Preparation Of Aldehyde Dehydrogenase 2 Agonist Alda-1 Injection And Its Effect

BackgroundIschemia reperfusion injury(I/R)injury refers to the partial or complete acute obstruction of the coronary arteries,and when it regains recanalization within a certain period of time,although the ischemic myocardium can return to normal perfusion,its tissue damage appears instead Progressive pathological process.A series of damaging changes such as myocardial uLtrastructure,energy metabolism,cardiac function,and electrophysiology caused by the ischemic phase are more prominent after recanalization of the blood vessels,and even severe arrhythmias can cause sudden death.Studies have confirmed that oxidative stress is an important cause of myocardial isdiemia-reperfusion injury[2.26.27.55.61].Alda-1,which is N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide,was selected by high-throughput screening method for ALDH2 Small molecuLe activators can increase the activity of ALDH2 through allosteric activation,can increase the activity of wild-type ALDH2 by 2 times,and increase the activity of mutant ALDH2 by 11 times.Administration before myocardial ischemia in rats can reduce the infarct size of the heart by about 60%,improve ventricuLar function after acute myocardial infarction and reduce mitochondrial dysfunction[2.26.27.55.61].Previous studies have shown that Alda-1 plays an important role in acute myocardial infarction and myocardial cell protection in animal experiments,but there is no relevant research report on its clinical.Acetaldehyde dehydrogenase 2,ALDH2 is located in mitochondria and is mainly distributed in liver,heart,brain and other tissues[2.3.4.16.22].ALDH2 participates in the alcohol metabolism pathway,catalyzes the conversion of acetaldehyde to acetic acid,relieves the symptoms of alcoholism,and reduces the damage of ethanol to muLtiple organs.ALDH2 has dehydrogenase activity and can metabolize a variety of aldehydes.Previous studies have shown that ALDH2 activation can reduce heart damage and brain damage caused by myocardial ischemia[5.10.16.46].Acute myocardial infarction and cardiac arrest are two of the most common clinically critical cardiovascuLar diseases that cause severe myocardial injury,cardiac dysfunction,and even death.However,the current methods for protecting myocardium are extremely scarce.This project intends to clearly clarify the protective effect and internal mechanism of acetaldehyde dehydrogenase 2(ALDH2)agonist Alda-1 on myocardial injury in many years of research work,and continue to explore the medicinal properties of Alda-1.This experiment intends to prepare clinical application injections,investigate its pharmacological effects and pharmacokinetic characteristics,and systematically evaluate the drugability of Alda-1 to provide a new type of compound with potential for drug protection for myocardial protection and to continue in-depth preclinical research Or clinical drug trials lay a certain foundation for research and development.Objectives1.Exploring the clinical application of Alda-1,a possible way for clinical translational research of Alda-1.2.To explore the effect of Alda-1 injection on myocardial ischemia-reperfusion injury.MethodsIn vivo study1.Establish a mouse ischemia-reperfusion modelSixty male C57BL/6 mice were selected and divided into six groups:sham operation group(Sham group),ischemia reperfusion injury(Ischemia reperfusion injury(I/R)),I/R+DMSO group,I/R+DMSO+Alda-1 group,I/R+ALC+PEG+NS group,I/R+ALC+PEG+NS+Alda-1 group.In the I/R group,the anesthesia was performed,the trachea was cut,and the ventilator was used to support breathing.At the same time,the chest was opened.The left anterior descending coronary artery was ligated for 45 minutes,and then the blood vessel was released for 4 hours.The mice in the Sham group were anesthetized and only threaded after thoracotomy,and no blood vessels were ligated.I/R+DMSO+Alda-1 mice:DMSO-dissolved Alda-1 was injected intraperitoneally at 20 mg/kg.Mice in I/R+DMSO group:the same dose of DMSO was injected intraperitoneally.I/R+ALC+PEG+NS+Alda-1 mice:ALC+PEG+NS dissolved Alda-1 was injected into the tail vein at 20 mg/kg.Mice in I/R+ALC+PEG+NS group:the same dose of ALC+PEG+NS was injected intraperitoneally.2.TTC staining was used to determine the area of myocardial infarction in each group of mice.3.Western blot(WB):The mice in each group were sacrificed to collect cardiomyocytes,extract cardiomyocyte total proteins,and detect the expression levels of BAX,Bcl-2,β-actin,and 4-HNE proteins.4.Small animal cardiac uLtrasound evaluation of cardiac function:measured under M uLtrasound,and calcuLated Left VentricuLar Ejection Fractions(LVEF)and Fractional shortening(FS).5.Rabbit vascuLar irritation testThree healthy rabbits were taken,and the left ear margin vein was injected with Alda-1 injection stock solution or diluent in an amount of 1 mL/kg.Using its own control,the right ear margin vein was injected with an equal volume of 0.9%sodium chloride injection.1 time/day for 3 consecutive days.H&E staining was then used to detect the pathological status of rabbit ear marginal veins.In vitro study1.Establishment of UV-Vis analysis method for Alda-1 injection1.1 Determination of detection wavelength10.0mg of Alda-1 was precisely weighed,and then added to a 10mL volumetric flask to make up to volume,to obtain a stock solution of 1mg/mL.Another 40uL of Alda-1 stock solution was taken and diluted with ethanol to 10mL to obtain a 4ug/mL Alda-1 detection solution.The ethanol was used as a blank control solution,and the Alda-1 detection solution was subjected to uLtraviolet scanning in the range of 200-700 nm.1.2 Establishment of standard curvePrecisely measure 10,20,30,40,50,60,70uL of Alda-1 stock solution under "1.1"into a 10mL volumetric flask,and make up to volume with ethanol to obtain a concentration of 1,2,Alda-1 standard solutions at 3,4,5,6,and 7 ug/mL.Using ethanol as a blank control solution,the drug absorption peak of each Alda-1 standard solution was detected at a detection wavelength of 285 nm.2.Alda-1 injection Alda-1 solubility determination and program selection2.1 According to the 2015 version of "Chinese Pharmacopoeia",2 Alda-1 injection solutions were formulated(1)Solvent:soybean oil for injection,glycerin for injection,saline Emulsifier:soybean phospholipid(2)Solvent:ethanol,polyethylene glycol 400,physiological saline Solubilization method:ultrasonic vibration2.2 Using HPLC to detect the solubility of Alda-1 in Alda-1 injection3.Hemolysis test of injection3.1 Preparation of red blood cell suspensionApproximately 20 mL of blood was collected from the heart of the rabbit.After treatment,the resuLting red blood cell volume was diluted with physiological saline into a 2%suspension for later use.3.2 Hemolytic test of Alda-1 injectionTake a clean centrifuge tube,add different volumes of Alda-1 injection stock solution in sequence,each concentration is in 2 tubes in parallel,and set a negative control(physiological saline)and a positive control(purified water).After all is added,mix well and immediately incubate in a 37℃ incubator.Results1.Preparation of Alda-1 injection1.1 Alda-1 UV absorption peak at 285nm1.2 Alda-1 injection solution selectionA preparation scheme using ethanol as the main solvent,polyethylene glycol 400 and physiological saline as the auxiliary solvent.1.3 Alda-1 drug concentration of Alda-1 injection is 4.98g/mL20 mg of Alda-1 was accurately weighed,added to a clean 5 mL EP tube,1 mL of ethanol was added to the EP tube,and sonicated for 3 minutes.Add 600uL of PEG400 and 400uL of saline to mix.2.Alda-1 injection safety experiment2.1 Alda-1 injection has no obvious vascuLar irritationSlight dilation of the blood vessels occasionally occurred during intravenous injection after dilution,and no obvious congestive edema or thrombosis was observed in the proximal cardiac vein at the acupuncture site.Histopathological examination under light microscopy showed that the vascuLar structure at the injection site was complete and vascuLar endothelial cells Obviously degeneration and necrosis,no thrombosis in the lumen.2.2 Alda-1 injection has no obvious hemolytic irritationIn the experiment,2%of the erythrocyte suspension showed no obvious hemolysis,no obvious agglutination reaction,and the upper liquid was clear.3.Efficacy experiment of Alda-1 injection3.1 Alda-1 injection can improve cardiac function in mice after I/R3.2 Alda-1 injection can reduce myocardial infarction caused by ischemia-reperfusion injuryTTC staining resuLts showed that Alda-1 injection can reduce myocardial infarction by about 15%.3.3 Alda-1 injection can induce cardiomyocyte apoptosisWestern Blot detection showed that tail vein injection of Alda-1 injection increased the expression of apoptotic pathway protective protein Bcl-2 and decreased the expression of apoptosis marker BAX.3.4 Alda-1 injection can reduce the accumuLation of cardiotoxic aldehydesWestern Blot tests showed that tail vein injection of Alda-1 injection reduced the accumuLation of toxic aldehydes 4-HNE.Conclusions1.Alda-1 injection with ethanol as the main solvent and polyethylene glycol 400 and saline as the auxiliary solvent has better Alda-1 solubility.2.Alda-1 injection has good safety.Rabbits are less irritating when injected intravenously and will not cause hemolysis of red blood cells.3.Alda-1 injection can improve the cardiac function of mice after ischemia-reperfusion and reduce ischemia-reperfusion injury.