Application of combination of gemcitabine and brinzolamide in preparation of pancreatic cancer chemotherapeutic drugs

Abstract

The invention provides application of gemcitabine combined with brinzolamide in preparation of pancreatic cancer chemotherapeutic drugs, and belongs to the technical field of chemotherapeutic drugs. According to the technical scheme, the brinzolamide is orally taken in the conventional administration process of gemcitabine, and the tumor inhibition effect of gemcitabine can be remarkably improved. Experimental research shows that in the gemcitabine chemotherapy process, the inhibition rate of pancreatic tumors can reach 85.29% by continuously taking brinzolamide orally at the dosage of 30 mg / kg / d, which is obviously superior to that of a gemcitabine and albumin combined paclitaxel scheme or a gemcitabine and capecitabine scheme which is commonly used at present. In addition, the combination scheme of the invention does not show obvious visceral organ toxicity and adverse reaction in the experiment process. Besides, immunological studies show that the combination scheme can regulate the tumor microenvironment, activate immune inflammation type TME and CD8 + T cell infiltration and inhibit G-MDSC infiltration, which may be one of the mechanisms of the invention for exerting the tumor inhibition effect, and lays a foundation for subsequent pharmacological studies.

Description

Technical Field

[0001] The present invention relates to the technical field of chemotherapy drugs, and in particular to an application of gemcitabine combined with brinzolamide in preparing a chemotherapy drug for pancreatic cancer. Background Art

[0002] Pancreatic cancer chemotherapy regimens are tailored to the patient's stage, physical condition, and genetic profile. Commonly used drugs include gemcitabine, 5-fluorouracil (5-FU), nab-paclitaxel, and oxaliplatin. Single-agent or combination regimens, such as FOLFIRINOX (oxaliplatin + irinotecan + 5-FU) or gemcitabine combined with nab-paclitaxel (AG regimen), are often used clinically. Treatment goals include postoperative adjuvant chemotherapy, preoperative neoadjuvant chemotherapy, and palliative chemotherapy in the advanced stage.

[0003] Adjuvant chemotherapy is suitable for patients who have undergone surgery to reduce the risk of recurrence. Gemcitabine alone or 5-FU + folinic acid are commonly used regimens, and the course of treatment is usually 6 months. For those in good physical condition, modified FOLFIRINOX may be used, but side effects such as bone marrow suppression need to be closely monitored. Preoperative neoadjuvant chemotherapy is used for the preoperative treatment of resectable or borderline resectable tumors to shrink the lesions and improve the success rate of surgery. The AG regimen (gemcitabine + albumin-bound paclitaxel) or FOLFIRINOX is the first choice, and the decision to perform surgery needs to be made after assessing the patient's tolerance. Late-stage palliative chemotherapy is aimed at patients with metastatic or locally advanced disease who are unable to undergo surgery, with the main goal of prolonging survival and relieving symptoms. The AG regimen and FOLFIRINOX are first-line regimens; second-line options include regimens based on liposomal irinotecan + 5-FU or oxaliplatin.

[0004] Gemcitabine is an antimetabolite chemotherapy drug that inhibits cancer cell proliferation by interfering with DNA synthesis and repair. Its core mechanism of action is its intracellular conversion into active metabolites, which are incorporated into DNA chains, leading to chain termination and inhibiting key enzymes involved in DNA repair, ultimately inducing apoptosis in tumor cells. After entering the cell, gemcitabine undergoes phosphorylation to produce its active metabolites, gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP). As a nucleoside analog, dFdCTP competitively incorporates into DNA chains, replacing normal nucleotides and preventing DNA chain elongation. Gemcitabine inhibits ribonucleotide reductase (RNR), reducing the production of deoxycytidine triphosphate (dCTP), thus reducing the raw materials required for DNA repair. The low dCTP concentration promotes more efficient incorporation of gemcitabine metabolites into DNA. Gemcitabine is widely used in chemotherapy for malignant tumors such as non-small cell lung cancer, pancreatic cancer, ovarian cancer, and breast cancer. Although it does have anti-tumor activity, its clinical efficacy in pancreatic cancer is still not ideal. Even when used in combination with platinum and taxane drugs, the median survival time of advanced patients is only 8-11 months, and the recurrence rate of patients receiving adjuvant chemotherapy is only reduced by about 8% to 14%.

[0005] Brinzolamide is a topical, clinically used, anti-ocular pressure medication. As a carbonic anhydrase inhibitor, brinzolamide inhibits carbonic anhydrase activity in the ciliary body, reducing aqueous humor production and thereby lowering intraocular pressure. Existing pharmacological studies have shown that brinzolamide, whose substrate is CA-II, can reduce aqueous humor secretion. By reducing bicarbonate ion production, it reduces sodium and water transport, lowering aqueous humor osmotic pressure and reducing aqueous humor production, thereby lowering intraocular pressure. Brinzolamide can also increase retinal CO2 levels, which acts on blood vessels, dilating vasodilation and increasing ocular perfusion pressure. Prior art has not yet demonstrated the use of brinzolamide as a chemotherapy agent. Summary of the Invention

[0006] The first technical problem to be solved by the present invention is: how to improve the chemotherapy regimen for pancreatic cancer to improve the efficacy.

[0007] The second technical problem to be solved by the present invention is how to further expand the medical uses of brinzolamide.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: Application of gemcitabine combined with brinzolamide in the preparation of pancreatic cancer chemotherapy drugs.

[0009] Preferably, the form of gemcitabine in the drug is gemcitabine hydrochloride.

[0010] Preferably, the dosage form of gemcitabine hydrochloride is an injection.

[0011] Preferably, the dosage of gemcitabine hydrochloride is 800-1250 mg / m per week. 2 .

[0012] Preferably, the dosage form of brinzolamide in the medicine is an oral dosage form.

[0013] Preferably, the dosage of brinzolamide is 30 mg / kg / d.

[0014] Preferably, the pancreatic cancer chemotherapy drug is an adjuvant chemotherapy drug after pancreatic cancer resection, a neoadjuvant chemotherapy drug before pancreatic cancer surgery, or a palliative chemotherapy drug for pancreatic cancer.

[0015] Preferably, the pancreatic cancer chemotherapy drug is a drug that promotes CD8 + T cell infiltration and drugs that inhibit G-MDSC infiltration.

[0016] As a preference, the pancreatic cancer chemotherapy drug is a drug that promotes CD4 + Drugs that infiltrate T cells, NK cells, and DC cells.

[0017] Preferably, the pancreatic cancer chemotherapy drug is a pancreatic solid tumor inhibitory drug.

[0018] The present invention provides the use of gemcitabine combined with brinzolamide for the preparation of pancreatic cancer chemotherapy drugs. This technical solution involves orally administering brinzolamide during the conventional administration of gemcitabine. Although brinzolamide does not have a direct tumor inhibitory effect as a carbonic anhydrase inhibitor, the combined administration can significantly enhance the tumor inhibitory effect of gemcitabine. Experimental studies have shown that continuous oral administration of brinzolamide at a dose of 30 mg / kg / d during gemcitabine chemotherapy can achieve a pancreatic tumor inhibition rate of 85.29%, which is significantly better than the currently commonly used gemcitabine + albumin-bound paclitaxel regimen, or gemcitabine + capecitabine regimen. Moreover, the combination regimen of the present invention did not show obvious organ toxicity and adverse reactions during the experiment. In addition, immunological studies have shown that the combination regimen of the present invention can regulate the tumor microenvironment, activate immune inflammatory TME, CD8 + T cell infiltration inhibits G-MDSC infiltration, which may be one of the mechanisms by which the present invention exerts its tumor inhibitory effect and lays the foundation for subsequent pharmacological studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a statistical result diagram of the tumor inhibition rate of each experimental group in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0020] The following is a detailed description of specific embodiments of the present invention. To avoid excessive unnecessary detail, well-known structures or functions will not be described in detail in the following examples. Approximate language used in the following examples can be used for quantitative expression to indicate that a certain amount of variation is allowed without changing the basic function. Unless otherwise defined, technical and scientific terms used in the following examples have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0021] 1. Efficacy Experiment 1. Experimental Materials Human pancreatic cancer cells HPAF-II were purchased from Shanghai Kanglang Biotechnology Co., Ltd. and authenticated by Wuhan Punosai Life Science Technology Co., Ltd.

[0022] Fifty SPF-grade female BALB / c nude mice, 6 weeks old, weighing 21-25 g, were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0023] Brinzolamide API was purchased from Wuhan Kemik Biopharmaceutical Technology Co., Ltd. Gemcitabine hydrochloride for injection was purchased from Qilu Pharmaceutical (Hainan) Co., Ltd. Sodium hydroxide was purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium carboxymethyl cellulose was purchased from Merck Life Sciences Co., Ltd. DMEM high-glucose medium was purchased from Merck Life Sciences Co., Ltd.; fetal bovine serum was purchased from Thermo Fisher Scientific; penicillin-streptomycin was purchased from Merck Life Sciences Co., Ltd.; 0.25% trypsin-EDTA solution was purchased from Shanghai Kanglang Biotechnology Co., Ltd.; PBS was purchased from Sinopharm Chemical Reagent Co., Ltd.; cell-grade dimethyl sulfoxide was purchased from Sinopharm Chemical Reagent Co., Ltd.; and MTT kit was purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0024] 1mL syringe, 12-gauge gavage needle, 20-gauge trocar, surgical scissors, surgical forceps, animal marking pen, vernier caliper, etc.; 10cm cell culture dish; 15 and 50mL centrifuge tubes; cell counting chamber.

[0025] 2. Instruments and equipment Pure water preparation system (Merck Milli-Q), pH meter (Shanghai Yidian Scientific Instrument Co., Ltd.); 1 / 100,000 analytical balance (Mettler Toledo); 2.5 μL, 10 μL, 100 μL, and 1000 μL pipettes (RAININ); flow cytometer (Beckman Coulter); biological safety cabinet (Shanghai Beiyin Biotechnology); CO2 cell incubator (Rayward); microscope (Olympus); automatic cell counter (Beckman Coulter); vortex mixer (Thermo Fisher Scientific); low-speed desktop centrifuge (Hunan Kaida); BRF-25V318 medical refrigerator-freezer (BIOBASE); electric blast drying oven (Tianjin Test); HW-12 electric constant temperature water bath (Shanghai Haozhuang Instrument Co., Ltd.).

[0026] 3. Experimental methods BALB / c nude mice were housed in a barrier environment IVC system with 18 ventilation changes per hour. The animal room temperature was 25 ± 1°C, the relative humidity was 50–60%, and the animals were maintained on a 12-hour day / night cycle. The animals had free access to sterile tap water, cobalt-60 irradiated feed, and sterilized corncob bedding.

[0027] HPAF-II cells in good condition and in the logarithmic growth phase were selected, digested, centrifuged, and the cell concentration was adjusted to 5.5×10 7 ~6×10 7 The cell suspension was inoculated into the subcutaneous tissue of the axilla of nude mice (0.2 mL / mouse) using a 1 mL syringe to establish a first-generation tumor-bearing mouse model.

[0028] When the tumor volume (TV) reaches 450-470 mm 3 The nude mice were killed by cervical dislocation, and the tumor tissue was removed. The well-grown tumor tissue blocks were selected and cut into approximately 1 mm pieces in pre-cooled sterile saline under ice bath conditions on a sterile operating table. 3 The uniform small pieces were inoculated into the right armpit of nude mice using a trocar to establish a second-generation tumor-bearing mouse model.

[0029] The average tumor volume of the second generation tumor-bearing mice was 100 mm 3 Nude mice were randomly divided into four groups based on tumor volume and body weight: model group (model), gemcitabine group (injection of gemcitabine hydrochloride injection), brinzolamide group (oral administration of brinzolamide), and brinzolamide and gemcitabine combination group (oral administration of brinzolamide + injection of gemcitabine hydrochloride injection), with 12 animals in each group. The dosing schedule was as follows.

[0030] The day of grouping was recorded as D0. Starting from D1, the first day after grouping, the animals in each group were treated as follows: Model group: blank solvent (0.5% CMC-Na) was first gavaged every day, and then physiological saline solution was injected into the abdominal cavity 30 minutes later.

[0031] Gemcitabine group: blank solvent (0.5% CMC-Na) was gavaged every day; on the first day of each week, gemcitabine hydrochloride injection (1000 mg / m 2 ) (administered once a week); Brinzolamide group: Brinzolamide suspension (30 mg / kg / d) was first gavaged daily, and then normal saline solution was injected intraperitoneally 30 minutes later.

[0032] Brinzolamide and gemcitabine combination group: Brinzolamide suspension (30 mg / kg / d) was administered orally every day; on the first day of each week, gemcitabine hydrochloride injection (1000 mg / m 2 ) (administered once a week); Mouse body weight and tumor size were monitored daily, and the dosing dose was calculated based on daily body weight. For three weeks, blood was collected from the eye sockets 1 hour after dosing on Day 22, and the animals were sacrificed. Tumor tissue was excised, weighed, and photographed. After the blood samples rested at room temperature for 1 hour, they were centrifuged at 14,000 rpm for 10 minutes at 4°C. The supernatant serum was collected, aliquoted, and stored at -80°C until needed.

[0033] 4. Evaluation method 4.1 Tumor growth inhibition rate The body weight of the animals was measured every day during the experimental period, the tumor diameter was measured, and the tumor volume was calculated according to the formula TV = A / 2×B2, where A is the longest axis of the tumor and B is the shortest axis of the tumor. The relative tumor volume (RTV) was calculated based on TV, and the tumor growth inhibition rate (TGI) was calculated to evaluate the anti-tumor activity of the drug. The calculation formulas are as follows: RTV = TVt / TV0 (where V0 is the tumor volume at D0; Vt is the tumor volume on the tth day); TGI = [1 - (RTVT / RTVc)]×100% (RTVT is the relative tumor volume of the drug administration group; RTVc is the relative tumor volume of the model group / negative control group).

[0034] 4.2 Organ index Organ index (%) = [organ weight (g) / body weight (g)]×100%.

[0035] 4.3 Evaluation index for drug synergistic effect The Jin Zhengjun Q value method was used to evaluate the drug synergistic effect: Q = EAB / (EA + EB - EA×EB). In the formula, the numerator represents the measured combined effect, and the denominator is the expected combined effect. Among them, EA and EB represent the growth inhibition rates of the two drugs when used alone, and EAB represents the growth inhibition rate of the two drugs when used in combination at the current dose. Q≥1.15 indicates a synergistic effect, 0.85 < Q < 1.15 indicates an additive effect, and Q≤0.85 indicates an antagonistic effect.

[0036] 4.4 HE staining Three tumor tissues were randomly taken from nude mice in each group, quickly placed in 4% paraformaldehyde, dehydrated, paraffin-embedded, sectioned, the sections were dewaxed, stained with hematoxylin-eosin (HE), and examined under an optical microscope.

[0037] 4.5 Immunohistochemistry Three tumor tissues were randomly taken from nude mice in each group. After paraffin embedding and sectioning, immunohistochemistry experiments were used to detect the protein expression of Ki67 and Cleaved Caspase-3 in the tumor tissues of mice.

[0038] 4.6 Data processing Graphpad Prism 9.5 software was used to process the experimental data, and the experimental results were expressed as Mean±SD. One-way analysis of variance was used for statistical analysis among multiple group samples; compared with the model group, *P < 0.05, **P < 0.01, ***p < 0.001, ****P < 0.0001; compared with the gemcitabine group, #P < 0.05, ##P < 0.01, P < 0.001.

[0039] 5. Experimental results 5.1 Body weight of nude mice The body weight of nude mice in each group changed slowly during the experimental period, and the body weight of animals in the model group decreased slowly during the dosing period, showing a trend of cancer-related emaciation; after intraperitoneal injection of gemcitabine on the first day, the body weight of animals in the gemcitabine group and the brinzolamide + gemcitabine group showed an overall downward trend, and the rate of decline was faster than that of the model group, indicating that gemcitabine has certain toxicity at the dosed concentration; there was no significant difference in the body weight of animals in the brinzolamide + gemcitabine group compared with the gemcitabine group, suggesting that the combination of brinzolamide and gemcitabine has no obvious effect on body weight.

[0040] 5.2 Tumor Growth The tumor volumes of animals in the model group and the brinzolamide group increased rapidly over time during the experimental period, while compared with the model group, the tumor volumes of the gemcitabine group and the brinzolamide + gemcitabine group increased more slowly.

[0041] At the end of the experiment, the RTV in the gemcitabine group was significantly lower than that in the model group. Tumor volume in the brinzolamide group was reduced, but not significantly, compared with the model group. The RTV in the brinzolamide + gemcitabine group was significantly lower than that in the model group. The RTV in the brinzolamide + gemcitabine group was significantly lower than that in the gemcitabine group. Gemcitabine inhibited pancreatic cancer by 33.27% in each group. Compared with the gemcitabine group, the tumor growth inhibition rate in the brinzolamide + gemcitabine group was significantly higher, reaching 85.29%, while no tumor inhibition was observed in the brinzolamide group. These results suggest that brinzolamide alone does not exhibit a definitive antitumor effect, but its combination with gemcitabine significantly enhances the antitumor effect of gemcitabine.

[0042] The synergistic effect of brinzolamide and gemcitabine was evaluated by Kim Jung-kyun's Q value method. The Q value of brinzolamide and gemcitabine combined was 1.31 according to the inhibition rate, indicating that the inhibitory effect of brinzolamide and gemcitabine on HPAF-II subcutaneous transplanted tumors in nude mice was synergistic.

[0043] 5.3 Histopathological examination HE staining revealed more pronounced fibrosis, intratumoral necrosis, and myxoid degeneration in the tumor tissues of the gemcitabine and brinzolamide + gemcitabine groups compared with the model group. Localized necrosis and fibrosis were more pronounced in the brinzolamide + gemcitabine group, suggesting a stronger antitumor activity in the brinzolamide + gemcitabine group. To further confirm these findings, immunohistochemistry was used to assess the antiproliferative or pro-apoptotic effects of gemcitabine alone or the brinzolamide + gemcitabine group on tumor tissue. Immunohistochemical analysis of the proliferation marker Ki67 demonstrated significantly higher expression in the model group compared with the other treatment groups, indicating high proliferative activity in the tumor tissues. The brinzolamide + gemcitabine group exhibited lower proliferative activity compared with the gemcitabine group. Immunohistochemical staining for cleaved caspase-3 revealed a reduced number of apoptotic cells in the model group tumor tissues, while the expression of apoptosis-related proteins in the other treatment groups was significantly increased, with the brinzolamide + gemcitabine group exhibiting a stronger pro-apoptotic effect.

[0044] 5.4 Organ Index To evaluate the overall effects of gemcitabine and its combination on the body, the present invention conducted statistical analysis of relevant organ indices. The results showed that there was some fluctuation in organ indices among the groups, but no significant differences were observed between the groups, suggesting that brinzolamide has no significant organ toxicity at this dosage.

[0045] 6. Conclusion The tumor volume results of each group of animals showed that brinzolamide could significantly enhance the anti-tumor activity of gemcitabine, and showed a synergistic effect when combined with gemcitabine (Q=1.31).

[0046] 2. Immunology Experiments Tumor tissue from mice at the endpoint of the efficacy study was collected, and appropriate-sized pieces of tissue were cut into 5 ml EP tubes. Mince the pieces and add 2 ml of tumor tissue digestion buffer. Digest the mixture on a shaker at 37°C, 250 rpm, for 30 minutes. Place a 70 μm cell sieve over a centrifuge tube and pour the digested tumor suspension through the sieve. Rinse the sieve with PBS to prepare a single-cell suspension. Centrifuge at 2000 rpm for 5 minutes to obtain a cell pellet. Discard the supernatant and resuspend the pellet in approximately 1 ml of erythrocyte lysis buffer. After 3 minutes, add 3 ml of PBS to terminate erythrocyte lysis. Centrifuge again, discard the supernatant, and resuspend the pellet in an appropriate amount of PBS.

[0047] The cell density was approximately 10 7Cells were plated at 400 cells / ml. Approximately 100 μl of each sample was transferred to a 96-well V-shaped plate and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded. CD16 / 32 antibody working solution was prepared at a 1:100 dilution, and 100 μl of the antibody working solution was added to resuspend the tumor tissue cells. The cells were incubated at room temperature for 10 minutes to prevent nonspecific staining. The cells were centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed once with 200 μl of PBS. Fixable Viability Stain antibody working solution was prepared at a 1:1000 dilution, and 100 μl of the working solution was added to each well to resuspend the cells. The cells were incubated at 4°C for 15 minutes for cell viability staining. The cells were washed once with 200 μl of PBS, centrifuged, and the supernatant discarded. Cell surface protein antibody working solution was prepared at a 1:400 dilution, and the cells were resuspended in 100 μl of the antibody working solution. The cells were incubated at room temperature for 30 minutes for cell surface protein staining. After staining, the cells were washed once with PBS, centrifuged, and the supernatant discarded. The cells were resuspended in PBS.

[0048] After staining, transfer the samples in the 96 V-shaped plate to a 5 ml round-bottom flow tube, vortex for 2 seconds, and then perform flow cytometry detection.

[0049] The test results showed that compared with the model group, brinzolamide group and gemcitabine group, the brinzolamide + gemcitabine combination group could promote the expression of CD8 + T cell infiltration and inhibition of G-MDSC infiltration indicate that the combination of brinzolamide and gemcitabine has the effect of regulating the tumor microenvironment.

[0050] Flow cytometry was used to further analyze the immune cell population in the tumor microenvironment (TME). The results showed that the combined approach of the present invention can promote the expression of immune cells (CD45 + cells) infiltration, increased CD8 + The proportion of T cells and the proportion of G-MDSC were reduced. In addition, the combination regimen further activated the immune inflammatory TME and promoted CD4 + Infiltration of T cells, NK cells and DC cells.

[0051] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the scope of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Application of gemcitabine combined with brinzolamide in the preparation of chemotherapy drugs for pancreatic cancer.

2. The use according to claim 1, characterized in that The form of gemcitabine in the drug is gemcitabine hydrochloride.

3. The use according to claim 2, characterized in that The dosage form of the gemcitabine hydrochloride is an injection.

4. The use according to claim 3, characterized in that The dosage of gemcitabine hydrochloride is 800-1250 mg / m per week. 2 .

5. The use according to claim 1, characterized in that In the medicine, the dosage form of brinzolamide is an oral dosage form.

6. The use according to claim 5, characterized in that The dose of brinzolamide was 30 mg / kg / d.

7. The use according to claim 1, characterized in that The pancreatic cancer chemotherapy drug is an adjuvant chemotherapy drug after pancreatic cancer resection, a neoadjuvant chemotherapy drug before pancreatic cancer surgery, or a palliative chemotherapy drug for pancreatic cancer.

8. The use according to claim 1, characterized in that The pancreatic cancer chemotherapy drug promotes CD8 + T cell infiltration and drugs that inhibit G-MDSC infiltration.

9. The use according to claim 1, characterized in that The pancreatic cancer chemotherapy drug is a drug that promotes CD4 + Drugs that infiltrate T cells, NK cells, and DC cells.

10. The use according to claim 1, characterized in that The pancreatic cancer chemotherapy drug is a pancreatic solid tumor inhibitory drug.

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