阅读说明：本技术 一种用于治疗新型冠状病毒感染肺炎的药物组合物 (Pharmaceutical composition for treating novel coronavirus infection pneumonia ) 是由 陈依军 杨勇 叶俊梅 赵维俊 于 2021-08-27 设计创作，主要内容包括：本发明涉及医药技术领域,本发明提供了一种联合应用的药物组合物,包括艾地苯醌与氯喹/羟氯喹或其药学上可成的盐。艾地苯醌能显著降低氯喹/羟氯喹引起的心脏毒性,可以大幅提高氯喹/羟氯喹的临床用药剂量。本发明联合用药的新用途将有助于在提高氯喹/羟氯喹用药剂量的同时确保安全性,可用于临床治疗新冠病毒感染肺炎。(The invention relates to the technical field of medicines, and provides a pharmaceutical composition for combined application, which comprises idebenone and chloroquine/hydroxychloroquine or pharmaceutically acceptable salts thereof. Idebenone can remarkably reduce cardiotoxicity caused by chloroquine/hydroxychloroquine, and can greatly improve clinical application dosage of chloroquine/hydroxychloroquine. The new application of the combined medicine is beneficial to improving the dosage of chloroquine/hydroxychloroquine and simultaneously ensuring the safety, and can be used for clinically treating the pneumonia infected by the new coronavirus.)

1. A pharmaceutical composition characterized by comprising idebenone and chloroquine/hydroxychloroquine or pharmaceutically acceptable salts thereof.

2. The pharmaceutical composition according to claim 1, wherein the molar ratio of idebenone to chloroquine/hydroxychloroquine or pharmaceutically acceptable salts thereof is from 0.05 to 0.2: 1.

3. the pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is an oral formulation or an injectable formulation.

4. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutically acceptable salt of chloroquine/hydroxychloroquine is an acid salt.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable chloroquine/hydroxychloroquine salt is selected from the group consisting of sulfate, hydrochloride, phosphate, nitrate, oxalate, formate, benzoate, acetate, trifluoroacetate, succinate, tartrate, malate, citrate, sulfonate, benzenesulfonate, and benzylsulfonate.

6. The pharmaceutical composition of claim 5, wherein said pharmaceutically acceptable chloroquine/hydroxychloroquine salt is chloroquine phosphate/hydroxychloroquine sulfate.

7. Use of a pharmaceutical composition according to any one of claims 1 to 6 for the preparation of a medicament for the prevention or treatment of pneumonia caused by a novel coronavirus infection.

8. The use according to claim 7, characterized in that the pharmaceutical composition is administered in a dose of chloroquine/hydroxychloroquine or a pharmaceutically acceptable salt thereof which is: 12-24mg/kg per person per day.

Technical Field

The invention relates to the field of biological medicine, and relates to a pharmaceutical composition and application thereof, in particular to a combination of idebenone and chloroquine/hydroxychloroquine or pharmaceutically acceptable salts thereof, which is applied to the treatment of new coronavirus infection and can relieve cardiotoxicity caused by chloroquine/hydroxychloroquine.

Background

The current triple variant virus is a new variant virus named b.1.618, characterized by a chromosomal rearrangement of 6 nucleotides (H146del and Y145del) in addition to the E484K and D614G mutations in the spike protein (the "delta" variant strain). Although some of the diagnosed cases have been vaccinated, b.1.618 variant infection still occurs. This situation is referred to as "breakthrough infection", i.e., the vaccinated person is also infected and can infect others. Indeed, the situation in which "delta" variant viruses remain infected after complete vaccination has emerged in several countries. Although vaccinated patients generally have less symptoms, less chance of becoming severe and a relatively shorter course of disease, the emergence and prevalence of the delta variant strains are undeniably leading to the breakthrough of the immune barrier established by the new coronary vaccines and also to more serious challenges in the prevention and treatment of new coronary pneumonia. With the continuous and accelerated mutation of new coronaviruses, the situation of breakthrough infection becomes more and more severe. Therefore, there is an urgent need for safe and effective therapeutic drugs.

While the development of vaccines is tightened all over the world, no effective drug which directly aims at COVID-19(corona virus disease-2019) exists so far. Chloroquine has shown a superior clinical effect in small sample clinical trials for the treatment of novel coronavirus pneumonia (COVID-19) over lopinavir/ritonavir (Clostrich) (Gautret P et al. int J Antinic Agents,2020,20: 105949; Horby P, et al., Lancet,2020,396: 1345-. The multicenter cooperative group of the science and technology hall of Guangdong province and the health and health committee of chloroquine phosphate for treating the new coronavirus pneumonia has also presented expert consensus on chloroquine phosphate for treating the new coronavirus pneumonia. Hydroxychloroquine (HCQ) is a 4-aminoquinoline antimalarial drug developed on the basis of chloroquine in 1946, and has pharmacological effects such as antimalarial, immunomodulatory, antiviral, antibacterial, and antifungal effects. Since hydroxychloroquine is a metabolite of chloroquine, they have similar structures and mechanisms of action, but are safer than chloroquine in clinical practice for the treatment of malaria and immune diseases (Rainsford, k.d., et al, inflmmopharmacography, 2015, 23: 231-69). In vitro studies show that hydroxychloroquine sulfate (HCQ) or Chloroquine (CQ) has the effect of remarkably inhibiting the replication of new coronavirus, and the immunosuppressive effect of the hydroxychloroquine or chloroquine can also reduce the occurrence of inflammatory factor storm.

Multiple clinical trial results suggest that early heavy use of HCQ can significantly reduce viral load, promote pneumonia absorption, reduce mortality, improve prognosis in patients with new coronary pneumonia (Gao, j.et al, Biosci Trends,2020,14: 72-73; Oscanoa, t.j., et al, Int J Antimicrob Agents,2020,56: 106078.). Notably, the clinical oral dosage of hydroxychloroquine sulfate tablets generally does not exceed 400 mg/day (Fiehn, C., et al., Z Rheumatotol, 2021,80(Suppl 1): 1-9). However, clinical data indicate that the low dose (or safe dose) of hydroxychloroquine sulfate does not achieve effective inhibition of serum levels of new coronaviruses in vivo, and thus higher tissue or blood levels of HCQ or CQ need to be achieved by increasing oral doses of HCQ or CQ to achieve the purpose of inhibiting viral replication. In vitro studies also show that the higher serum drug concentrations of hydroxychloroquine sulfate are, the better the clearance of the new coronaviruses (Watson, j.a., et al, eife, 2020,9: e 58631). The dosage of HCQ used for clinically effective treatment of new coronary pneumonia is reported to be in the range of 800-1200mg/d (Oscanoa, T.J., et al., Int J Anthrob Agents,2020,56: 106078.). Unfortunately, clinical use of large doses of hydroxychloroquine sulfate or chloroquine phosphate results in severe cardiotoxicity in patients, including arrhythmias such as prolonged Q-T intervals in the cardiac cycle, broadening of QRS waves, torsades de pointes, and sudden death from heart failure in patients when severe (Burrell, Z.L., Jr.et al., New Engl J Med,1958,258: 798;. Tosmann, E.et al., Immunopharmacol Immunotoxicol,2013,35: 434-42). Also, clinical data show that patients with new coronary pneumonia in excess of 1/3 have varying degrees of myocardial damage and may further increase the risk of cardiovascular disease in patients if treated with HCQ or CQ (Naksuk, N.et al., Eur Heart J Acute Cardiovasc Care,2020,9: 215-.

Disclosure of Invention

The present invention has been made to solve the above-mentioned problems occurring in the prior art, and an object of the present invention is to provide a pharmaceutical combination composition capable of reducing or reversing chloroquine/hydroxychloroquine cardiotoxicity. Through a large number of experiments, different anti-oxidative stress drugs including vitamin E, vitamin C, folic acid, coenzyme Q10 and idebenone are respectively combined with chloroquine phosphate/hydroxychloroquine sulfate for use, so as to reduce or eliminate the cardiotoxicity of the chloroquine phosphate/hydroxychloroquine sulfate. Finally, the combined use of idebenone and chloroquine phosphate/hydroxychloroquine sulfate can greatly reduce the cardiotoxic effect caused by chloroquine phosphate/hydroxychloroquine sulfate, so that the safe dose of the chloroquine phosphate/hydroxychloroquine sulfate can be greatly increased. The invention is expected to provide a new treatment way for patients infected by the new coronavirus in clinic.

In order to achieve the above object, the technical solution of the present invention is as follows:

a pharmaceutical composition comprises idebenone and chloroquine/hydroxychloroquine or pharmaceutically acceptable salts thereof.

In the technical scheme of the invention, in the combined medicine composition, the effective component idebenone can relieve the cardiotoxic effect of chloroquine phosphate/hydroxychloroquine sulfate. The invention discovers that idebenone can remarkably reduce cardiotoxicity caused by chloroquine phosphate/hydroxychloroquine, and is dose-dependent in a certain range: in the myocardial cells, the molar ratio of idebenone to chloroquine phosphate/hydroxychloroquine sulfate is (0.05-0.2): 1, (0.1-0.2): 1, can obviously reverse the decrease of the myocardial cell metabolic rate caused by chloroquine phosphate/hydroxychloroquine sulfate and maintain the normal metabolic rate of the myocardial cell.

The pharmaceutical composition is preferably an oral formulation or an injectable formulation.

The pharmaceutically acceptable salt of chloroquine/hydroxychloroquine is a salt formed by chloroquine/hydroxychloroquine and an organic acid or an inorganic acid, such as sulfate, hydrochloride, phosphate, nitrate, oxalate, formate, benzoate, acetate, trifluoroacetate, succinate, tartrate, malate, citrate, sulfonate, benzenesulfonate, benzylsulfonate and the like.

In a specific embodiment of the present invention, a pharmaceutical combination of idebenone and chloroquine phosphate/hydroxychloroquine sulfate is selected.

The invention also aims to provide application of the pharmaceutical composition in preparing a medicament for treating the pneumonia infected by the novel coronavirus.

When the composition is used for treating pneumonia caused by novel coronavirus infection, the dosage of chloroquine/hydroxychloroquine or pharmaceutically acceptable salts thereof in the pharmaceutical composition is 12-24mg/kg per person per day (2-4 times of safe dosage of hydroxychloroquine sulfate used alone clinically).

In the technical scheme of the invention, the medicine combination provides a basis for clinical application in treatment of the novel coronavirus infection pneumonia.

In the invention, hydroxychloroquine sulfate is abbreviated as HCQ, chloroquine phosphate is abbreviated as CQ, idebenone is abbreviated as IDE, vitamin C is abbreviated as VitC, vitamin E is abbreviated as VitE, folic acid is abbreviated as FA, and coenzyme Q10 is abbreviated as CoQ 10.

The invention has the advantages that:

the research of the invention finds that hydroxychloroquine sulfate has no direct inhibition effect on myocardial potassium, sodium and calcium ion channels, and the myocardial cell damage is caused by enhancing the mitochondrial oxidative stress of the myocardial cell. According to the invention, different antioxidant stress drugs including vitamin E, vitamin C, folic acid, coenzyme Q10 and idebenone are respectively combined with hydroxychloroquine sulfate for use, so as to reduce or eliminate the cardiotoxicity of hydroxychloroquine sulfate. The results show that the vitamin E, the vitamin C, the folic acid and the coenzyme Q10 have no effect of remarkably reversing the reduction of the cell metabolic rate caused by the hydroxychloroquine sulfate and can not inhibit the oxidative stress caused by the hydroxychloroquine sulfate. The idebenone combined with chloroquine phosphate/hydroxychloroquine sulfate can obviously reduce the increase of myocardial cell oxidative stress level caused by the chloroquine phosphate/hydroxychloroquine sulfate, and the combined use of the idebenone and the chloroquine phosphate/hydroxychloroquine sulfate can greatly reduce the cardiotoxic effect caused by the chloroquine phosphate/hydroxychloroquine sulfate, so that the safe use dose of the chloroquine phosphate/hydroxychloroquine sulfate can be greatly increased. Is expected to provide a new treatment option for patients infected by the new coronavirus in clinic.

Drawings

FIG. 1 is a graph showing the effect of hydroxychloroquine sulfate on the cardiac function of rats in example 1 of the present invention. (A) Rat Q-T interval before and 5min after tail vein injection of HCQ with different doses; (B) comparing the Q-T interval of the rats before and 5min after the injection of the low dose HCQ; (C) comparing the Q-T interval of the rats before and 5min after the injection of the HCQ with the medium dose; (D) the Q-T interval of rats was compared before and 5min after the high dose HCQ injection. ns, no significant difference; p < 0.05; p < 0.001.

FIG. 2 is a graph showing the effect of hydroxychloroquine sulfate on cardiomyocyte function in example 2 of the present invention. (A-C) effects of HCQ on the hERG channel; (D-E) Effect of HCQ on Cav1.2 ion channels; (F-G) HCQ on Nav1.5 ion channels.

FIG. 3 is a graph showing the effect of chloroquine phosphate/hydroxychloroquine sulfate on mitochondrial oxidative stress in cardiomyocytes in example 3 of the present invention. (A) Rho123 staining was performed after different concentrations of HCQ or CQ treated cardiomyocytes for 6 h; (B) detecting Mito SOX by a flow cytometer after the myocardial cells are treated for 6 hours by 100 mu M HCQ or CQ; (C) detecting DHE by a flow cytometer after treating the myocardial cells for 6 hours by 100 mu M HCQ or CQ; (D) detecting JC-1 by a flow cytometer after treating the myocardial cells for 6 hours by 100 mu M of HCQ or CQ; (E) the ATP content of the cells was detected 6h after the cardiomyocytes were treated with 100. mu.M HCQ or CQ. P < 0.01; p < 0.001.

FIG. 4 shows the effect of the combination of different antioxidant stress agents and hydroxychloroquine sulfate on the rate of myocardial cell metabolism in rats in example 4 of the present invention. NRCMs were treated with 100. mu.M HCQ and different concentrations of anti-oxidative stress drugs VitC (A), VitE (B), FA (C), CoQ10(D) and IDE (E) for 6h, and then CCK8 was used to detect cell activity. ns, no significant difference; p < 0.001.

FIG. 5 shows the effect of different anti-oxidative stress agents in combination with chloroquine phosphate/hydroxychloroquine sulfate on the oxidative stress of myocardial cell mitochondria in example 5 of the present invention. (A) After cardiomyocytes were treated with 100. mu.M HCQ, 100. mu.M HCQ + 25. mu.M VitC, 100. mu.M HCQ +24mM VitE, 100. mu.M HCQ + 20. mu.M FA, 100. mu.M HCQ + 40. mu.M CoQ10, 100. mu.M HCQ + 20. mu.M IDE for 6h, Mito SOX was detected by flow cytometry; (B) after cardiomyocytes were treated with 100. mu.M HCQ, 100. mu.M HCQ + 25. mu.M VitC, 100. mu.M HCQ +24mM VitE, 100. mu.M HCQ + 20. mu.M FA, 100. mu.M HCQ + 40. mu.M CoQ10, 100. mu.M HCQ + 20. mu.M IDE for 6h, Rho123 was detected by flow cytometry; (C) after the myocardial cells are treated for 6 hours by 100 mu M CQ and 100 mu M CQ +20 mu M IDE, the Mito SOX is detected by a flow cytometer; (D) the cardiomyocytes were treated with 100. mu.M CQ, 100. mu.M CQ + 20. mu.M IDE for 6h, and then Rho123 was detected by flow cytometry. P < 0.001.

FIG. 6 is a graph showing the effect of combinations of idebenone and a lethal dose of hydroxychloroquine sulfate on mortality in mice in example 6 of the present invention. Mice were injected with a lethal dose of HCQ (2 mg/mouse) intravenously at the tail, or HCQ (2 mg/mouse) + IDE (0.018 mg/mouse), 10 mice per group simultaneously, and the mortality of mice was counted after administration.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included within the invention. The combination of idebenone and chloroquine phosphate/hydroxychloroquine sulfate and the use thereof in the present invention are further illustrated by the following specific examples in which no specific techniques or conditions are indicated, according to the techniques or conditions described in the literature in the art or according to the product specifications.

Example 1: effect of Hydroxychloroquine sulfate on rat cardiac function

SD rats were given different doses of HCQ, respectively: the low dose is 12.5mg/kg (corresponding to human body dose of 2mg/kg, 120 mg/human), the medium dose is 25mg/kg (corresponding to human body dose of 4mg/kg, 240 mg/human), and the high dose is 37.5mg/kg (corresponding to human body dose of 6mg/kg, 360 mg/human).

In order to eliminate the influence of different HCQ oral preparations due to bioavailability difference, tail vein injection is selected, namely, the absorption rate of each animal to the medicine is ensured to be 100%.

The effect of different doses of HCQ on the Q-T interval in rats was compared by electrocardiographic examination of the rats before and 5 minutes after injection.

The results showed that the rats in all three doses of HCQ-administered group exhibited different degrees of prolongation of Q-T interval compared to the control group (vehicle) (FIG. 1A). Analysis of each group revealed that the Q-T interval was not statistically different before and after dosing in the 12.5mg/kg group (68.27ms vs.73.09ms, p ═ 0.3364) (fig. 1B); the Q-T interval was significantly prolonged after administration in the 25mg/kg group (61.79ms vs.72.42ms, p ═ 0.0155) (fig. 1C); the 37.5mg/kg group had the greatest effect on Q-T intervals in three doses (59.97ms VS.81.85ms, p <0.001) (FIG. 1D). These results indicate that HCQ administration results in an extended Q-T interval in rats, consistent with clinical dosing for patients with new coronary pneumonia.

Example 2: effect of Hydroxychloroquine sulfate on myocardial cell function

Detection of the Effect of HCQ on myocardial Potassium ion channels

(1) After overexpression of the hERG potassium channel using HEK-293 cells, membrane currents were recorded using a HEKA EPC-10 patch clamp amplifier and PATCHMASTER collection system (HEKA Instruments Inc., D-67466 Lambrrecht, Pfalz, Germany).

(2) The membrane voltage is clamped at-80 mV, cells are given continuous 2s, +20mV voltage stimulation to activate the hERG potassium channel, repolarization is carried out to-50 mV for 5s, outward tail current is generated, and the stimulation frequency is once every 15 s. The current value is the peak value of the tail current. Quinidine (Quinidine) was a positive control.

The results show that different concentrations of hcq (h) have no significant inhibitory effect on the hERG potassium channel (fig. 2A-C).

Detection of the Effect of HCQ on myocardial calcium ion channels

(1) And (3) separating the guinea pig ventricular myocytes by using a Langendorff perfusion device, transferring the acutely separated primary guinea pig cardiac myocytes into a cell bath of an inverted microscope platform in an electrophysiological experiment system, and recording the Cav1.2 channel current by adopting a whole-cell recording mode.

(2) And (3) carrying out cell perfusion on extracellular fluid containing HCQ with different concentrations, and continuously recording until the inhibition effect of the drug on Cav1.2 current reaches a stable state, wherein the peak value of the inward current is the current value after the drug is added, and the positive result is Verapami (Verapamil) with the concentration of 30 mu M.

The results show that HCQ at different concentrations had no significant inhibitory effect on cav1.2 channels (fig. 2D-E).

Detection of the Effect of HCQ on myocardial sodium ion channels

(1) Human Nav1.5 sodium ion channels were overexpressed in HEK293 cells, and round slides with HEK293 hNav1.5 cells attached were transferred to a cell bath on an inverted microscope platform in an electrophysiological experimental system.

(2) And (3) perfusing extracellular fluid containing HCQ with different concentrations, and continuously recording until the inhibition effect of the drug on Nav1.5 current reaches a stable state, wherein the peak value of the inward current is the current value after drug addition. The steady state criterion is determined by whether the nearest consecutive 3 current traces coincide. Amitriptyline (Amitriptyline) at 10. mu.M served as a positive control.

(3) In the experiment, the effect of HCQ on hNav1.5 sodium ion channel current at different concentrations is evaluated by measuring the maximum current values of the control group and the HCQ treatment group, and calculating the ratio (Mean +/-SE) of the maximum current value (absolute value) of the HCQ treatment group to the maximum current value (absolute value) of the control group.

The results show that although HCQ has a weak inhibitory effect on the nav1.5 ion channel (IC50 ═ 36.42 μ M) (fig. 2F-G), the effect on this ion channel was clinically insignificant when IC50 was greater than 20 μ M according to FDA guidelines.

Example 3: effect of Hydroxychloroquine sulfate and chloroquine phosphate on myocardial cell mitochondrial oxidative stress

1. Fluorescence staining detection of primary rat myocardial cells

(1) Inoculation: inoculating primary milk rat myocardial cells into a 24-well plate with a density of 6 × 10 per well⁴Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air and culturing at 37 ℃ for 12 h;

(2) cells were treated with 25. mu.M, 50. mu.M, 100. mu.M HCQ or 25. mu.M, 50. mu.M, 100. mu.M CQ, and stained with Rho123 and Hoechst 6 hours after the treatment, specifically Rhodamine 123 (Biyunyun day, C2007) and Hoechst 33342 (Biyunyun day, C1022) according to the instructions, and recorded by photography. Normally, the inner and outer membranes of mitochondria maintain a certain potential difference, and when mitochondria are damaged, the potential difference of the inner and outer membranes of mitochondria is destroyed, so that the membrane potential is reduced. When the membrane potential decreases, Rho123 enters mitochondria and fluoresces. Therefore, the fluorescence intensity of Rho123 can be used as an indirect indication of the degree of mitochondrial membrane potential impairment.

The experimental results show that as the concentration of HCQ and CQ increases, the mitochondrial membrane potential decreases more significantly (fig. 3A), suggesting mitochondrial oxidative stress injury.

2. Flow cytometry detection of primary milk rat myocardial cells Mito SOX and DHE

(1) Inoculation: inoculating primary milk rat myocardial cells into 12-well plates with a density of 4 × 10 per well⁵Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air and culturing at 37 ℃ for 12 h;

(2) treating cells with HCQ or CQ at 100 μ M concentration, and performing Mito SOX and DHE staining after 6h treatment;

(3) removing the cell culture medium, digesting the cells with pancreatin without EDTA, and centrifuging at 2000rpm for 5 min;

(4) each sample was resuspended in 500. mu.l PBS and centrifuged at 2000rpm for 5 min;

(5) resuspend the cells with 500. mu.l Mito SOX and DHE dye working solutions, incubate at 37 ℃ in the dark for 30min, and centrifuge at 2000rpm for 5 min;

(6) resuspend the cells with 500. mu.l PBS, centrifuge at 2000rpm for 5 min;

(7) the cells were resuspended in 500. mu.l PBS and filtered through a 200 mesh cell screen and detected immediately by flow cytometry injection. When the oxidative stress level in the cell is increased, the content of Reactive Oxygen Species (ROS) is increased, and the mitoSOX can emit fluorescence after being combined with ROS in mitochondria, so that the oxidative stress level of the mitochondria of the cell can be judged according to the relative value of the fluorescence intensity of the mitoSOX. DHE can be combined with ROS generated in cell nucleus and emit fluorescence, so that the relative value of MitoSOX fluorescence intensity can be used for judging the oxidation stress level of cell mitochondria; and judging the oxidation stress level of the mitochondria of the cell according to the relative value of the DHE fluorescence intensity.

The results showed that the fluorescence intensity of Mito SOX and DHE was significantly increased in HCQ or CQ-treated cardiomyocytes (FIGS. 3B-C), suggesting that both HCQ and CQ can cause significant increases in the level of oxidative stress in cardiomyocytes.

3. Primary rat myocardial cell JC-1 flow detection method

(1) Inoculation: inoculating primary milk rat myocardial cells into 12-well plates with a density of 4 × 10 per well⁵Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air and culturing at 37 ℃ for 12 h;

(2) cells were treated with HCQ or CQ at a concentration of 100. mu.M, administered for 6 hours, and then JC-1 staining was carried out according to the method of a mitochondrial membrane potential detection kit (Biyun, C2006), and the green fluorescence intensity (cells with low mitochondrial membrane potential, the same principle as Rho123) was detected by flow cytometry.

The results showed that the proportion of cardiomyocytes with low mitochondrial membrane potential was significantly increased compared to the control group (fig. 3D), confirming that HCQ and CQ can cause a decrease in the mitochondrial membrane potential of cardiomyocytes, suggesting mitochondrial damage.

4. ATP content detection of primary rat myocardial cells

(1) Inoculation: inoculating primary milk rat myocardial cells into a 60mm culture dish, wherein the density of each well is 3 multiplied by 10⁶Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air and culturing at 37 ℃ for 12 h;

(2) treating cells with HCQ or CQ with concentration of 100 μ M, administering for 6h, and detecting ATP content according to enhanced ATP detection kit (Biyunyan, S0027).

The experimental results show that both HCQ and CQ cause a decrease in ATP content in cardiomyocytes (fig. 3E), suggesting that both HCQ and CQ cause impairment of mitochondrial function in cardiomyocytes.

Example 4: effect of combined administration of different antioxidant stress drugs and hydroxychloroquine sulfate on rat myocardial cell metabolic rate

(1) Inoculation: inoculating primary milk rat myocardial cells into a 96-well plate, wherein the density of each well is 1 multiplied by 10⁴Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air and culturing at 37 ℃ for 12 h;

(2) after the primary milk rat cells to be extracted beat, 100 mu M HCQ + VitC (different concentrations), 100 mu M HCQ + VitE (different concentrations), 100 mu M HCQ + FA (different concentrations), 100 mu M HCQ + CoQ10 (different concentrations) and 100 mu M HCQ + IDE (different concentrations) are given, and each group of cells are continuously cultured for 6 hours after being added with medicine;

(3) after 6h of drug treatment, 10 uL CCK8 is added into each hole to detect the cell metabolic rate;

(4) the plates were incubated in an incubator for 4 hours and the absorbance at 450nm was measured using a microplate reader.

The results show that HCQ causes the metabolic rate of the myocardial cells to be reduced by 50 percent, and after 100 mu M HCQ and 12.5 mu M, 25 mu M and 50 mu M VitC are respectively applied to the myocardial cells for 6 hours, the cell metabolic rate is not obviously changed, and the cell metabolic rate of each group is close to that of HCQ single administration group cells (figure 4A); after 100 mu M HCQ and 6mM, 12mM and 24mM VitE are respectively carried out on the myocardial cells for 6h, the cell metabolic rate is not obviously changed, and the cell metabolic rate of each group is close to that of cells of a single HCQ administration group (figure 4B); after 100 mu M HCQ and 10 mu M, 20 mu M and 40 mu M FA are applied to the myocardial cells for 6 hours simultaneously, the cell metabolism rate is not obviously changed, and the cell metabolism rate of each group is close to that of HCQ single administration group cells (figure 4C); after 100 mu M HCQ and 20 mu M, 40 mu M and 80 mu M CoQ10 are respectively carried out on the myocardial cells for 6h, the cell metabolism rate is not obviously changed, and the cell metabolism rate of each group is close to that of HCQ single administration group cells (figure 4D); after 100 μ M HCQ was applied to cardiomyocytes for 6h simultaneously with 5 μ M, 10 μ M and 20 μ M IDE, respectively, the cell metabolic rate gradually increased and was dose-dependent with IDE, and 10 μ M and 20 μ M IDE significantly reversed the decrease in cardiomyocyte metabolic rate caused by HCQ (fig. 4E).

Example 5: effect of combination of different antioxidant stress drugs and hydroxychloroquine sulfate or chloroquine phosphate on myocardial cell mitochondrial oxidative stress

1. Mito SOX flow cytometry detection of primary breast rat myocardial cells

(1) Inoculation: inoculating primary milk rat myocardial cells into 12-well plates with a density of 4 × 10 per well⁵Culturing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air at 37 ℃ for 12 h;

(2) administration of 100 μ M HCQ to cardiomyocytes alone or in combination with different antioxidant stress drug treatments: 100 μ M HCQ, 100 μ M HCQ +25 μ M VitC, 100 μ M HCQ +24mM VitE100 μ M HCQ +20 μ M FA, 100 μ M HCQ +40 μ M CoQ10, 100 μ M HCQ +20 μ M IDE, and CQ single dose or CQ-combined IDE (100 μ M CQ +20 μ M IDE), each group was treated for 6h before Mito SOX staining;

(3) removing the cell culture medium, digesting the cells with pancreatin without EDTA, and centrifuging at 2000rpm for 5 min;

(4) each sample was resuspended in 500. mu.l PBS and centrifuged at 2000rpm for 5 min;

(5) resuspend the cells with 500. mu.l Mito SOX dye working solution, incubate in the dark at 37 ℃ for 30min, and centrifuge at 2000rpm for 5 min;

(6) resuspend the cells with 500. mu.l PBS, centrifuge at 2000rpm for 5 min;

(7) the cells were resuspended in 500. mu.l PBS and filtered through a 200 mesh cell screen and detected immediately by flow cytometry injection.

The experimental results show that HCQ and CQ both cause significant increase in mitochondrial oxidative stress level and decrease in mitochondrial membrane potential (FIGS. 5A and C), and that the myocardial cell mitochondrial oxidative stress level after treatment with 100. mu.M HCQ + 25. mu.M VitC, 100. mu.M HCQ +24mM VitE, 100. mu.M HCQ + 20. mu.M FA, and 100. mu.M HCQ + 40. mu.M CoQ10 was reduced compared with that of HCQ single-dose group, but the difference was not significant (FIG. 5A), but the oxidative stress in the myocardial cells of HCQ + group IDE was reduced to a level close to normal (FIG. 5A). Therefore, the effect of reducing the myocardial cell oxidative stress injury caused by HCQ by IDE is obviously superior to that of VitC, VitE, FA and CoQ 10. The level of oxidative stress in CQ + IDE cardiomyocytes was similar to that of the control group (fig. 5C), indicating that IDE was effective in reducing the oxidative stress damage of cardiomyocytes caused by CQ.

2. Detecting the primary rat myocardial cell Rho123 by flow cytometry: the cell treatment process was the same as in (1) to (7) of FIG. 1.

Experimental results of the fructification experiment show that HCQ and CQ both cause mitochondrial membrane potential damage (FIGS. 5B and D), and that the mitochondrial membrane potential damage of the cardiomyocytes after treatment with 100. mu.M HCQ + 25. mu.M VitC, 100. mu.M HCQ +24mM VitE, 100. mu.M HCQ + 20. mu.M FA and 100. mu.M HCQ + 40. mu.M CoQ10 is relieved compared with that of the HCQ single administration group, but the difference is not significant compared with that of the HCQ single administration group (FIG. 5B). The mitochondrial membrane potential of the cardiomyocytes in the HCQ + IDE group was close to normal (FIG. 5B). Therefore, the effect of IDE in reducing myocardial cell mitochondrial membrane potential damage caused by HCQ is obviously better than that of VitC, VitE, FA and CoQ 10. CQ + IDE myocardial cell mitochondrial membrane potential was also close to the control group (FIG. 5D), indicating that IDE was effective in reducing CQ-induced myocardial cell mitochondrial membrane potential damage.

Example 6: effect of combinations of idebenone and lethal amounts of hydroxychloroquine sulfate on mortality of mice

(1) The clinical oral daily dose of idebenone is 90mg, the mouse (C57BL/6) equivalent dose is 0.0234mg, calculated according to oral bioavailability 70%, and the mouse intravenous dose is 0.018 mg.

(2) According to the above dose, mice were given a lethal dose of HCQ (2 mg/mouse, equivalent to 686 mg/mouse dose of human body (60kg body weight)) in the tail vein, or HCQ (2 mg/mouse) + IDE (0.018 mg/mouse) in the tail vein at the same time, and the mortality rate was observed after administration for 10 mice per group.

The experimental results show that when HCQ is independently administrated, the mortality of mice is 90%, and the mortality of mice in HCQ + IDE group is 40% (figure 6), and is remarkably reduced compared with the mortality of HCQ group, which suggests that IDE can remarkably reduce the mortality of mice caused by HCQ when IDE and HCQ are jointly administrated.

In conclusion, idebenone can remarkably reduce cardiotoxicity caused by chloroquine phosphate or hydroxychloroquine sulfate, and has obvious advantages in action compared with other anti-oxidative stress drugs including vitamin C, vitamin E, folic acid and coenzyme Q10. Idebenone maintains mitochondrial membrane potential mainly by reducing the increase of myocardial cell oxidative stress level caused by chloroquine phosphate or hydroxychloroquine sulfate, thereby keeping myocardial cell metabolism and function at normal level.

While the foregoing is directed to embodiments of the present invention, it will be appreciated that the foregoing is illustrative and not limiting, and that numerous modifications may be made by those skilled in the art without departing from the principles of the invention, which are intended to be within the scope of the appended claims.