 inhibitor (thymidine 5-fluoro), TS inhibitor (fluorouridine), TK1 / TS inhibitor combination (mass ratio 1:1), with a total of 1 control group and 3 treatment groups, 6 mice in each group; Dosing (25 mg / kg for each drug), plotting, and statistical analysis were the same as in Example 1; Results: On day 15, the efficacy of each group was compared and evaluated. The tumor inhibition rate of TK1 inhibitor was 46.12% (p<0.05), the tumor inhibition rate of TS inhibitor was 56.8% (p<0.05), and the tumor inhibition rate of TK1 / TS inhibitor combination was 77.23% (p<0.05). The synergy index CI = 0.7723 - 0.7672>0, confirming synergy. The toxicity indicators (body weight, blood routine) of TK1 / TS inhibitor combination were not significantly different from those of single drug (P > 0.05).

[0021] References 1. Jiawei Zhang, Jingsong Shi, Liangge Wang, Xinjie Liu, Zemin Cao, Cihan Ruan, Guangzhi Ning, Shiqing Feng, Xue Yao, Shan Gao. Re-analysis of single-cell RNA-seq data reveals the origin and roles of cycling myeloid cells. Stem Cells 2024, 42(7): 593-606 2.Gmeiner WH, Okechukwu CC. Review of 5-FU resistance mechanisms in colorectal cancer: clinical significance of attenuated on-target effects. Cancer Drug Resist. 2023;6(2):257-272. 3. Ozawa Yoichi et al. Pharmaceutical composition for treating tumors [P]. Invention patent: CN 120813358A; application date: 2023-10-03. 4.Roell KR, Reif DM, Motsinger-Reif AA. An Introduction toTerminology and Methodology of Chemical Synergy-Perspectives from AcrossDisciplines. Front Pharmacol. 2017;8:158.

Claims

1. A pharmaceutical composition for treating tumors, characterized in that, The pharmaceutical composition comprises: (1) a therapeutically effective amount of a TK1 inhibitor; and (2) a therapeutically effective amount of a TS inhibitor; wherein the TK1 inhibitor is thymidine 5-fluoro, i.e., 2'-deoxythymidine-5'-fluoro, abbreviated as dT5F, whose structure corresponds to R1=F in formula (I); the TS inhibitor is selected from 5-fluorouracil (5-FU) or its pharmaceutically acceptable salts, pemetrexed, capecitabine, or floxuridine; the tumor is selected from colorectal cancer, non-small cell lung cancer, breast cancer, or liver cancer; wherein the mass ratio of the TK1 inhibitor to the TS inhibitor is (0.2–5):

1. （I）。 2. The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier or excipient, and formulated as a lyophilized powder for injection, tablet, capsule, or liposome injection.

3. Use of the pharmaceutical composition according to any one of claims 1 or 2 in the preparation of a medicament for treating tumors, wherein, The tumor is selected from colorectal cancer, non-small cell lung cancer, breast cancer, or liver cancer.

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