WO2020215481A1 - Applications of psoralen in preparing nrf2 inhibitor, medicament for nrf2 inhibition-related diseases, and anticancer medicament - Google Patents

Abstract

Applications of psoralen in preparing an Nrf2 inhibitor, a medicament for Nrf2 inhibition-related diseases, and an anticancer medicament. Psoralen reduces the level of Nrf2 protein expression, reduces the antioxidative stress ability of human non-small cell lung carcinoma A549 cells, and increases the proapoptotic effects of chemotherapeutic medicament cisplatin with respect to A549. Psoralen has no significant impact on animal liver and kidneys when used as a medicament.

Description

Application of Buguzhining in the preparation of Nrf2 inhibitors, drugs for diseases related to Nrf2 inhibition, and anticancer drugs Technical field

The invention belongs to the technical field of medicine, and specifically relates to the application of Buguzhining in the preparation of Nrf2 inhibitors, drugs for diseases related to Nrf2 inhibition, and anticancer drugs.

Background technique

Psoralen is the mature fruit of the leguminous plant Psoralea corylifolia L., has a variety of pharmacological activities, and has gradually attracted attention in anti-tumor aspects. Studies have shown that the psoralen and isopsoralen in psoralen can inhibit the proliferation of human gastric cancer cells BGG-823. Bakuchiol also has a significant inhibitory effect on the proliferation of liver cancer HepG2 cells. It can inhibit the proliferation of tongue squamous cell carcinoma Tca8113 cells and induce the apoptosis of human liver cancer HepG2 cells.

Although psoralen has good anti-tumor effects, the clear mechanism of action has not yet been fully elucidated. Li Pengzhan (Clinical study of Xiaojiyin combined with erlotinib in the treatment of non-small cell lung cancer [D]. Guangzhou University of Traditional Chinese Medicine, 2016:15.) pointed out that the ingredients of psoralen can inhibit topoisomerase II and DNA polymerization Enzymes, cytotoxicity and other different ways to exert anti-tumor activity. Psoralen can induce tumor cell mitochondrial degeneration, decrease the expression of apoptosis-suppressing gene Bcl-2 and promote cell apoptosis; Wu Xia Research on tumor angiogenesis of small cell lung cancer [D]. Guangzhou University of Traditional Chinese Medicine, 2013:7.) pointed out that psoralen mainly relies on psoralen for its anti-cancer effect, and psoralen can effectively kill mouse sarcoma cells The principle of the effect is that "psoralen forms complexes and molecular cross-links with mouse sarcoma cell DNA, thereby inhibiting DNA synthesis".

Existing studies have shown that activation of the Nrf2-ARE signaling pathway can promote the occurrence and development of lung cancer and resistance to radiotherapy and chemotherapy, reduce tumor cell apoptosis and promote tumor cell proliferation, and the accumulation of Nrf2 in the nucleus can lead to tumor cells gaining resistance. One of the important reasons for drug efficacy, therefore, it is necessary to develop new Nrf2 inhibitory drugs in lung cancer treatment strategies aimed at activation of the Nrf2-ARE pathway. Buguzhining (Corylin) is an isoflavone compound isolated from psoralen. Existing literature shows that Buguzhining can treat atherosclerosis (patent CN107737121A), cardiovascular disease, obesity, Aztec Hymer's disease, diabetes (CN105343051A), etc., but Buguzhining as an anti-cancer drug and the therapeutic effect of Buguzhining on cancer based on Nrf2 signal inhibition has not been proposed.

Summary of the invention

The purpose of the present invention is to provide a new application of Buguzhining, that is, its application in the preparation of Nrf2 inhibitors, drugs for diseases related to Nrf2 inhibition, and anticancer drugs to solve one or more of the above problems .

According to one aspect of the present invention, there is provided the application of Buguzhining in the preparation of Nrf2 inhibitors, wherein the structural formula of Buguzhining is as follows:

Experiments show that different concentrations of Buguzhining can inhibit the activity of ARE-dependent luciferase, and the inhibitory ability increases with the increase of the dose of Buguzhining, suggesting that Buguzhining can inhibit Nrf2-ARE-related oxidative reactions. Stimulate the transduction pathway.

According to another aspect of the present invention, there is provided the application of Buguzhining in the preparation of drugs for diseases related to Nrf2 inhibition, wherein the structural formula of Buguzhining is as follows:

In some embodiments, the drug is composed of Buguzhining and pharmaceutically acceptable excipients, wherein the mass percentage of Buguzhining in the drug is 20±5%.

In some embodiments, the drug is a lipid nanobody, capsule, granule, tablet, dripping pill, or oral liquid.

Among them, when the drug is a lipid nanobody, it is prepared as follows: according to the weight ratio of psoralen and soybean lecithin 0.03～0.05:1, take 0.06～0.1g of psoralen and dissolve in 30ml Mix well in water and ethanol, then add 20ml PBS buffer, the pH of PBS buffer is 7.0, mix well to obtain 50ml psoralen mixture, and mix it with 3 to 5 glass beads and 0.1ml Tween-80 After mixing, place the membrane in a rotary evaporator water bath at constant temperature and rotate for 6 hours, and the membrane washing temperature is 37°C to obtain a liposome suspension; the suspension is sonicated at room temperature for 20 minutes and passed through a 0.22 μm microporous membrane to obtain a supplement Guzhining lipid nanobody membrane filtration solution.

When the drug is a tablet, it is prepared as follows: dissolve 150 g of psoralen and 40 g of hydroxypropyl cellulose SSL in 400 mL of ethanol, add the above solution to 500 ml of liquid paraffin, dry under reduced pressure to remove the ethanol, Guzhining and hydroxypropyl cellulose form microparticles to precipitate, and the precipitated microparticles are dried at 50°C; the dried microparticles are mixed with 10g of cross-linked sodium hydroxymethylcellulose and 1g of magnesium stearate that have passed through a 100-mesh sieve, and then compressed. Each tablet contains 30mg of psoralen, and each tablet has a net weight of 0.13g. It can be used for oral use. It can be taken once a day, 2 tablets each time, or 2 times a day, 1 tablet each time.

In some embodiments, the disease is cancer. Specifically, the cancer is lung cancer. Experiments show that Buguzhining can down-regulate the expression level of Nrf2 protein and reduce the ability of human non-small cell lung cancer cell A549 to resist oxidative stress.

According to another aspect of the present invention, there is provided the use of psoralen and cisplatin in the preparation of lung cancer drugs, wherein the structural formula of psoralen is as follows:

In some embodiments, the drug is composed of the psoralen and cisplatin and pharmaceutically acceptable excipients.

In some embodiments, the drug is a capsule, granule, tablet, dripping pill, or oral liquid.

In some embodiments, anti-cancer specifically refers to anti-lung cancer.

The present invention is based on the applicant’s screening, extraction and research based on more than fifty Chinese herbal medicines. It is found through experiments that Buguzhining alone has an inhibitory effect on human lung cancer cell line A549 subcutaneously transplanted tumors and is equivalent to DDP and cisplatin The combined use is more effective in inhibiting tumors, thus providing a new use of Buguzhining, which can down-regulate the expression level of Nrf2 protein, reduce the anti-oxidative stress ability of human non-small cell lung cancer cells A549, and improve the effect of the chemotherapy drug cisplatin The pro-apoptotic effect of A549 can be used as a Nrf2 inhibitor and as an anti-cancer drug, especially lung cancer. When Buguzhining is used as a drug, animal experiments show that Buguzhining has no obvious effect on the liver and kidney of animals.

Description of the drawings

Figure 1 is a comparison chart of luciferase activity of different concentrations of Buguzhining;

Figure 2 shows the electrophoresis spectrum of the effect of Buguzhining on the expression of Nrf2, HO-1 and AKR1 protein;

Figure 3 shows the inhibition of the growth of subcutaneous transplanted tumors in nude mice in each test group;

Figure 4 shows the HE slices of liver and kidney of mice in each test group.

Detailed ways

The present invention will be further elaborated below in conjunction with specific examples, and combined with the accompanying drawings and experiments, the Nrf2 inhibitory effect, treatment and its inhibition of related diseases and anti-lung cancer effects of Buguzhining provided by the present invention will be further described in detail.

Example 1

Buguzhining is used to prepare drugs for diseases related to Nrf2 inhibition, and when the drug is lipid nanosomes, it can be obtained as follows:

According to the weight ratio of psoralen to soybean lecithin 0.05:1, take 0.1g of psoralen, dissolve it in 30ml absolute ethanol and mix well, then add 20ml PBS buffer solution, the pH value of PBS buffer solution is 7.0, fully Mix well to obtain 50ml psoralen mixture, mix it with 3 to 5 glass beads and 0.1ml Tween-80, then place it in a rotary evaporator water bath for 6 hours to rotate and wash the membrane at a temperature of 37℃ to obtain Liposome suspension. The suspension was sonicated at room temperature for 20 minutes and passed through a 0.22 μm microporous filter membrane to obtain a psoralen lipid nanobody membrane filtration solution.

The particle size of the liposome prepared by the above method is within 100 nm, the liposome loading capacity is 28%, and the content of psoralen is 5 mg/ml.

Example 2

Buguzhining is used to prepare drugs for diseases related to Nrf2 inhibition. When the drug is a tablet, it can be obtained by the following preparation method:

Dissolve 150g of psoralen and hydroxypropylcellulose SSL40g in 400mL of ethanol, add the above solution to 500ml of liquid paraffin, and dry under reduced pressure to remove the ethanol. The particles of psoralen and hydroxypropylcellulose are separated out. The precipitated particles are dried at 50°C; the dried particles are uniformly mixed with 10 g of cross-linked sodium hydroxymethyl cellulose and 1 g of magnesium stearate that have passed through a 100-mesh sieve, and compressed into tablets.

5000 tablets were prepared by the above method, each with a net weight of 0.13 g, and each tablet containing 30 mg of psoralen. The tablet can be taken for oral use, and can be taken once a day, 2 tablets each time; or can be taken 2 times a day, 1 tablet each time.

In the following, combined with the experiment, the effect of Buguzhining on Nrf2 inhibition, treatment and its inhibition of related diseases, and anti-lung cancer effects will be further explained in detail.

1. The inhibitory effect of Buguzhining on Nrf2

1. Test materials

1) Test cells

Human breast cancer cell line (MDA-MB-231-ARE-Luc) transfected with antioxidant response element-luciferase plasmid, provided by the Department of Pharmacology and Toxicology, University of Arizona School of Pharmacy.

2) Experimental medicinal material: Buguzhining, purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd.

3) Test reagent

DMSO was purchased from Sigma Company, fetal bovine serum was purchased from Hyclone Development Company, DMEM high glucose medium was purchased from Gibco Company, Phosphate Buffer Saline (PBS) was purchased from Hyclone Development Company, and Luciferase Quantitative Detection Kit was purchased From the ThermoFisher company.

4) Test equipment

The model of the automatic cell counter is Countstar IC100, and the GloMax 20/20 chemiluminescence detector was purchased from Promega.

2. Test method

Culture the MDA-MB-231-ARE-Luc cell line in a 48-well plate. When the confluence reaches 50%-70%, add different concentration gradients of Buguzhining (dissolved in DMSO) and harvest the cells within 24 hours. Dilute the lysis solution 5X PLB (passive lysis buffer) according to the kit instructions, remove the cell culture medium, wash it with PBS to remove the remaining medium, and add 80ul 1X PLB to each well. Shake the culture plate on a shaker for 20 min at room temperature to fully lyse the cells. Put 20ul cell lysate and 50ul Luciferase assay reagent in the EP tube, beat up and down with the pipette tip 2-3 times, measure in the luminometer, read, record the data and perform statistical analysis.

3. Test results

The test results are shown in Figure 1, and the data are shown in Table 1. Comparisons show that different concentrations of Buguzhining can inhibit the activity of ARE-dependent luciferase, and the inhibitory capacity increases with the increase of the dose of Buguzhining. Rising, suggesting that Buguzhining can inhibit Nrf2-ARE-related oxidative stress transduction pathways, and studies have shown that Nrf2-ARE is a key pathway for cellular oxidative stress response, which is effective in anti-tumor, anti-inflammatory, liver disease, and nerve damage. It plays an important role in many aspects.

Table 1 Effects of the compounds of the present invention on ARE-dependent luciferase activity

Concentration (umol/L)	Fluorescence value (A.U)
1	6.28±0.11
2.5	3.2±0.21
5	2.31±0.19
10	1.69±0.22
15	0.39±0.04
20	0.24±0.01

The results in Table 1 show that Buguzhining can effectively inhibit ARE-dependent luciferase activity, and the degree of inhibition increases with increasing dose.

2. Buguzhining down-regulates the level of Nrf2 protein

1. Test materials

1) Test cells

Human non-small cell lung cancer cell line A549, provided by the Center for Regenerative and Translational Medicine, Guangdong Academy of Chinese Medicine.

2) Experimental medicinal material: Buguzhining, purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd.

3) Test reagent

DMSO was purchased from Sigma, fetal calf serum was purchased from Hyclone Development Company, DMEM high glucose medium was purchased from Gibco Company, Phosphate Buffer Saline (PBS) was purchased from Hyclone Development Company, and NP-40 cell lysate was purchased from Hyclone Development Company. In Biyuntian, the BCA protein concentration determination kit was purchased from ThermoFisher; Anti-GAPDH antibody (ab8245), Anti-Nrf2 antibody (ab62352), Anti-HO-1 antibody (ab52947), Anti-AKR1C1 antibody (ab192785) ), Goat Anti-Rabbit IgG (ab6721).

4) Test equipment

The model of the automatic cell counter is Countstar IC100, and the GloMax 20/20 chemiluminescence detector was purchased from Promega.

2. Test method

A549 cells were inoculated in a 6-well plate, and when the confluence reached about 50%-70%, different concentrations of DMSO were added to dissolve Psoralen. The cells were collected after 24 hours of treatment, the culture medium was discarded, and the cells were washed with PBS, and the cells were digested with appropriate trypsin and cultured. The digestion is terminated by the base, and the cell suspension is collected in a centrifuge tube. Centrifuge at 1100rpm for 5min, discard the supernatant, resuspend and wash with PBS twice (each time at 1100rpm, 5min).

Add 1ml of diluted NP-40 lysate to each tube, pipette thoroughly, and place on ice to lyse for 30-45min. The low-temperature centrifuge is pre-cooled at 4°C, and the cells are fully lysed at 12000g for 15min. Centrifuge the supernatant carefully with a pipette and put it in a new EP tube for labeling. Perform protein quantitative detection according to the instructions of the BCA kit and calculate the WB Load the sample volume, add 4×loading buffer to the protein sample, heat the protein at 100°C for 5 minutes, and store it at -20°C for later use.

Configure the SDS-PAGE gel, assemble the solidified gel plate into the electrophoresis tank, fill the inner layer with fresh electrophoresis solution, add a proper amount of electrophoresis solution to the outer layer to submerge the bottom of the gel plate, and pull out the comb vertically. Calculate the volume after quantification by BCA and load each group sequentially. Add 5ul protein Maker to the comb holes on both sides of the sample.

After loading the sample, connect the interfaces of the electrophoresis tank correctly, and perform electrophoresis at a constant voltage of 110V for about 1 hour. Stop the electrophoresis when the protein sample is close to running out of the separation gel. Take out the gel and soak it in the transfer buffer, and cut the filter paper and PVDF membrane of appropriate size. The filter paper is soaked in the buffer solution, the PVDF membrane is activated in methanol, and then placed on the electrode plate of the membrane transfer instrument in the order of filter paper, membrane, glue, and filter paper to drive out the bubbles, seal the electrode plate clamp, and assemble the membrane transfer instrument. Press 90V to transfer the film for 1h.

Configure the blocking solution (5% skimmed milk powder), and place the PVDF membrane in the blocking solution on a shaker and incubate at room temperature for 1 hour. The PVDF membrane was cut according to the molecular weight of the detected protein, the antibody was diluted in the blocking solution according to the instructions, and the PVDF membrane was placed in the primary antibody dilution solution and blocked overnight at 4°C. After the primary antibody incubation, the membrane was washed with TBST, and then the secondary antibody was incubated. After 1 hour of incubation, the membrane was washed with TBST. After the developer is configured, the film is placed in a chemiluminescence imaging system for development and exposure.

3. Test results

The results are shown in Figure 2. Buguzhining began to show a significant inhibitory effect on Nrf2 in the A549 cell line at 10uM, and with the increase of the dose, the inhibitory effect was more obvious, and Nrf2 was regulated as a nuclear transcription factor. The protein levels of downstream genes such as HO-1 and AKR1CI were also significantly reduced. It shows that Buguzhining can inhibit Nrf2 in A549 cells.

3. The therapeutic effect of Buguzhining and its combination with cisplatin on human lung cancer cell A549 transplanted into nude mice

1. Test materials

1) Experimental animals

BALB/c male nude mice aged 6 weeks, weighing 18-22 g, were provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine.

2) Test cells

Human non-small cell lung cancer cell line A549, provided by the Center for Regenerative and Translational Medicine, Guangdong Academy of Chinese Medicine.

3) Test medicinal material: Buguzhining, purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd.

4) Test reagent

Cisplatin for injection (DDP, freeze-dried) was purchased from Qilu Pharmaceutical Co., Ltd.; corn oil was purchased from Guangdong Changxing Food Trading Co., Ltd., 0.9% sodium chloride injection was purchased from Beijing Shuanghe Pharmaceutical Co., Ltd., DMSO It was purchased from Sigma, the source of fetal bovine serum was Hyclone Development Company, DMEM high glucose medium was purchased from Gibco, Phosphate Buffer Saline (PBS) was purchased from Hyclone Development Company, and trypsin-EDTA digestion solution was purchased from Gibco. .

5) Test equipment: The model of the automatic cell counter is Countstar IC100.

2. Test method

After resuscitation, the A549 cells are cultured in a 37°C cell incubator. When the cells grow to more than 90% confluence, they are subcultured. They are inoculated into new culture flasks according to the first pass three or four pass, and the subculture is repeated to expand the culture to a suitable number of models.

Discard the medium in the culture flask, wash with PBS and add an appropriate amount of trypsin digestion solution, add an appropriate amount of medium to terminate the digestion when the cells are rounded or fall off under the microscope, pipet and suck into the centrifuge tube, centrifuge at low speed for 5 minutes to discard the supernatant , Wash 2-3 times with PBS, count the cells, dilute the cell suspension according to the number of cells 107/100ul PBS, the whole process is carried out on ice.

Then use a 1ml pre-cooled syringe to suck the mixed cell suspension into the right armpit of the nude mouse, with 100ml cell suspension per mouse. When the tumor grows to 80mm ^3, the mice are randomly grouped and labeled.

Randomly divided into 4 groups, at least 5 animals in each group, respectively, intraperitoneal injection of corn oil-soluble DMSO, corn oil-soluble psoralen (10mg/kg), physiological saline soluble cisplatin (2mg/kg), and psoralen combined with cis Platinum is injected once every other day and repeated 5 times. Weigh the weight of each mouse and observe the tumor growth state each time, measure the longest diameter (a) and shortest diameter (b) of the tumor with a vernier caliper, and calculate and record the tumor volume according to V=1/2ab ² .

The nude mice were sacrificed the next day after the last administration.

3. Test results

As shown in Figure 3, compared with the control group, the Buguzhining group and the cisplatin group can inhibit the growth of subcutaneous transplanted tumors in nude mice, and compared with the Buguzhining single agent group and the cisplatin single agent group, the combined The inhibitory effect of the administration group on the transplanted tumor was more obvious. This indicates that Buguzhining can improve the inhibitory effect of cisplatin on A549.

Table 2 The effect of the compound of the present invention on the size of human lung cancer cell line A549 subcutaneously transplanted tumor

Group	dose	Tumor volume (mm 3 )	Tumor inhibition rate (%)
blank	0	309±38.91	To
DDP	2mg/kg	99.16±1.8	67.91
Buguzhining group	10mg/kg	99.41±2.42	67.83
Buguzhining + DDP group	-	68.87±5.69	77.71

The results of the effects of each group on the size of human lung cancer cell line A549 subcutaneously transplanted tumor are shown in Table 2, indicating that Buguzhining alone has the same inhibitory effect on human lung cancer cell line A549 subcutaneously transplanted tumor as DDP, and combined with cisplatin to inhibit The tumor effect is stronger.

The above test indicates that Buguzhining can be used to prepare Nrf2 inhibitors, drugs related to Nrf2 inhibition of diseases (especially lung cancer), and anti-lung cancer drugs (if combined with existing drugs, better anti-lung cancer effects can be achieved) The medicine mentioned here means that its main active ingredient is psoralen, and then pharmaceutically acceptable excipients are added to form capsules, granules, tablets, dripping pills or oral liquids.

4. Experiment of Buguzhining on liver and kidney damage in mice

1. Test materials

1) Experimental animals

6-week-old male BALB/c nude mice, weighing 18-22g, were provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine, and the certificate number was 11400700340882.

2) Test medicinal materials and reagents

Buguzhining was purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd.; corn oil was purchased from Guangdong Changxing Food Trading Co., Ltd., DMSO was purchased from Sigma Company, phosphate buffered saline (PBS) was purchased from Hyclone Development Company, and HE stained The liquid was purchased from Google Bio, and absolute ethanol and xylene were purchased from Tianjin Damao Chemical Reagent Company;

3) Test equipment:

Inverted phase contrast microscope (CKX41 Olympus), automatic cell counter (Countstar IC100); biological safety cabinet (BSC-1000IIAC); automatic staining and sealing integrated workstation (LEICAST5020), tissue embedding machine (HisTOSTAR), all Automatic closed tissue dehydrator (SHANDON PATACENTRE), paraffin microtome (LEICA RM2245).

2. Test method

Hematoxylin-eosin (Hematoxylin-eosin, HE) staining: use a paraffin microtome to slice the paraffin samples, the thickness is 3.5μm, serial sections, the staining procedure is baking slices drying for 20 minutes, xylene twice × 10 minutes, absolute ethanol 2 times × 2 minutes, 95% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, water washing for 1 minute, hematoxylin for 8 minutes, hematoxylin for 10 minutes, water washing for 2 times for 1 minute, 0.5% hydrochloric acid alcohol for 10 seconds, Wash with water for 10 minutes, eosin for 2 minutes, wash with water for 1 minute, 80% ethanol for 5 seconds, 85% ethanol for 5 seconds, 90% ethanol for 5 seconds, 95% ethanol for 1 minute, anhydrous ethanol 2 times × 2 minutes, anhydrous ethanol 3 Minutes, xylene 2 times × 2 minutes, after finishing dyeing, directly use neutral gum to seal.

3. Test results

The results are shown in Figure 4, indicating that Buguzhining has no significant effect on the liver and kidney of animals. Among them, liver slices showed complete morphology and structure of liver sinusoids and hepatic cords, no nuclear edema in the liver, and no cytoplasmic lesions; kidney slices showed intact glomeruli, and the number was consistent with the control group, and no lesions were seen in cytoplasm and nucleus.

What has been described above are only some embodiments of the present invention. For those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, and these all fall within the protection scope of the present invention.

Claims (10)

1. Application of Buguzhining in the preparation of Nrf2 inhibitors. The structural formula of Buguzhining is as follows:

   
2. Application of Buguzhining in the preparation of drugs for diseases related to Nrf2 inhibition, wherein the structural formula of Buguzhining is as follows:

   
3. The application according to claim 2, wherein the medicine is composed of the psoralen and pharmaceutically acceptable excipients, and the mass percentage of the psoralen in the medicine is 20%. ±5%.
4. The application according to claim 2, wherein the medicine is a capsule, granule, tablet, dripping pill or oral liquid.
5. The use according to any one of claims 2 to 4, wherein the disease is cancer.
6. The use according to claim 5, wherein the cancer is lung cancer.
7. Application of Buguzhining and Cisplatin in the preparation of anticancer drugs, wherein the structural formula of Buguzhining is as follows:

   
8. The application according to claim 7, wherein the medicine is composed of the psoralen, cisplatin and pharmaceutically acceptable excipients.
9. The application according to claim 7, wherein the medicine is a capsule, granule, tablet, dripping pill or oral liquid.
10. The use according to any one of claims 7-9, wherein the anti-cancer refers to anti-lung cancer.

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