Antitumor combination of a cyclic pentapeptide galaxamide and doxorubicin and use thereof

Abstract

The present application belongs to the technical field of medicine, and particularly relates to an antitumor combination drug of cyclopentapeptide Galaxamide and doxorubicin and application thereof. In order to increase the efficacy or reduce the toxic side effects of doxorubicin, the present application can increase the efficacy of doxorubicin by combination of Galaxamide and doxorubicin. Compared with the respective single drug, the combination of the two drugs can enhance the inhibition effect on tumor cell proliferation, can enhance the effect of inducing tumor cell cycle arrest and promoting apoptosis, and can more significantly inhibit the growth of subcutaneous transplanted tumors. The present application uses Galaxamide in combination with doxorubicin as an antitumor combination drug, has a stronger antitumor effect, and does not bring additional toxic side effects, thereby providing a new method for increasing the efficacy or reducing the toxic side effects of doxorubicin.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an antitumor combination drug of cyclic pentapeptide Galaxamide and doxorubicin and its application. Background Technology

[0002] Cancer, a malignant tumor originating from epithelial tissue, seriously threatens human health and life, and is one of the leading causes of death. As our understanding of cancer continues to grow, treatment options are gradually increasing, from traditional surgery, radiotherapy, and chemotherapy to today's molecularly targeted drugs and cellular immunotherapy. Humanity has never given up its efforts to conquer cancer. Unfortunately, the high cost and limited applicability of molecularly targeted drugs and cellular immunotherapy severely restrict their application, meaning that the vast majority of patients still rely on surgery and radiotherapy / chemotherapy to treat cancer.

[0003] Doxorubicin is a broad-spectrum antibiotic that inhibits RNA and DNA synthesis. Since its FDA approval in 1974 for use alone or in combination therapy, it has been widely used as a first-line treatment for various cancers. However, its significant cardiotoxicity limits its use. To enhance the efficacy of doxorubicin or reduce its side effects, the combination of doxorubicin with other drugs has received increasing attention.

[0004] Marine cyclic peptides are a class of promising lead compounds among marine natural products. Studies have shown that marine cyclic peptides from different sources possess unique chemical structures and pharmacological properties, such as antitumor, antifungal, antifouling, anti-inflammatory, and antihypertensive activities. In the field of antitumor therapy, the discovery and development of marine cyclic peptides and their derivatives in recent years have made significant contributions to the treatment of various cancers, and many have already been applied in clinical practice. Galaxamide is a novel cyclic pentapeptide isolated by the applicant from the seaweed *Galaxaura filamentosa* in the Xisha Islands of the South China Sea. Its molecular weight was determined to be 593.8 g / mL using mass spectrometry and other methods, and its molecular formula is C1. 32 H 59N5O5, with the molecular structure cyclo(Leu-Me-Leu-Leu-Leu-Me-Leu), is a cyclic pentapeptide formed by the condensation and cyclization of leucine and N-methylleucine, with a molecular configuration of SSSSS. Meanwhile, the applicant has completed the total synthetic route design for Galaxamide and further improved the synthetic process to scale up the reaction. The maximum yield of a single linear peptide is now 3.5 g, and the maximum yield of a single linear peptide cyclization is 200 mg, meeting the requirements for laboratory-scale amplification (Xu WJ, Liao XJ, Xu SH, et al. Isolation, structure determination, and synthesis of galaxamide, a rare cytotoxic cyclic pentapeptide from a marinealgae Galaxaura filamentosa[J]. Organic letters, 2008, 10(20):4569-4572.). However, there are currently no reports of using Galaxamide to enhance the efficacy of doxorubicin. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides an antitumor combination drug of cyclic pentapeptide Galaxamide and doxorubicin, using Galaxamide and its derivatives as sensitizers for the antitumor chemotherapy drug doxorubicin, which has the effects of inhibiting tumor cell proliferation, inducing cell cycle arrest and apoptosis, and can improve the antitumor effect and efficacy of doxorubicin.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides the use of a pharmaceutical composition in the preparation of an antitumor drug, the pharmaceutical composition comprising component A and component B, wherein component A is selected from any one of cyclic pentapeptide Galaxamide and its derivatives, and component B is selected from any one of doxorubicin hydrochloride, liposomal doxorubicin and its derivatives.

[0008] A second aspect of the present invention provides the use of a pharmaceutical composition in the preparation of a drug for inhibiting tumor cell proliferation, the pharmaceutical composition comprising component A and component B, wherein component A is selected from any one of cyclic pentapeptide Galaxamide and its derivatives, and component B is selected from any one of doxorubicin hydrochloride, liposomal doxorubicin and its derivatives.

[0009] The structural formula of the cyclic pentapeptide Galaxamide is as follows:

[0010]

[0011] In a preferred embodiment of the present invention, the pharmaceutical composition comprises cyclic pentapeptide Galaxamide and doxorubicin hydrochloride.

[0012] Galaxamide is a novel marine cyclic peptide with antitumor activity and has the potential to become a new type of anticancer drug; Doxorubicin is a first-line chemotherapy drug that has been proven in practice and recognized in clinical practice, but the prominent cardiotoxicity of doxorubicin limits its use.

[0013] This invention has found that the combined use of Galaxamide and doxorubicin can enhance the efficacy of doxorubicin. Compared with individual administration, the combined use of the two drugs can enhance the inhibitory effect on tumor cell proliferation, enhance the induction of tumor cell cycle arrest and promote apoptosis, and more significantly inhibit the growth of subcutaneous xenografts in mice. This suggests that using Galaxamide as a potentiator for doxorubicin can improve its antitumor effect and efficacy, providing a new method for enhancing the efficacy of doxorubicin.

[0014] In a preferred embodiment of the present invention, the tumor includes cervical cancer, breast cancer, liver cancer, prostate cancer, lung cancer, colorectal cancer, and stomach cancer.

[0015] In a more preferred embodiment of the present invention, the tumor includes cervical cancer, prostate cancer, colorectal cancer, and gastric cancer.

[0016] In a preferred embodiment of the present invention, the tumor cells include cervical cancer cells, breast cancer cells, liver cancer cells, prostate cancer cells, lung cancer cells, colorectal cancer cells, and gastric cancer cells.

[0017] In a more preferred embodiment of the present invention, the tumor cells include cervical cancer cells HeLa, breast cancer cells MDA-MB-231, liver cancer cells SK-HEP1, prostate cancer cells PC-3, prostate cancer cells DU145, lung cancer cells LLC, prostate cancer cells RM-1, colorectal cancer cells CT26, and gastric cancer cells MFC.

[0018] In a more preferred embodiment of the present invention, the tumor cells include cervical cancer cells HeLa, prostate cancer cells RM-1, prostate cancer cells PC-3, colorectal cancer cells CT26, and gastric cancer cells MFC.

[0019] As a preferred embodiment of the present invention, the dosage form of the anti-tumor drug or drug that inhibits the proliferation of tumor cells includes oral liquid, tablet, injection, capsule, granule, and powder.

[0020] In a preferred embodiment of the present invention, the antitumor drug or drug that inhibits tumor cell proliferation is administered orally or by injection. The drug formulation can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally). If certain drugs are unstable under gastric conditions, they can be prepared as enteric-coated tablets.

[0021] In a preferred embodiment of the present invention, the antitumor drug or drug for inhibiting tumor cell proliferation further includes a pharmaceutically acceptable carrier. The carrier is a functional pharmaceutical excipient available in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some novel pharmaceutical polymers, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention discloses an antitumor combination drug of the cyclic pentapeptide Galaxamide and doxorubicin. The combined use of Galaxamide and doxorubicin enhances the efficacy of doxorubicin. Compared to using either drug alone, the combined use strengthens the inhibitory effect on tumor cell proliferation, enhances the induction of tumor cell cycle arrest and promotes apoptosis, and more significantly inhibits the growth of subcutaneous xenografts. This invention utilizes Galaxamide synergistically with doxorubicin as an antitumor combination drug, resulting in a stronger antitumor effect and providing a new method to enhance the efficacy of doxorubicin. Attached Figure Description

[0024] Figure 1 The inhibition rate curves and combination drug index (CI) of Galaxamide and doxorubicin at different concentration gradients on various tumor cell lines are shown.

[0025] Figure 2 The effects of Galaxamide and doxorubicin at different concentration gradients on cell cycle arrest in HeLa and RM-1 cells, and bar charts.

[0026] Figure 3 The effects of combined administration of Galaxamide and doxorubicin at different concentration gradients on the expression levels of cell cycle-related proteins in HeLa and RM-1 cells;

[0027] Figure 4 The effects of combined administration of Galaxamide and doxorubicin at different concentration gradients on apoptosis in HeLa and RM-1 cells and bar charts.

[0028] Figure 5The effects of combined administration of Galaxamide and doxorubicin at different concentration gradients on the expression levels of apoptosis-related proteins in HeLa and RM-1 cells;

[0029] Figure 6 Comparison of tumor size in the control group, the two single-drug groups, and the combined-drug group in HeLa nude mouse tumor-bearing, RM-1 nude mouse tumor-bearing, and RM-1C57 mouse tumor-bearing experiments;

[0030] Figure 7 The changes in tumor volume in HeLa nude mice, RM-1 nude mice, and RM-1C57 mice with tumors were shown in the experiments.

[0031] Figure 8 Scatter plot of transplanted tumors in HeLa nude mice, RM-1 nude mice, and RM-1C57 mice tumor-bearing experiments;

[0032] Figure 9 This is a comparison of serum ALT, AST, Cr, and BUN levels in the control group, the two single-drug groups, and the combined-drug group in the tumor-bearing experiment of RM-1C57 mice. Detailed Implementation

[0033] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0035] Example 1: Inhibitory effect of Galaxamide synergistic with doxorubicin on tumor cell proliferation in vitro

[0036] The inhibitory effects of different concentration gradients of Galaxamide and Doxorubicin hydrochloride on the proliferation of human cervical cancer cells (HeLa), human breast cancer cells (MDA-MB-231), human liver cancer cells (SK-HEP1), human prostate cancer cells (PC-3), human prostate cancer cells (DU145), mouse lung cancer cells (LLC), mouse prostate cancer cells (RM-1), mouse colorectal cancer cells (CT26), and mouse gastric cancer cells (MFC) were investigated. All cell lines were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum. Specific procedures are as follows:

[0037] (1) Collect cells growing in the logarithmic phase. Calculate the total number of cells required based on the cell proliferation rate and cell adhesion area, using a ratio of 2000-8000 cells per well. Add the required cell suspension to the pipette trough and replenish the required culture medium according to the standard of 100 μL of culture medium per well of a 96-well plate. After mixing by pipetting, use a pipette to evenly add the cell suspension to the 96-well plate and then incubate in a 5% CO2, 37℃ incubator until the cells adhere (approximately 12 hours).

[0038] (2) Treatment was performed with different concentration gradients of Galaxamide (0, 3, 10 μM) and doxorubicin (0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 μM). Galaxamide was added first and incubated for 1 h, followed by doxorubicin. Each drug concentration was set up with 3-4 replicates, and the mixture was incubated in a 5% CO2, 37℃ incubator for 72 h.

[0039] (3) Add 10 μL of MTT solution to each well under light-protected conditions and continue incubation in a cell culture incubator for 4 hours in the dark. After carefully aspirating the culture medium from the wells using a vacuum pump, add 50 μL of DMSO to each well and shake on a shaker at low speed for 10 minutes. After the crystals have completely dissolved, measure the absorbance of each well at OD 570 nm using an ELISA reader and calculate the inhibition rate of each drug concentration gradient on tumor cells. The test results are shown in […]. Figure 1 Table 1.

[0040] Figure 1 Table 1 shows the inhibition rate curves and combination index (CI) of Galaxamide and doxorubicin at various concentration gradients on different tumor cell lines; Table 1 shows the inhibitory effect of Galaxamide and doxorubicin combination on tumor cell proliferation. The IC50 values ​​of Galaxamide and doxorubicin combination on tumor cells are shown in Table 1. 50 and Figure 1 The inhibition rate curves and the combined drug index (CI) show that among these nine tumor cell types, HeLa, RM-1, PC-3, CT26, and MFC are more sensitive to the combined action of Galaxamide and Doxorubicin, exhibiting a strong synergistic effect; however, LLC, MDA-MB-231, DU145, and SK-HEP-1 are less sensitive to the combined action, showing a strong synergistic effect only at certain concentration gradients.

[0041] Table 1. IC50 of various tumor cell lines after combined administration of Galaxamide and Doxorubicin at different concentration gradients. 50 Summary

[0042]

[0043] Example 2: The synergistic effect of Galaxamide and doxorubicin on the induction of HeLa and RM-1 cycle arrest

[0044] (1) Collect HeLa and RM-1 cells in the logarithmic growth phase and seed them in appropriate numbers into 6-well plates (HeLa: 3000 cells / well, RM-1: 5000 cells / well). After the cells adhere overnight, treat them with different concentration gradients of Galaxamide (0, 3, 10 μM) and doxorubicin (0, 0.03, 0.1, 0.3, 1 μM). First, add Galaxamide and incubate for 1 h, then add doxorubicin, and incubate in a 5% CO2, 37℃ incubator for 48 h.

[0045] (2) After 48 h of drug treatment, 1 mL of trypsin cell digestion solution containing 0.25% trypsin and 0.02% EDTA was added, and the cells were incubated at 37 °C for three minutes. The cells were then mixed with the old culture medium in a 6-well plate, centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. After washing twice with PBS, 200 μL of 70% ethanol pre-chilled at 4 °C was added, and the cells were fixed overnight at 4 °C. Cells were collected by centrifugation and washed twice with 1 mL of PBS pre-chilled at 4 °C. 200 μL of PI staining solution was added, and the cells were gently resuspended by pipetting and incubated at 4 °C in the dark for 30 minutes. After filtering through a 400-mesh nylon mesh, the cells were analyzed using a flow cytometer. Finally, the experimental data were analyzed using ModFit LT 5.0 software.

[0046] like Figure 2 As shown, when used alone, Galaxamide dose-dependently reduces the proportion of HeLa and RM-1 cells in the G2 / M and S phases, and induces cell arrest in the G0 / G1 phase; Doxorubicin dose-dependently induces cell arrest in the G2 / M phase for both HeLa and RM-1 cells. When Galaxamide and Doxorubicin are used in combination, HeLa and RM-1 cells are significantly arrested in the G2 / M phase, leading to apoptosis. Figure 3 As shown, the higher the protein expression level, the darker the protein band. These results indicate that Galaxamide can effectively increase the sensitivity of HeLa and RM-1 to Doxorubicin. Furthermore, compared to monotherapy, HeLa and RM-1 were more prone to cell cycle arrest and apoptosis under low concentrations of Doxorubicin treatment.

[0047] Example 3: The synergistic effect of Galaxamide and Doxorubicin on inducing apoptosis in HeLa and RM-1 cells.

[0048] (1) Collect HeLa and RM-1 cells in the logarithmic growth phase and seed them in appropriate numbers into 6-well plates (HeLa: 3000 cells / well, RM-1: 5000 cells / well). After the cells adhere overnight, treat them with different concentration gradients of Galaxamide (0, 3, 10 μM) and doxorubicin (0, 0.03, 0.1, 0.3, 1 μM). First, add Galaxamide and incubate for 1 h, then add doxorubicin, and incubate in a 5% CO2, 37℃ incubator for 48 h.

[0049] (2) After 48 h of drug treatment, 1 mL of trypsin cell digestion solution containing 0.25% trypsin and 0.02% EDTA was added, and the cells were incubated at 37 °C for three minutes. The cells were then mixed with the old culture medium in a 6-well plate, centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. After washing twice with PBS, the cells were treated with an apoptosis detection kit (Shanghai Sangon Biotech Co., Ltd., I305FA0003). The cells were first washed with 200 μL of 1×Binding Bufffer buffer, centrifuged to remove the supernatant, and then resuspended in 195 μL of 1×Binding Bufffer buffer. 5 μL of Annexin V-FITC and 10 μL of PI staining solution were added, and the mixture was gently pipetted and incubated at 4 °C in the dark for 10 minutes. After filtration through a 400-mesh nylon screen, the cells were analyzed using a flow cytometer. Finally, the experimental data were analyzed using FlowJo V10 software.

[0050] The results are as follows Figure 4 As shown, with increasing concentrations of Galaxamide and Doxorubicin alone, the proportion of apoptosis in HeLa and RM-1 cells gradually increased; however, under combined drug treatment, the increase in the proportion of apoptosis in HeLa and RM-1 cells was even greater, and it was mainly early apoptosis. Figure 5 As shown, the higher the protein expression level, the darker the protein band.

[0051] Example 4: Inhibitory effect of Galaxamide synergistic with doxorubicin on the growth of subcutaneous xenografts in mice.

[0052] (1) The test animals were SPF-grade 4-week-old female BALB / C-Nude nude mice and 4-week-old female C57BL / 6 mice (Guangdong Jicui Yaokang Biotechnology Co., Ltd.). In order to more strongly demonstrate that Galaxamide and doxorubicin have a synergistic effect on the growth inhibition of transplanted tumors in mice, three animal experiments were conducted: xenografting of HeLa in nude mice, allogeneic transplantation of RM-1 in nude mice, and allogeneic transplantation of RM-1 in C57 mice.

[0053] (2) Mouse tissue block xenografts were selected as mouse tumor models. Compared with subcutaneous cell inoculation, tissue block xenografts have a higher tumor formation rate, more uniform tumor development speed, and are closer to the tumor growth state in clinical patients.

[0054] For the transplanted tumor tissue, several nude mice and C57 mice were selected in advance and inoculated with 400W HeLa and RM-1 cells. After the tumor matured, the mice were euthanized by cervical dislocation. The skin, scab, pseudomembrane, etc., attached to the tumor were peeled off, and the subcutaneous tumor was completely removed and placed in a sterile culture dish. DMEM culture medium was added, and necrotic tissue in the tumor was removed with a scalpel. The more viable tissue on the outer side of the tumor was selected and cut into tissue blocks of about 3×3×3mm. The chest skin of normal mice was disinfected and anesthetized. A small incision was made at the costal arch, and the subcutaneous tissue was bluntly dissected with forceps until it reached the axilla. The tumor tissue block was pushed to the axilla with forceps, the incision was sutured, and the skin was disinfected.

[0055] (3) About four days after the transplanted tumor surgery, the tumor volume grew to about 100 mm. 3 Mice were randomly divided into four groups: a blank control group (Control group), a Galaxamide monotherapy group (GLD group), an adriamycin monotherapy group (DOX group), and a Galaxamide and adriamycin combination therapy group (GLD+DOX group).

[0056] (4) After grouping, intraperitoneal administration was performed every two days. In the three animal experiments, the dosage of Galaxamide in the GLD group and the GLD+DOX group was 6 mg / kg, and the dosage of doxorubicin in the DOX group and the GLD+DOX group was 1 mg / kg in the HeLa tumor-bearing experiment. Because RM-1 cells are more sensitive to doxorubicin, the dosage of doxorubicin in the DOX group and the GLD+DOX group was 0.5 mg / kg in the RM-1 tumor-bearing experiment.

[0057] (5) When there is a statistically significant difference in tumor volume among the groups, the mice were euthanized by cervical dislocation, and then dissected, the tumors and organs were separated, and the tumors were weighed and their morphology was observed. After taking pictures, the corresponding tissues were frozen in an ultra-low temperature freezer at -80℃.

[0058] This example investigated the in vivo growth inhibitory effect of Galaxamide in combination with doxorubicin on human cervical cancer cell line HeLa and mouse prostate cancer cell line RM-1 xenografts. The results are as follows: Figure 6 As shown, the cyclic pentapeptide Galaxamide has a certain inhibitory effect on xenografts in nude mice. Figure 7 , 8As shown, compared with the control group and the single-drug experimental group, the tumor volume and weight increased at a slower rate in the combined drug group, indicating that the combined drug group had a synergistic effect in mice and could significantly inhibit tumor growth. The inhibition rates of GLD and DOX groups against HeLa xenografts were 23.2% and 12.6%, respectively, while the inhibition rate of GLD+DOX group against HeLa xenografts reached 51.3%, which was significantly different from the control group (P<0.01) and also significantly different from the single-drug experimental group (P<0.05). There was no statistically significant difference in the body weight of nude mice among the four groups, suggesting that the combined administration of 6 mg / kg Galaxamide and 1 mg / kg Doxorubicin did not have significant side effects on nude mice when improving the tumor inhibition rate.

[0059] In RM-1 nude mouse and normal mouse tumor-bearing experiments, considering that RM-1 cells are more sensitive to doxorubicin than HeLa cells, the doxorubicin concentration in both mouse experiments was 0.5 mg / kg in the DOX monotherapy group and the GLD+DOX combination therapy group. All other drug concentrations and experimental methods remained consistent with the HeLa nude mouse tumor-bearing experiments. As shown in Table 2, at the same drug concentration, the tumor inhibition rates of RM-1 xenografts in C57 cells (35.8% and 34.3%, respectively) were higher than those in nude mice (15.7% and 22.8%, respectively). Furthermore, the GLD+DOX group achieved an inhibition rate of 56.7% on RM-1 xenografts in nude mice and 56.4% on RM-1 xenografts in normal mice. This indicates that the synergistic effect of combined Galaxamide and Doxorubicin is present not only in nude mice with immunodeficiency due to the lack of T cells, but also in C57 mice with normal immunity. There was no statistically significant difference in body weight between nude mice and C57 tumor-bearing mice, suggesting that the combined administration of 6 mg / kg Galaxamide and 0.5 mg / kg Doxorubicin has no significant side effects in nude mice.

[0060] Table 2. Summary of mouse body weight, tumor weight, and tumor inhibition rate in HeLa nude mouse tumor-bearing, RM-1 nude mouse tumor-bearing, and RM-1C57 mouse tumor-bearing experiments.

[0061]

[0062] Example 5: Biochemical detection of drug hepatotoxicity and nephrotoxicity in C57 mouse xenograft tumor experiments.

[0063] There was no statistically significant difference in mouse body weight before and after the three batches of animal experiments, suggesting that the combination of drugs had no significant side effects compared to the single use of the drugs. In order to further study the in vivo toxicity of the combination of Galaxamide and Doxorubicin, blood was collected from the orbital cavity of C57BL / 6 mice and serum was separated for biochemical detection of liver and kidney toxicity.

[0064] (1) In the C57 mouse xenograft experiment, whole blood obtained by collecting blood through the orbital cavity was placed at 4°C overnight and allowed to stand. After initial serum precipitation, the blood was pre-cooled and centrifuged at 3000 rpm for 30 min at 4°C. The upper serum layer was aspirated and the lower blood cell precipitate was discarded. The serum was stored in an ultra-low temperature freezer at -80°C and biochemical indicators were detected the next day.

[0065] (2) Using ALT and AST detection kits (Nanjing Jiancheng Bioengineering Institute, C009-3-1; C010-3-1), add 200 μL of reagent A (Tris, LDH, NADH) and 50 μL of reagent B (Tris, L-alanine, α-ketoglutarate) to a 96-well plate, mix well, preheat to 37℃, add 10 μL of serum, and incubate at 37℃ for 1 min. Analyze the results using an ELISA reader at OD500. 340nm Measure the absorbance A1, then incubate at 37℃ for 2 min and measure the absorbance A2. The formulas for calculating ALT and AST values ​​are as follows:

[0066]

[0067] In the formula, TV = total reaction volume (mL), SV = sample volume (mL), 6.22 = millimolecular absorptivity of NADH at 340 nm, P = cuvette path length (cm). In this determination, TV = 260 μL, SV = 10 μL, P = 1 cm, that is, K value is 4180.

[0068] (3) Using the Cr assay kit (Nanjing Jiancheng Bioengineering Institute, C011-2-1), 180 μL of enzyme solution A (creatinine amide hydrolase, creatine aminohydrolase) was added to a 96-well plate. Then, 6 μL of ultrapure water, 6 μL of creatinine standard (442 μmol / L), and 6 μL of serum were added to the blank group, standard group, and sample group, respectively. The plates were incubated at 37℃ for 5 min, and the chromatograms were read using an OD500 reader. 546nm Measure absorbance A1, add 60 μL of enzyme solution B (sarcosine oxidase, peroxidase), and incubate at 37℃ for 5 min. Measure absorbance A2. The formula for calculating the Cr value is:

[0069]

[0070]

[0071] In the formula, TV = total reaction volume (mL), SV = sample volume (mL), and V A =Enzyme solution A (mL), V B = Enzyme solution B volume (mL), standard concentration C 标准 =442 μmol / L. In this determination, SV = 6 μL, V A =180μL, V B =60μL, i.e., K value is 0.756.

[0072] (4) Using the BUN assay kit (Nanjing Jiancheng Bioengineering Institute, C013-2-1), 250 μL of buffer enzyme solution (urease) was added to a 24-well plate. Then, 20 μL of ultrapure water, 20 μL of urea standard (10 mmol / L), and 20 μL of serum were added to the blank group, standard group, and sample group, respectively. The plates were incubated at 37℃ for 10 min. Then, 1 mL of phenol chromogenic reagent and 1 mL of alkaline sodium hypochlorite were added, and the plates were incubated at 37℃ for another 5 min. The assay was performed using an ELISA reader with an OD value of [missing value]. 640nm The formula for calculating the BUN value is as follows: (This is based on the measurement of absorbance A.)

[0073]

[0074] In the formula, the concentration of the standard is C. 标准 =10mmol / L.

[0075] The results are as follows Figure 9 As shown in Table 3, the four biochemical indicators indicate that neither 6 mg / kg Galaxamide nor 0.5 mg / kg Doxorubicin alone caused significant liver and kidney toxicity, and the combined use of these drugs did not cause additional liver and kidney toxicity. The differences in the levels of each biochemical indicator were not statistically significant.

[0076] Table 3 Summary of Four Liver and Kidney Biochemical Indicators

[0077]

[0078] In summary, using Galaxamide as an adjuvant to doxorubicin, the combined use of the two drugs can enhance the antitumor effect and efficacy of doxorubicin without bringing additional toxic side effects. This invention provides a new method to increase the efficacy of doxorubicin and has broad application prospects.

[0079] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The use of a pharmaceutical composition in the preparation of an antitumor drug, characterized in that, The pharmaceutical composition comprises cyclic pentapeptide Galaxamide and doxorubicin hydrochloride, and the tumor is cervical cancer or prostate cancer.

2. The use of a pharmaceutical composition in the preparation of a drug for inhibiting tumor cell proliferation, characterized in that, The pharmaceutical composition comprises cyclic pentapeptide Galaxamide and doxorubicin hydrochloride, and the tumor cells are cervical cancer cells or prostate cancer cells.

3. The application according to claim 2, characterized in that, The tumor cells are cervical cancer cells HeLa, prostate cancer cells RM-1, or prostate cancer cells PC-3.

4. The application according to claim 1 or 2, characterized in that, The dosage forms of the antitumor drugs or drugs that inhibit the proliferation of tumor cells include oral liquids, tablets, injections, capsules, granules, and powders.

5. The application according to claim 1 or 2, characterized in that, The antitumor drug or drug that inhibits the proliferation of tumor cells is administered orally or by injection.

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