Application of ginsenoside Rf in preparation of enhancer of PD-1 inhibitor anti-tumor drugs

Abstract

The application discloses application of ginsenoside Rf in preparation of an enhancer of a PD-1 inhibitor antitumor drug, and the ginsenoside Rf has the effects of improving a PD-1 inhibitor antitumor response rate, prolonging a survival period, and reducing toxicity, and has a significant advantage in enhancing PD-1 inhibitor treatment of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmacy, and particularly relates to an application of ginsenoside Rf in preparation of an enhancer of a PD-1 inhibitor anti-tumor drug, especially an anti-non-small cell lung cancer drug. BACKGROUND

[0002] Lung cancer is a common malignant tumor in the world. The GLOBOCAN 2018 report provided by the International Agency for Research on Cancer indicates that there were 1810 million new cases of cancer and 960 million deaths from cancer in the world in 2018. Lung cancer is the most commonly diagnosed cancer (accounting for 11.6% of the total cases) and is the main cause of cancer death (accounting for 18.4% of the total cancer deaths) [1] . Lung cancer is divided into small cell lung cancer and non-small cell lung cancer. Among them, non-small cell lung cancer (NSCLC) accounts for more than 80% of the total number of lung cancer, which includes squamous cell carcinoma and non-squamous carcinoma (adenocarcinoma, large cell carcinoma and other types of cancer) [2] .

[0003] The treatment method for early NSCLC is surgical resection, but since 80% of patients are diagnosed with advanced NSCLC, the cancer cells have already metastasized and cannot be removed, so the main treatment methods are chemotherapy, radiotherapy, molecular target therapy, etc. At present, the U.S. Food and Drug Administration (FDA) has approved the PD-1 (Programmed cell death protein 1, PD-1) inhibitor single therapy or combined with chemotherapy for first-line treatment of patients with advanced NSCLC.

[0004] However, even though the PD-1 / PD-L1 immune checkpoint inhibitor has become a standard treatment method for NSCLC and other malignant tumors, a large part of patients do not respond to this therapy, and the overall objective response rate (ORR) of single drug treatment of this inhibitor is still low, of which the ORR of NSCLC is only 20%, and some patients have developed drug resistance [3,4] . In addition, anti-PD-1 / PD-L1 immunotherapy, while blocking immune checkpoints to enhance tumor-specific immune responses, will also non-specifically activate the immune system, disrupt immune homeostasis, produce serious inflammatory adverse reactions, including hepatitis, pneumonia, etc., affect these important organs, and even produce fatal adverse consequences [5] . Therefore, it is an urgent need in the clinic to further find a combined drug method that can improve the anti-tumor response rate of the immune checkpoint inhibitor, increase the ORR, and reduce the toxic side effects.

[0005] Ginsenoside Rf is a glycoside derived from Panax ginseng C.A.Meyer of Araliaceae, and its molecular formula is C 42 H 72 O 14 , and its relative molecular mass is 801.01. Ginsenoside Rf, as an important monomer component in ginseng, has various therapeutic effects, mainly including anti-inflammatory, anti-allergic, pain relief, neuroprotection and other effects [6-9] . Although ginsenoside Rf has various effects, there is a lack of research on ginsenoside Rf in treating non-small cell lung cancer, and there is a lack of research on the combination of ginsenoside Rf and PD-1 inhibitor in treating cancer.

[0006] The present application combines ginsenoside Rf and PD-1 inhibitor, significantly improves the efficacy of PD-1 inhibitor in treating non-small cell lung cancer, and reduces the toxicity, which proves that ginsenoside Rf has the effect of enhancing the PD-1 inhibitor in treating non-small cell lung cancer, and has important significance for NSCLC treatment.

[0007] [1] Bray, F., et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries [J]. CA Cancer J Clin, 2018. 68(6): p. 394-424.

[0008] [2] Ettinger, D.S., et al., Non-Small Cell Lung Cancer, Version 5.2017, NCCN Clinical Practice Guidelines in Oncology [J]. J Natl ComprCanc Netw, 2017. 15(4): p. 504-535.

[0009] [3] Yarchoan, M., A. Hopkins, and E.M. Jaffee, Tumor Mutational Burden and Response Rate to PD-1 Inhibition [J]. N Engl J Med, 2017. 377(25): p. 2500-2501.

[0010] [4] Gide, T.N., et al., Distinct Immune Cell Populations Define Response to Anti-PD-1 Monotherapy and Anti-PD-1 / Anti-CTLA-4 Combined Therapy [J]. Cancer Cell, 2019. 35(2): p. 238-255 e6.

[0011] [5] Boutros, C., et al., Safety profiles of anti-CTLA-4 and anti-PD-1 antibodies alone and in combination [J]. Nat Rev Clin Oncol, 2016. 13(8): p. 473-86.

[0012] [6] Lee W J, Lee G H, Hur J, et al. Taurine and Ginsenoside Rf Induce BDNF Expression in SH-SY5Y Cells: A Potential Role of BDNF in Corticosterone-Triggered Cellular Damage [J]. Molecules (Basel, Switzerland), 2020, 25(12).

[0013] [7] Li Y, Chen C, Li S, et al. Ginsenoside Rf relieves mechanical hypersensitivity, depression-like behavior, and inflammatory reactions in chronic constriction injury rats [J]. Phytotherapy research: PTR, 2019, 33(4): 1095-103.

[0014] [8]Kim M K,Kang H,Baek C W,et al.Antinociceptive and anti-inflammatory effects of ginsenoside Rf in a rat model of incisional pain[J].Journal of ginseng research,2018,42(2):183-91.

[0015] [9]Song L.P.Inhibitory effects of red ginseng and ginsenosides Rg3, Rf and Rh2 on passive cutaneous anaphylaxis and contact dermatitis in mice. Foreign Medicine (Plant Medicine) [J]. 2007, (05): 220. SUMMARY

[0016] The application aims to provide an application of ginsenoside Rf in preparation of an enhancer of a PD-1 inhibitor anti-tumor drug, in particular, an anti-non-small cell lung cancer drug, so as to solve the problems of low anti-tumor response rate of the PD-1 inhibitor, low overall objective remission rate of single drug treatment and high toxicity.

[0017] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme:

[0018] The application provides an application of ginsenoside Rf in preparation of an enhancer of a PD-1 inhibitor anti-tumor drug.

[0019] The molecular formula of the ginsenoside Rf is C 42 H 72 O 14 , the molecular weight is 801.01, and the structure is:

[0020]

[0021] According to an embodiment, the ginsenoside Rf enhances the PD-1 inhibitor anti-non-small cell lung cancer, that is, the ginsenoside Rf enhances the PD-1 inhibitor to inhibit the growth of lung cancer cells.

[0022] Therefore, the tumor in the application is the same as the tumor targeted by the PD-1 inhibitor. The PD-1 inhibitor is mainly used for the treatment of melanoma and non-small cell lung cancer, urothelial carcinoma, and also has certain effects on renal cell carcinoma, bladder cancer, Hodgkin's lymphoma and the like.

[0023] In the embodiment of the application, the tumor is lung cancer, in particular, non-small cell lung cancer, such as lung squamous cell carcinoma or lung adenocarcinoma.

[0024] Furthermore, the tumor cell is an NCI-H226 cell (human lung squamous cell carcinoma cell).

[0025] In one embodiment of the present application, the PD-1 inhibitor is pembrolizumab, and in particular, Keytruda® is used. PD-1 inhibitor.

[0026] Further, the PD-1 inhibitor, the anti-tumor drug and the enhancer can be used in a kit. The ginsenoside Rf and the PD-1 inhibitor are configured in a mass ratio of 10-40:1 (preferably 15-30:1, and more preferably 20:1). Preferably, the concentration of the PD-1 inhibitor is above 10 μg / mL, and the concentration of the ginsenoside Rf is above 100 μg / mL (in one embodiment of the present application, the concentration is 100-400 μg / mL, and preferably 200 μg / mL). In particular, the concentration of the ginsenoside Rf is 400 μg / mL and 200 μg / mL, which can significantly enhance the effect of the PD-1 inhibitor on non-small cell lung cancer.

[0027] According to one embodiment, the ginsenoside Rf enhances the effect of the PD-1 inhibitor on non-small cell lung cancer, which is that the ginsenoside Rf enhances the inhibition of the PD-1 inhibitor on the size of the tumor and prolongs the survival of the mice.

[0028] The mice are C57BL / 6 mice, and the Lewis lung cancer model is constructed by inoculating Lewis lung cancer cells in the right front limb armpit of the mice.

[0029] For reference, in one embodiment of the present application, the single dose of the ginsenoside Rf is 10 mg / kg of the organism (1 time / day), and the single dose of the PD-1 inhibitor is 10 mg / kg of the organism (2 times / week).

[0030] Further, the enhancer is a synergistic and attenuating agent.

[0031] Compared with the prior art, the present application has the following advantages:

[0032] The inventors have found that the ginsenoside Rf can enhance the effect of the PD-1 inhibitor on non-small cell lung cancer, and this effect has been verified in the in vitro model of human lung squamous cell carcinoma NCI-H226 and the in vivo model of Lewis lung cancer C57 / BL6 mice. The main effects are that the survival rate of tumor cells is significantly reduced, the growth of tumor is inhibited, and the survival period is prolonged. The results prove that the ginsenoside Rf can improve the anti-tumor response rate of the PD-1 inhibitor and reduce the toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1Effect of ginsenoside combined with PD-1 inhibitor on survival rate of NCI-H226 human lung squamous carcinoma cells.(A) Effect of ginsenoside Rf, Rc, Rb3 combined with PD-1 inhibitor on survival rate of NCI-H226 human lung squamous carcinoma cells.(B) Effect of different concentrations of ginsenoside Rf combined with PD-1 inhibitor on survival rate of NCI-H226 human lung squamous carcinoma cells.n = 3, *P < 0.05 compared with PD-1 inhibitor group, P < 0.0001 compared with isotype control IgG group, #### P < 0.0001.

[0034] Figure 2 Effect of ginsenoside Rf combined with PD-1 inhibitor on LLC tumor volume in mice.(A) Diagram of LLC tumor volume change in tumor-bearing mice.(B) LLC tumor volume in tumor-bearing mice on the 22nd day after tumor inoculation.(C) LLC tumor weight in tumor-bearing mice on the 22nd day after tumor inoculation.IgG: isotype control group; PD-1: PD-1 inhibitor monotherapy group; PD-1 + Rf: PD-1 inhibitor combined with ginsenoside Rf administration group; Rf: ginsenoside Rf monotherapy group.n = 8, *P < 0.05, **P < 0.01.

[0035] Figure 3 Diagram of LLC tumor-bearing mouse survival.IgG: isotype control group; PD-1: PD-1 inhibitor monotherapy group; PD-1 + Rf: PD-1 inhibitor combined with ginsenoside Rf administration group; Rf: ginsenoside Rf monotherapy group.IgG, Rf, PD-1 + Rf groups, n = 8; PD-1 group, n = 7, *P < 0.05, **P < 0.01. DETAILED DESCRIPTION

[0036] The method provided by the present application will be further illustrated by the following examples. The described examples are only part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by the skilled person without creative labor are within the scope of protection of the present application.

[0037] Example 1

[0038] This example relates to the study of ginsenoside Rf enhancing the effect of PD-1 inhibitor on inhibiting lung cancer cell growth. The PD-1 inhibitor used in the following examples is The PD-1 inhibitor is pembrolizumab.

[0039] I. Experimental method

[0040] (I) Experimental grouping

[0041] Isotype control IgG group: the final concentration of isotype control IgG is 10 μg / mL;

[0042] PD-1 inhibitor group: the final concentration of PD-1 inhibitor was 10 μg / mL PD-1 inhibitor: 25 mg / mL, purchased from Merck Sharp & Dohme Corp, USA, item number S007468;

[0043] Ginsenoside Rc group: the final concentration of ginsenoside Rc was 200 μg / mL

[0044] Ginsenoside Rc combined with PD-1 inhibitor: the final concentration of ginsenoside Rc was 200 μg / mL, and the final concentration of PD-1 inhibitor was 10 μg / mL

[0045] Ginsenoside Rb3 group: the final concentration of ginsenoside Rb3 was 200 μg / mL

[0046] Ginsenoside Rb3 combined with PD-1 inhibitor: the final concentration of ginsenoside Rb3 was 200 μg / mL, and the final concentration of PD-1 inhibitor was 10 μg / mL

[0047] Ginsenoside Rf group: the final concentration of ginsenoside Rf was 400 μg / mL, 200 μg / mL, 150 μg / mL, and 100 μg / mL, respectively

[0048] Ginsenoside Rf combined with PD-1 inhibitor: the final concentration of ginsenoside Rf was 400 μg / mL, 200 μg / mL, 150 μg / mL, and 100 μg / mL, respectively, and the final concentration of PD-1 inhibitor was 10 μg / mL

[0049] (II) Cell administration and statistical analysis

[0050] Human PBMC cells (human peripheral blood cells) were inoculated in a 96-well plate at a density of 1.0 × 10 5 After 48 h of culture in a 37℃, 5% CO2 incubator, the logarithmic growth phase NCI-H226 cells (human lung squamous carcinoma cells) were digested and centrifuged, and then inoculated in a 96-well plate at a density of 1.0 × 10 4Cells were seeded at a density of 100 μL per well in 96-well plates containing PBMC cells (PBMC cells: NCI-H226 cells = 10:1) and co-cultured at 37°C in a 5% CO2 incubator. After 24 h, 200 μL of the PBMC-NCI-H226 co-culture system was added. The final volume for each group was 210 μL, with 3 replicates per group. After drug administration, cells were cultured at 37°C in a 5% CO2 incubator for another 72 h. The experiment was repeated in triplicate. After 72 h of drug administration, the culture medium in the 96-well plates was discarded, and each well was washed twice with 100 μL of PBS solution to remove PBMC cells. Then, 100 μL of 10% CCK-8 solution was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator for 4 h. The absorbance (OD) value of each well at 450 nm was measured using a microplate reader, and the viability of NCI-H226 cells was calculated according to Formula 1.

[0051]

[0052] Differences between cell experimental groups were analyzed using Dunnett's multiple comparisons test. P < 0.05 indicated that the differences were statistically significant.

[0053] II. Experimental Results

[0054] Experimental results are as follows Figure 1 As shown. Figure 1 As shown in Figure A, compared with the isotype control IgG group, the survival rate of lung squamous cell carcinoma cells in the PD-1 inhibitor group was significantly decreased. The survival rate of lung squamous cell carcinoma cells in the ginsenoside Rf and PD-1 inhibitor groups also showed a significant decreasing trend. However, the combination of ginsenoside Rb3 and ginsenoside Rc, structural analogs of ginsenosides, with PD-1 inhibitors did not show any inhibitory effect on the growth of NCI-H226 human lung squamous cell carcinoma cells. Based on this, further research was conducted on the effect of ginsenoside Rf combined with PD-1 inhibitors on the survival rate of NCI-H226 human lung squamous cell carcinoma cells. The results are as follows... Figure 1 As shown in Figure B, compared with the PD-1 inhibitor group, the survival rate of NCI-H226 human lung squamous cell carcinoma cells in the 400 μg / mL and 200 μg / mL ginsenoside Rf combined with PD-1 inhibitor groups was significantly reduced. These results indicate that, compared with PD-1 inhibitors alone, 400 μg / mL and 200 μg / mL ginsenoside Rf combined with PD-1 inhibitors can more effectively inhibit the growth of NCI-H226 human lung squamous cell carcinoma cells. However, the structural analogues of ginsenoside Rf, ginsenoside Rb3 and ginsenoside Rc, regardless of whether they were used in combination with PD-1 inhibitors, did not show any inhibitory effect on the growth of human lung squamous cell carcinoma cells. This suggests that ginsenoside Rf is a potential active substance against non-small cell lung cancer and can enhance the anti-cancer effect of PD-1 inhibitors.

[0055] Example 2

[0056] This example relates to a study on inhibition of tumor growth in LLC tumor-bearing mice by ginsenoside Rf in combination with a PD-1 inhibitor.

[0057] I. Experimental Methods

[0058] (I) Experimental grouping:

[0059] Isotype control IgG group (IgG): intraperitoneal injection of isotype IgG antibody 10 mg / kg twice a week, and 1.0% carboxymethylcellulose sodium solution 0.01 mL / g once a day by gavage (as a solvent control for the combination administration group Rf);

[0060] PD-1 inhibitor monotherapy group (PD-1): intraperitoneal injection of PD-1 inhibitor 10 mg / kg twice a week, and 1.0% carboxymethylcellulose sodium solution 0.01 mL / g once a day by gavage;

[0061] Rf monotherapy group (Rf): intraperitoneal injection of isotype IgG antibody 10 mg / kg twice a week, and ginsenoside Rf monomer 10 mg / kg once a day by gavage;

[0062] PD-1 inhibitor combined with Rf group (PD-1+Rf): intraperitoneal injection of PD-1 inhibitor 10 mg / kg twice a week, and ginsenoside Rf monomer 10 mg / kg once a day by gavage.

[0063] (II) Animal administration and statistical analysis

[0064] After 7 days of environmental adaptation, Lewis lung cancer cells (mouse lung adenocarcinoma cells) in the logarithmic growth phase were taken, centrifuged, diluted with PBS to a cell suspension of 5×10 6 / mL, and kept on ice to maintain cell viability. 0.1 mL was inoculated into the right front limb axilla of C57BL / 6 mice. After 7 days of inoculation, the mice were randomly grouped according to body weight, and administration was started on the 8th day of inoculation. During the administration period, the body weight of the mice was monitored and recorded every day. During the administration period, the length and width of the mouse tumor were measured once and recorded using a vernier caliper, and the tumor volume was calculated. The tumor volume calculation formula is:

[0065] Formula two: Tumor volume (mm3) = length (mm) x width (mm) 2 / 2.

[0066] After 24 hours of the last administration, the mice were anesthetized with 1.5% sodium pentobarbital, and the tumor was isolated and weighed. The differences between the tumor volume and tumor weight of the mice were analyzed using t-test. P<0.05 indicates that the difference is significant.

[0067] II. Experimental Results

[0068] The tumor volume of LLC tumor-bearing mice was recorded during the 14-day administration period, and the results are shown in Figure 2 It can be seen that, on the 22nd day after tumor transplantation, the subcutaneous tumor volume of the mice in the ginsenoside Rf combined with PD-1 inhibitor administration group (PD-1+Rf) was significantly reduced (P<0.01) compared with the isotype control IgG group, while the PD-1 inhibitor monotherapy group (PD-1) could reduce the tumor volume but the difference was not significant. Compared with the PD-1 inhibitor monotherapy group (PD-1), the tumor volume of the mice in the combined administration group (PD-1+Rf) was also significantly reduced (P<0.01); compared with the ginsenoside Rf monotherapy group (Rf), the tumor volume of the mice in the combined administration group (PD-1+Rf) was smaller (P<0.01). Moreover, the tumor weight results of each group Figure 2 C) were similar to the tumor volume results of B. In summary, ginsenoside Rf assisted PD-1 inhibitor can significantly inhibit the growth of subcutaneously transplanted tumors in LLC tumor-bearing mice, and the effect is stronger than that of PD-1 inhibitor monotherapy. Figure 2

[0069] Example 3:

[0070] This example relates to ginsenoside Rf combined with PD-1 inhibitor to prolong the survival of LLC tumor-bearing mice.

[0071] I. Experimental methods

[0072] The animals were grouped as in Example 2, and the administration of PD-1 inhibitor was stopped after four administrations, and the ginsenoside Rf monomer was administered continuously every day. The body weight of the mice was monitored every day during the administration period, and their mental state, fur color, food intake, defecation, and death were observed and recorded.

[0073] The difference in survival between groups of mice was analyzed using the Log-rank (Mantel-Cox) test. P<0.05 indicates that the difference is significant.

[0074] II. Experimental results

[0075] The number of LLC tumor-bearing mice was statistically analyzed, and the survival curve results are shown in Figure 3 ​As shown, there was no significant difference in survival time between the PD-1 inhibitor monotherapy group (PD-1) and the ginsenoside Rf monotherapy group (Rf) compared with the isotype control IgG group, indicating that the PD-1 inhibitor monotherapy and the ginsenoside Rf monotherapy had no obvious effect on the survival time of LLC mice. However, compared with the isotype control IgG group, the survival time of the PD-1 inhibitor combined with Rf administration group (PD-1+Rf) was significantly prolonged (P<0.05); compared with the ginsenoside Rf monotherapy, the survival time of the combined administration group (PD-1+Rf) was also significantly prolonged (P<0.05); and compared with the PD-1 inhibitor monotherapy group, the survival time of the combined administration group (PD-1+Rf) was also significantly prolonged (P<0.01), which indicated that the ginsenoside Rf assisted PD-1 inhibitor could significantly prolong the survival time of LLC tumor-bearing mice.

Claims

1. Application of ginsenoside Rf in preparation of a PD-1 inhibitor anti-non-small cell lung cancer drug enhancer, wherein the PD-1 inhibitor is pembrolizumab, and the mass ratio of ginsenoside Rf to the PD-1 inhibitor is 10-40:

1.

2. Use according to claim 1, characterized in that: The non-small cell lung cancer is lung squamous carcinoma or lung adenocarcinoma.

3. Use according to claim 1, characterized in that: The PD-1 inhibitor is KEYTRUDA® PD-1 inhibitor.

4. The use according to claim 1, characterized in that: The enhancer is a synergistic attenuator.

Images