CN114948941B

CN114948941B - New application of artemisinin and pharmaceutical composition

Info

Publication number: CN114948941B
Application number: CN202110198656.9A
Authority: CN
Inventors: 郑文华; 杨超
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2024-03-08
Anticipated expiration: 2041-02-22
Also published as: CN114948941A

Abstract

The invention relates to the technical field of new application of medicines, in particular to new application of artemisinin and a pharmaceutical composition. The invention provides an application of artemisinin in preparation of a medicine for preventing or treating amiodarone toxicity. Artemisinin can effectively improve the side effects of amiodarone, especially the pulmonary toxicity, and can reduce various toxic side effects of amiodarone by being combined with amiodarone.

Description

New application of artemisinin and pharmaceutical composition

Technical Field

The invention relates to the technical field of new application of medicines, in particular to new application of artemisinin and a pharmaceutical composition.

Background

Amiodarone (AM) is a highly effective drug for the treatment of cardiac arrhythmias. However, its use also causes adverse effects on organs such as thyroid, gastrointestinal tract, liver, eye, nerves, skin and lungs, so that the use of amiodarone is limited. Among them, pulmonary toxicity is the most harmful and the most fatal side effect. The pathogenesis of amiodarone-induced pulmonary toxicity may include direct cell damage (e.g., oxidative stress), and inflammatory mediator release, among others. Research and search for drugs that are effective in preventing and treating the pulmonary toxic effects of amiodarone are of potential and important value.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a novel application of artemisinin and a pharmaceutical composition. The invention provides a new application of artemisinin, which can effectively improve the side effect of amiodarone, especially the pulmonary toxicity, and can reduce the toxic side effect by combining with amiodarone.

The invention is realized in the following way:

in a first aspect, the invention provides the use of artemisinin for the preparation of a medicament for the prevention or treatment of amiodarone toxicity.

In alternative embodiments, the amiodarone toxicity is a variety of side effects of amiodarone, preferably, the side effects include pulmonary toxicity.

In alternative embodiments, the amiodarone toxicity includes side effects caused by oxidative damage and apoptosis of human bronchial epithelial cells.

In alternative embodiments, the amiodarone toxicity includes side effects caused by at least one of the following phenomena: (1) increased LDH release; (2) elevated ROS levels; (3) increased Caspase 3 activity; (4) mitochondrial membrane potential decrease.

In an alternative embodiment, the drug is at least one of the following drugs: (1) an agent that reduces LDH release; (2) an agent that reduces ROS levels; (3) agents that reduce Caspase 3 activity; (4) an agent that elevates mitochondrial membrane potential; (5) agents that up-regulate protein levels of CaMKK2 and Nrf 2; (6) an agent that increases the phosphorylation level of AMPK; (7) agents that activate the CaMKK2/AMPK/Nrf2 signaling pathway.

In a second aspect, the present invention provides a pharmaceutical composition comprising, as active ingredients, artemisinin and amiodarone.

In a third aspect, the present invention provides the use of artemisinin for the preparation of an improver which activates or promotes or upregulates at least one of the following; (1) mitochondrial membrane potential; (2) protein level of CaMKK 2; (3) protein level of Nrf 2; (4) phosphorylation level of AMPK; (5) CaMKK2/AMPK/Nrf2 signal paths.

In a fourth aspect, the present invention provides the use of artemisinin for the preparation of an inhibitor for inhibition of at least one of the following; (1) LDH release; (2) ROS levels; (3) Caspase 3 activity.

The invention has the following beneficial effects: the embodiment of the invention provides a novel application of artemisinin, which can effectively improve lung toxicity caused by oxidation injury and apoptosis of human bronchial epithelial cells, which are caused by the increase of LDH release, the increase of ROS level, the increase of Caspase 3 activity, the decrease of mitochondrial membrane potential and the like, caused by amiodarone, and can further relieve or improve the side effect of amiodarone, can be used as a medicine for preventing or treating amiodarone lung toxicity, and lays a theoretical foundation for the research and development of medicines for clinically treating amiodarone lung side effect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph of the detection result provided in Experimental example 1 of the present invention;

FIG. 2 is a graph of the detection result provided in Experimental example 2 of the present invention;

FIG. 3 is a graph showing the detection results provided in Experimental example 3 of the present invention;

FIG. 4 is a graph showing the detection results provided in Experimental example 4 of the present invention;

FIG. 5 is a graph of the detection result provided in Experimental example 5 of the present invention;

FIG. 6 is a graph showing the effect of artemisinin at various concentrations on the phosphorylation levels of AKT, ERK and AMPK as provided in Experimental example 6 of the present invention;

FIG. 7 is a graph showing the results of the detection of CaMKK2 and LKB1 with different concentrations of artemisinin provided in Experimental example 6 of the present invention;

FIG. 8 is a graph showing the results of detection of Nrf2 with artemisinin at various concentrations provided in Experimental example 6 of the present invention;

FIG. 9 is a graph showing the results of the detection of CaMKK2 and LKB1 at different treatment times for equiconcentration artemisinin provided in Experimental example 6 of the present invention;

FIG. 10 is a graph showing the results of the detection of SOD1 and Nrf2 at different treatment times of artemisinin at equal concentration provided in Experimental example 6 of the present invention;

FIG. 11 is a graph showing the results of the detection of p-AMPK by an equal concentration of artemisinin as provided in Experimental example 6 in combination with various doses of the CaMKK2 inhibitor STO-609;

FIG. 12 is a graph showing the results of detecting SOD1 and Nrf2 in which the activity of the AMPK inhibitor Compound C against artemisinin is reversed, provided in Experimental example 6 of the present invention;

FIG. 13 is a graph showing the results provided in Experimental example 7 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The embodiment of the invention provides a new application of artemisinin, in particular to an application of artemisinin in which toxic and side effects caused by amiodarone can be effectively improved, and then artemisinin can be used as a drug for preventing or treating amiodarone toxicity. In particular to the lung side effects caused by amiodarone, and the lung side effects are the side effects caused by oxidative damage and apoptosis of human bronchial epithelial cells. Or an increase in amiodarone-induced LDH release, an increase in ROS levels, an increase in Caspase 3 activity, a decrease in mitochondrial membrane potential, and side effects caused by apoptosis or pulmonary toxicity. Artemisinin is also indicated for use in the treatment of diseases caused by oxidative damage and apoptosis of human bronchial epithelial cells, or by increased LDH release, increased ROS levels, increased Caspase 3 activity, decreased mitochondrial membrane potential, and apoptosis.

Artemisinin is then the drug by decreasing LDH release; drugs that reduce ROS levels; drugs that reduce Caspase 3 activity and drugs that raise mitochondrial membrane potential, in turn improve amiodarone-induced toxicity, particularly pulmonary side effects. The inventor researches find that artemisinin is a medicine for up-regulating the protein levels of CaMKK2 and Nrf 2; drugs that elevate the phosphorylation level of AMPK; and agents that activate the CaMKK2/AMPK/Nrf2 signaling pathway can in turn ameliorate amiodarone-induced toxicity, particularly pulmonary side effects.

That is to say the use of artemisinin for the preparation of an improver which activates or promotes or upregulates at least one of the following substances; (1) mitochondrial membrane potential; (2) protein level of CaMKK 2; (3) protein level of Nrf 2; (4) phosphorylation level of AMPK; (5) CaMKK2/AMPK/Nrf2 signal paths. Or for preparing an inhibitor that inhibits at least one of the following; (1) LDH release; (2) ROS levels; (3) Caspase 3 activity.

Thus, pharmaceutical compositions having low pulmonary toxicity but still good therapeutic effect on arrhythmia can be prepared with artemisinin and amiodarone as active ingredients. The pharmaceutical composition can be a pharmaceutical pack or a pharmaceutically acceptable dosage form known in the prior art, and even patients can directly take the artemisinin preparation and the amiodarone preparation with the required dosage according to the required dosage.

Further, the molar ratio of artemisinin to amiodarone is: 25-100:3. The effect of the pharmaceutical composition can be further ensured by adopting the proportion.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Experimental example

Medicine and reagent: amiodarone was purchased from the pharmaceutical company of cinrofil and artemisinin was purchased from the biotechnology company of melons. BEAS-2B cells are offered by university of medical science, guangzhou, wittig. Fetal bovine serum, DMEM medium and diabodies were purchased from Gibco, usa, lactate dehydrogenase (Lactate dehydrogenase, LDH) cytotoxicity, reactive oxygen species (Reactive oxygen species, ROS), caspase 3 activity, mitochondrial membrane potential detection kit from Shanghai Biyun biotechnology institute, and apoptosis detection kit from bioengineering (Shanghai) stock.

Instrument: the fluorescence microplate reader, fluorescence microscope, cell incubator, biosafety cabinet were all purchased from us Thermo Fisher Scientific, and the flow cytometer was purchased from BD.

Cell culture: human bronchial epithelial cells (BEAS-2B cells) were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% diabody and placed at 37℃in 5% CO ₂ Is cultured in a constant temperature incubator.

Experimental grouping: blank Control (noted Control): culturing with common medium (i.e. without serum);

artemisinin control group (designated 50. Mu.M ART): pretreating with common culture medium containing 50 μm artemisinin for 2 hr, and culturing with common culture medium;

amiodarone group (recorded as 3 μm AM): culturing with common culture medium containing 3 μm amiodarone;

artemisinin pretreatment+amiodarone group: pretreatment with a common medium containing (25. Mu.M, 50. Mu.M, 100. Mu.M) artemisinin was performed for 2h, and then culture was performed with a common medium containing 3. Mu.M amiodarone, respectively. The corresponding group was designated 25. Mu.M ART+3. Mu.M AM in sequence; 50. Mu.M ART+3. Mu.M AM;100 μM ART+3 μM AM;

inhibitor + artemisinin pretreatment + amiodarone group (noted as Compound C + ART + AM): firstly, treating with a common culture medium containing 1 mu M of AMPK inhibitor Compound C for 10min, then changing to a common culture medium containing 50 mu M of artemisinin for 2h, and then changing to a common culture medium containing 3 mu M of amiodarone for culture;

inhibitor control (noted as Compound C): the culture was pretreated with a common medium containing 1. Mu.M Compound C for 10min and then replaced with the common medium.

Experimental example 1 cell proliferation Activity and toxicity detection

The method comprises the following steps: (1) BEAS-2B cells were seeded in 96-well plates at 5000 wells, the cells were treated in groups according to the above experiment, 6 replicates per group, 10. Mu.l of 5mg/ml MTT per well was added after culturing for 24 hours, the supernatant was discarded after culturing for 3 hours, 100. Mu.l of DMSO per well was added, and absorbance was measured at 570nm using an ELISA reader. (2) After cells were treated in the same manner, 120. Mu.l of supernatant from each well was placed in a new 96-well plate, 60. Mu.l of LDH detection working solution was added, incubated at room temperature for 30min under light-shielding conditions, and absorbance was measured at 490nm using an ELISA reader.

The detection results are shown in FIG. 1, wherein A in FIG. 1 is the detection result of cell proliferation activity, and B in FIG. 1 is the detection result of cytotoxicity.

As can be seen from fig. 1 a, the cell proliferation activity of amiodarone group was significantly reduced compared to the blank group, and the difference was statistically significant (# # p < 0.001). Compared with amiodarone group, the cell proliferation activity of artemisinin pretreatment and amiodarone group is obviously increased, and the difference is statistically significant (p <0.05, p <0.01, p < 0.001), which indicates that artemisinin can promote the decrease of cell proliferation activity caused by amiodarone.

As can be seen from fig. 1B, the amiodarone group showed significantly increased LDH release, increased cytotoxicity, and statistically significant differences compared to the blank group. Compared with amiodarone, the artemisinin pretreatment and the LDH release of amiodarone are obviously reduced, the cytotoxicity is reduced, and the difference is statistically significant, so that the artemisinin can reduce the increase of LDH release caused by amiodarone, and then the cytotoxicity is reduced.

Experimental example 2ROS level detection

The method comprises the following steps: the BEAS-2B cells were seeded in 96-well plates 10000 times per well, the cells were treated in groups of 6 replicates according to the above-described experiment, after culturing for 24 hours, the supernatant was aspirated, 50. Mu.l of diluted DCFH-DA (final concentration 10. Mu.M) was added, incubated in a 37℃cell incubator for 30 minutes, then washed 2 times with serum-free medium, 50. Mu.l of serum-free medium was further added, fluorescence intensity was detected at 488nm excitation wavelength and 525nm emission wavelength by means of a fluorescence microplate reader, and fluorescence was observed and photographed by means of a fluorescence microscope.

The detection result is shown in fig. 2, wherein a in fig. 2 is a fluorescent microscope photographing result diagram; in fig. 2, B is a graph of the detection result of the fluorescent microplate reader.

As can be seen from FIG. 2A, the green fluorescence of the amiodarone group was significantly enhanced, while the artemisinin pretreatment + amiodarone group was significantly reduced from the green fluorescence of the amiodarone group. As can be seen from fig. 2B, the fluorescence intensity of amiodarone group was significantly increased compared to the blank control group, the difference had a statistical significance (# # p < 0.001), whereas the artemisinin pretreatment + amiodarone group had significantly lower fluorescence intensity than the amiodarone group, the difference had a statistical significance (< 0.05, <0.01, < 0.001), indicating that artemisinin was able to reduce the rise in ROS levels caused by amiodarone.

Experimental example 3 mitochondrial Membrane potential detection

The method comprises the following steps: inoculating 10000 BEAS-2B cells in each well into a 96-well plate, treating the cells in groups according to the experiment, repeating 3 times in each group, sucking the supernatant after culturing for 24 hours, adding 50 μl JC-1 staining working solution, incubating for 30min in a 37 ℃ cell incubator, washing 2 times with JC-1 staining buffer solution, adding 50 μl cell culture solution, observing fluorescence by using a fluorescence microscope, and photographing; BEAS-2B cells were plated at about 4.5X10 cells per well ⁵ The cells were inoculated into 6-well plates, the following day of observation was performed until the cell density reached 80%, the drug treatment was performed, after the culture was completed for 24 hours, the supernatant was aspirated, the cells were digested with 0.25% pancreatin, the supernatant was discarded by centrifugation, the cells were resuspended with JC-1 staining working solution, incubated in a 37℃cell incubator for 30min, centrifuged and washed 2 times with JC-1 staining buffer, and finally the cells were resuspended with 200. Mu.l JC-1 staining buffer, and mitochondrial membrane potential changes were analyzed by flow cytometry.

The detection result is shown in fig. 3, wherein a in fig. 3 is a photograph result diagram of a fluorescence microscope; FIG. 3B is a graph showing the results of flow cytometry analysis.

As can be seen from FIG. 3A, the amiodarone group showed an increase in green fluorescence and a decrease in red fluorescence (indicating a decrease in mitochondrial membrane potential) compared to the blank control group, whereas the artemisinin pretreatment+amiodarone group (50 μMART+3 μM AM) improved the increase in green fluorescence caused by amiodarone, and the red fluorescence decreased, whereas the artemisinin control group, i.e., the pure artemisinin pretreatment, had no significant effect on the mitochondrial membrane potential of the cells.

As can be seen from fig. 3B, the amiodarone group and the blank group showed a significant decrease in mitochondrial membrane potential, whereas artemisinin pretreatment+amiodarone group (50 μm art+3 μm AM) improved the decrease in mitochondrial membrane potential caused by amiodarone, and the differences were statistically significant (p < 0.001).

Taken together, artemisinin alone has no effect on mitochondrial membrane potential, but artemisinin can raise the decrease in mitochondrial membrane potential caused by amiodarone.

Experimental example 4 detection of caspase 3 Activity

The method comprises the following steps: BEAS-2B cells were plated at about 4.5X10 cells per well ⁵ The cells were treated according to the above-mentioned experimental group after the next day of observation until the cell density reached about 80%, the cells were digested with 0.25% pancreatin, centrifuged to discard the supernatant, washed once with PBS, resuspended and precipitated by adding 200 μl lysate, lysed for 15min in ice bath, centrifuged for 15min at 4 ℃ at 12000r, the supernatant was transferred to a pre-chilled centrifuge tube, a suitable amount of the reaction solution was added for incubation at 37 ℃ for 2h, and absorbance was measured at 405 nm.

Referring to fig. 4, the activity of Caspase 3 in the amiodarone group is obviously increased compared with that in the blank control group, the artemisinin pretreatment+amiodarone group (50 μm art+3 μm AM) can inhibit the increase of Caspase 3 activity caused by amiodarone, the difference is statistically significant (p < 0.01), and the artemisinin control group, namely, the pure artemisinin pretreatment has no obvious effect on the activity of Caspase 3 in cells.

Taken together, it is demonstrated that artemisinin alone has no significant effect on Caspase 3 activity, but artemisinin inhibits the increase in Caspase 3 activity caused by amiodarone.

Experimental example 5 apoptosis detection

The method comprises the following steps: BEAS-2B cells were plated at about 4X 10 cells per well ⁵ The cells were treated according to the above-described experimental group after the next day of observation until the cell density reached about 70%, the supernatant was aspirated, digested with 0.25% pancreatin, centrifuged to discard the supernatant, washed 3 times with PBS, mixed with 195. Mu.l of 1 Xbinding Buffer resuspended cells with 5. Mu.l of Annexin V-FITC for 10min at room temperature in the absence of light, then added with 10. Mu. l Propidium Iodide, and flow-through assay was performed after mixing.

The results of the assay are shown in FIG. 5, wherein A in FIG. 5 is a flow chart of the results of the detection of normal cells (bottom left), early apoptotic cells and late apoptotic cells (bottom right and top right) and dead cells (top left), and B in FIG. 5 is a statistical chart of the differences in the proportion of apoptotic cells (early and late) in each group. The amiodarone group had significantly fewer apoptotic cells than the blank control group, whereas the artemisinin pretreatment+amiodarone group (50 μmart+3 μm AM) had statistically significant differences (p <0.01, p < 0.001), and the artemisinin control group, i.e., pure artemisinin pretreatment, had no significant effect on apoptosis. Taken together, it is shown that pure artemisinin has no effect on apoptosis, but artemisinin can reduce or decrease the increase of apoptosis caused by amiodarone, i.e., artemisinin can inhibit apoptosis caused by amiodarone.

Experimental example 6Western blot detection of protein expression

The method comprises the following steps: (1) BEAS-2B is 1.7 to 2X 10 per well ⁵ The cells are inoculated in a 12-well plate, when the cell density reaches 80% -90% in the next day, artemisinin (0, 6.25, 12.5, 25, 50 and 100 mu M) with different concentrations is treated for 2 hours, the culture solution is discarded, the cells are collected by adding cell lysate and are placed on ice for cracking for about 15 minutes, then the cells are heated in a metal bath at 100 ℃ for 10 minutes, and the cells are uniformly mixed by vortex and centrifuged briefly and then stored at-80 ℃ for standby. An equal volume of protein sample was subjected to SDS-PAGE gel electrophoresis (80V20min,120V 90min), then was electrotransferred to a methanol-activated PVDF membrane by a protein wet transfer method (90V 120 min), blocked with 3% BSA at room temperature for 1h, and after 3 times of PBST washing, the primary antibodies (anti-p-AKT, anti-p-ERK, anti-p-AMPK, anti-CaMKK2, anti-LKB1 and anti-Nrf 2) were incubated overnight at 4℃and after 3 times of PBST washing, the secondary antibodies labeled with horseradish peroxidase were incubated at room temperature for 1h, and after 3 times of PBST washing, developed with ECL developing solution.

(2) The detection is carried out by adopting the method (1) and the difference is that: 50. Mu.M artemisinin treatment was tested for different times (0, 7, 15, 30, 60 and 120 min), and primary antibodies were anti-CaMKK2, anti-LKB1, anti-SOD1 and anti-Nrf2.

(3) The detection is carried out by adopting the method (1) and the difference is that: BEAS-2B cells in 12-well plates were pre-treated with different doses of the CaMKK2 inhibitor STO-609 (0, 0.3, 1, 3 and 10. Mu.M) for 10min, followed by treatment with 50. Mu.M artemisinin for 2h; then testing is carried out; and the primary antibody is anti-p-AMPK.

(4) The detection is carried out by adopting the method (1) and the difference is that: cell treatment is carried out according to an artemisinin control group, an amiodarone group, an inhibitor+artemisinin pretreatment+amiodarone group and an inhibitor control group, and primary antibodies are anti-SOD1 and anti-Nrf2.

The detection results are shown in fig. 6-8, wherein fig. 6 is a graph showing the effect of artemisinin at different concentrations on AKT, ERK and AMPK phosphorylation levels, and fig. 7 is a graph showing the detection results of artemisinin at different concentrations on CaMKK2 and LKB 1; FIG. 8 is a graph showing the results of detection of Nrf2 with artemisinin at various concentrations; FIG. 9 is a graph showing the results of various treatment times of equiconcentration artemisinin for CaMKK2 and LKB 1; FIG. 10 is a graph showing the results of SOD1 and Nrf2 measurements at various treatment times with equiconcentration artemisinin; FIG. 11 is a graph showing the results of detection of p-AMPK by STO-609 at various doses in combination with equiconcentration artemisinin; FIG. 12 is a graph showing the results of detecting SOD1 and Nrf2 in which the activity of artemisinin is reversed by Compound C.

From fig. 6, it can be seen that the phosphorylation level of AMPK gradually increased with increasing artemisinin concentration, whereas the phosphorylation levels of AKT and ERK did not significantly increase.

From FIGS. 7 and 9, it can be seen that the protein level of CaMKK2 gradually increased with increasing artemisinin treatment concentration and time, while the protein level of LKB1 did not change significantly.

From FIGS. 8 and 10, it can be seen that the protein level of Nrf2 gradually increased with increasing concentration and time of artemisinin treatment, and the SOD1 protein level also gradually increased with increasing time of artemisinin treatment.

As can be seen from FIG. 11, pretreatment with various concentrations of the CaMKK2 inhibitor STO-609 (0, 0.3, 1, 3 and 10. Mu.M) for 10min blocked the activation of AMPK by artemisinin, indicating that artemisinin-mediated AMPK activation was dependent on CaMKK2 kinase rather than LKB1 kinase.

As can be seen from FIG. 12, the 2h artemisinin pretreatment can inhibit the decrease of the expression of the Nrf2 protein caused by the 24h amiodarone treatment, while the 10min Compound C pretreatment can block the promotion of the expression of the Nrf2 protein by the artemisinin, and the Compound C can block the promotion of the expression of the SOD1 protein by the artemisinin.

Taken together, artemisinin up-regulates protein levels of CaMKK2, nrf2 and activates AMPK in a dose and time dependent manner, demonstrating that artemisinin activates the CaMKK2/AMPK/Nrf2 signaling pathway to protect BEAS-2B cells from oxidative stress and apoptosis caused by amiodarone.

Experimental example 7 study of blocking of side effects of artemisinin on amiodarone

See experimental examples 1 and 2.

The results of the assay are shown in FIG. 13, wherein FIG. 13A is a fluorescence graph in which Compound C pretreatment blocks the reduction of cell viability caused by amiodarone, FIG. 13B is a reduction of AMPK blocks the reduction of cell viability caused by amiodarone, FIG. 13C is a fluorescence graph in which Compound C pretreatment blocks the inhibition of ROS caused by amiodarone, and FIG. 13D is a fluorescence intensity graph in which Compound C pretreatment blocks the inhibition of ROS caused by amiodarone.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of artemisinin for the preparation of a medicament for the prevention or treatment of amiodarone toxicity, which is a side effect of amiodarone, including pulmonary toxicity.