CN113633635B - Pharmaceutical composition for treating novel coronavirus infection pneumonia - Google Patents

Abstract

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.

Description

Pharmaceutical composition for treating novel coronavirus infection pneumonia

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 (H146 del and Y145 del) 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 likelihood of becoming severe and a relatively shorter course of disease, the emergence and prevalence of the delta variant strains are undeniably responsible for breaking the immune barrier established by the new corona vaccine and for providing more serious challenges in the prevention and treatment of new corona 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 preliminary clinical efficacy in small sample clinical trials for treatment of novel coronavirus pneumonia (COVID-19) superior to lopinavir/ritonavir (kresoxim) (Gautret P et al int J antisicrob Agents,2020,20, 105949 horby P, et al, lancet,2020,396 1345-1352), and has been incorporated into the national health care committee "new coronavirus pneumonia diagnosis and treatment protocol (trial sixth edition). The multi-center cooperation group for treating the novel coronavirus pneumonia by using chloroquine phosphate in science and technology halls of Guangdong province and the health committee of Guangdong province has also provided expert consensus on treating the novel coronavirus pneumonia by using chloroquine phosphate. 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, 23-69. Various in vitro studies show that hydroxychloroquine sulfate (HCQ) or Chloroquine (CQ) has the effect of obviously inhibiting the replication of new coronavirus, and the immunosuppressive effect of the hydroxychloroquine sulfate can also reduce the generation 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. 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 the serum drug concentration of hydroxychloroquine sulfate, the better the clearance effect on the new corona virus (Watson, j.a., et al, elife,2020, 9. HCQ is reported to be clinically effective in treating neocoronary pneumonia in the 800-1200mg/d range (Oscanoa, t.j., et al, int J Antimicrob Agents,2020, 56. 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, broadened QRS wave, 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. Also, clinical data show that more than 1/3 of patients with new coronary pneumonia have varying degrees of myocardial damage, which 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.

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 approach 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 to 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 day (2-4 times of safe dosage of clinically and independently used hydroxychloroquine sulfate) for each human.

In the technical scheme of the invention, the medicine composition provides a basis for clinically applying to the 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 CoQ10.

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 the 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 myocardial cells were treated with HCQ or CQ at different concentrations for 6h; (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. After NRCMs are given 100 mu M HCQ and are respectively added with different concentrations of antioxidant stress drugs of VitC (A), vitE (B), FA (C), coQ10 (D) and IDE (E) for treatment for 6h, CCK8 detects the 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 the 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 the 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 the combination 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 caused by the difference of bioavailability, 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 the data from each group revealed that the Q-T interval was not statistically different before and after dosing in the 12.5mg/kg group of rats (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-Ap 0.001) (FIG. 1D). These results indicate that administration of HCQ results in an extended Q-T interval in rats, consistent with clinical dosing in 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 a PATCHMASTER acquisition system (HEKA Instruments Inc., D-67466 Lambrright, 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) do not have significant inhibitory effect on the hERG potassium channel (FIGS. 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 (IC 50=36.42 μ M) on the nav1.5 ion channel (fig. 2F-G), the effect on this ion channel is clinically insignificant when the IC50 is 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 the density of 6 × 10 per well ⁴ Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air and culturing at 37 ℃ for 12h;

(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 concentrations of HCQ and CQ increase, the mitochondrial membrane potential decreases more significantly (fig. 3A), suggesting mitochondrial oxidative stress damage.

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 the density of 4 × 10 per well ⁵ Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air and culturing at 37 ℃ for 12h;

(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 5min;

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

(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 5min;

(6) Resuspend cells with 500. Mu.l PBS, centrifuge at 2000rpm for 5min;

(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 cardiac muscle 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 well ⁵ Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air at 37 ℃ for culturing for 12 hours;

(2) Cells are treated by HCQ or CQ with the concentration of 100 mu M, JC-1 staining is carried out after administration treatment for 6h according to the operation method of a mitochondrial membrane potential detection kit (Biyun day, C2006), and the green fluorescence intensity (cells with low mitochondrial membrane potential, the principle is the same as Rho 123) is detected by a flow cytometer.

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 10 ⁶ Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air at 37 ℃ for culturing for 12 hours;

(2) Cells are treated by HCQ or CQ with the concentration of 100 mu M, and ATP content detection is carried out according to an operation method of an enhanced ATP detection kit (Biyun day, S0027) after administration treatment for 6 hours.

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 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 10 ⁴ Placing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air and culturing at 37 ℃ for 12h;

(2) After the primary dairy 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 all groups of cells are continuously cultured for 6 hours after the drugs are added;

(3) After the drug treatment for 6h, adding 10 microliter CCK8 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 applied to 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 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 well ⁵ Culturing the cells in a cell culture box containing 5% of carbon dioxide and 95% of air at 37 ℃ for 12h;

(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), mito SOX staining was performed separately 6h after each group was treated;

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

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

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

(6) Resuspend cells with 500. Mu.l PBS, centrifuge at 2000rpm for 5min;

(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 obvious increase of mitochondrial oxidative stress level and reduction of mitochondrial membrane potential (figure 5A, C), and the mitochondrial oxidative stress level of the myocardial cells treated by 100 mu M HCQ +25 mu M VitC, 100 mu M HCQ +24mM VitE100 mu M HCQ +20 mu M FA and 100 mu M HCQ +40 mu M CoQ10 is reduced compared with that of the HCQ single administration group, but the difference is not significant (figure 5A), but the oxidative stress in the myocardial cells of the HCQ + IDE group is reduced to be close to the normal level (figure 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 CoQ10. 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 fructification results show that HCQ and CQ both cause mitochondrial membrane potential damage (FIGS. 5B, D), and that myocardial cell mitochondrial membrane potential damage 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, 100. Mu.M HCQ + 40. Mu.M CoQ10 is alleviated compared with HCQ single-dose group, but the difference is not significant compared with HCQ single-dose 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 on reducing myocardial cell mitochondrial membrane potential injury caused by HCQ is obviously better than that of VitC, vitE, FA and CoQ10.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 (C57 BL/6) equivalent dose is 0.0234mg, calculated according to oral bioavailability 70%, and the mouse intravenous dose is 0.018mg.

(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 (60 kg 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 superiority in action compared with other antioxidant stress medicaments 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.

Claims (9)

1. A pharmaceutical composition is characterized by comprising pharmaceutically acceptable salts of idebenone and chloroquine or chloroquine, or comprising pharmaceutically acceptable salts of idebenone and hydroxychloroquine or hydroxychloroquine.

2. The pharmaceutical composition according to claim 1, wherein the molar ratio of idebenone to chloroquine or a pharmaceutically acceptable salt of chloroquine is 0.05 to 0.2, or the molar ratio of idebenone to hydroxychloroquine or a pharmaceutically acceptable salt of hydroxychloroquine is 0.05 to 0.2.

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 or hydroxychloroquine is an acid salt.

5. The pharmaceutical composition according to claim 4, characterized in that said pharmaceutically acceptable salt of chloroquine or hydroxychloroquine is a sulfate, a hydrochloride, a phosphate, a nitrate, an oxalate, a formate, a benzoate, an acetate, a trifluoroacetate, a succinate, a tartrate, a malate, a citrate, a sulfonate.

6. The pharmaceutical composition according to claim 5, characterized in that the sulfonate is a benzenesulfonate or a benzylsulfonate.

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

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

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

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