CN117100696A - Pharmaceutical preparation for treating ischemic myocardial reperfusion injury and application thereof - Google Patents

Abstract

The invention provides a pharmaceutical preparation for treating ischemic myocardial reperfusion injury and application thereof, wherein the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 60 to 150 parts of oil phase, 8 to 20 parts of emulsifying agent, 10 to 30 parts of osmotic pressure regulator, 0 to 10 parts of stabilizing agent and 600 to 800 parts of water for injection. The emulsion for intravenous injection has the advantages that due to the wrapping of emulsion particles, the in vivo elimination time of vorinostat is prolonged, the half-life period of the medicine is prolonged, and the bioavailability is improved, and the emulsion particles have the targeting characteristic of being easily distributed to heart damaged parts, so that the effect of the vorinostat on inhibiting histone deacetylase of cardiac muscle is exerted, the mitochondrial steady state of cardiac muscle cells is maintained, the apoptosis of the cardiac muscle cells is reduced, and the myocardial infarction area is reduced.

Description

Pharmaceutical preparation for treating ischemic myocardial reperfusion injury and application thereof

Technical Field

The invention relates to a pharmaceutical preparation for treating ischemic myocardial reperfusion injury and application thereof, belonging to the field of new pharmaceutical dosage forms.

Background

It is well known that Acute Myocardial Infarction (AMI) has a high incidence and is fatal, and is one of the main causes of death in the population worldwide. For acute myocardial infarction treatment includes: intravenous drug thrombolysis therapy, direct percutaneous coronary intervention (PPCI), emergency coronary bypass (CABG), etc., which enables ischemic myocardium to rapidly resume blood perfusion and oxygen supply. Among them, PPCI is the first therapeutic strategy for ST elevation myocardial infarction (STEMI) reperfusion therapy. Although revascularization (PPCI, CABG, and drug thrombolysis, etc.), timely recovery of myocardial blood perfusion after myocardial ischemia is the most effective treatment strategy for alleviating myocardial ischemia injury, significantly reducing mortality in the acute phase of myocardial infarction patients, a significant portion of patients eventually move toward ventricular negative remodeling and heart failure due to myocardial ischemia reperfusion injury.

With the continuous improvement of clinical efficacy of PPCI for STEMI patients, although the mortality rate of myocardial infarction patients is obviously reduced compared with the prior art, the incidence rate of heart failure of myocardial infarction patients is higher and higher. Thus, there remains a need to explore new therapeutic approaches that can prevent myocardial reperfusion injury, and reduce myocardial infarction area, and can maintain left ventricular contractile function and prevent the occurrence of heart failure. (Luo Feng, su Jiang. Research status and hope for prevention and treatment of myocardial ischemia reperfusion injury [ J ]. Guangdong medical science, 2019,40 (2): 3)

Myocardial Ischemia Reperfusion Injury (MIRI) is an important issue in clinical cardiology, and ischemic myocardium causes more severe acute injury than when vascular is occluded after vascular patency, clinically leading to severe arrhythmia, enlarged necrotic area of myocardium, cardiac rupture and even death. Therefore, the mechanism of causing reperfusion injury is discussed, and an effective drug is searched for alleviating the injury process, so that the method has very important significance for the treatment of clinical acute myocardial infarction.

Currently, there are two main approaches to controlling the occurrence of myocardial ischemia/reperfusion injury (MIRI) during therapy for acute myocardial infarction patients (AMI), namely, surgical procedure blood flow control and drug therapy.

Blood flow control during operation adopts mechanical intervention means (including post-ischemia treatment and remote ischemia treatment), and measures such as a remote protection device, a thrombus aspiration catheter and the like are selectively applied when PPCI is carried out. Myocardial ischemia drug therapy goes through the development process of myocardial ischemia pre-adaptation-myocardial ischemia post-adaptation. The treatment time is divided into myocardial ischemia pre-adaptation drugs and post-adaptation drugs. The pre-adaptation medicine is to apply various medicines (such as adenosine, endogenous opioid peptide, etc.) to simulate the mechanism of body ischemia pre-adaptation to exert myocardial protection. The main mechanisms of pre-adaptation to protect the heart are potassium channel inhibition, activation of endogenous substances (e.g. adenosine, opioid peptides, bradykinin, etc.), enzyme signaling pathways, mitochondrial regulation, etc.

However, the time of acute myocardial infarction in clinic is not generally predicted, and the time of patient arrival at the medical facility is after ischemia has occurred, so the timing of the treatment of myocardial ischemia reperfusion injury with a medicament must be used at least when vascular opening reperfusion is performed during interventional procedures, and not possibly prior to a myocardial infarction event. Therefore, the clinical application value of the pre-adaptive medicine has great limitation from the aspect of clinical treatment.

Post-adaptation (Myocardial ischemic postconditioning, MIP) drugs can be used during and after ischemia reperfusion, and are more practical and potentially of clinical application than pre-adaptation. The ideal post-drug adaptation treatment can relieve myocardial ischemia reperfusion injury while recovering coronary blood flow, further improve the treatment effect of myocardial ischemia, obviously improve the life quality of patients, and has become one of the hot spots of current research.

Although post-study drug adaptation may have some effect on antagonism/antagonism of drug reperfusion injury, none of the drugs currently being administered into clinical studies have reached the end of clinical trial. For example, beta receptor blockers nebivolol, sodium-calcium exchange inhibitor amiloride, oxygen radical scavenger edaravone (ClinicalTrials. Gov ID: NCT 00265239), mitochondrial core pore MPTP modulator formulation TRO40303 (ClinicalTrials. Gov ID: NCT01379261, NCT 01374321), and the mitochondrial targeting small molecule peptide MTP-131 (ClinicalTrials. Gov ID: NCT 01572909) from Stealth Biotherapeutics have not reached clinical end points after the end of clinical phase 2-3, and have been examined.

Other mechanisms of drug delivery, such as HMG-CoA reductase selective inhibitors atorvastatin (ClinicalTrials. Gov ID: NCT 01780740), immunosuppressant cyclosporin A (ClinicalTrials. Gov ID: NCT 01650662), GLP-1 analog liraglutide (ClinicalTrials. Gov ID: NCT 02001363), etc., have been given promise in early stages, and their drug efficacy appears to be primarily effective only in small sample clinical trials or animal trials, and reliable clinical results have not been achieved in the population study of later clinical large sample queues. Therefore, the development of safer and more effective anti-myocardial ischemia/reperfusion injury drugs remains a common research hotspot for cardiovascular doctors worldwide.

With intensive studies on the mechanism of myocardial ischemia reperfusion injury, among many of the mechanisms thereof, autophagy has attracted attention from researchers in recent years.

Autophagy is a metabolic pathway in cells that degrades long-life proteins and damaged organelles by lysosomes, playing an important role in the process of cellular stress. The beneficial effects of autophagy in myocardial ischemia reperfusion injury include 1) autophagy relieves the energy crisis caused by ischemia by producing ATP; 2) Autophagy is activated to clear dysfunctional mitochondria, promote mitochondrial turnover, and maintain cardiomyocyte function; 3) Following ischemia reperfusion, autophagy is activated to clear ubiquitin, maintaining intracellular protein homeostasis.

Although the mechanism of autophagy during ischemia reperfusion has been greatly advanced, the regulation of autophagy by mTOR, atg, LC3 and AMPK has been studied clearly. However, the search for suitable autophagy-modulating drugs from a large number of autophagy-modulating targets is a challenging task.

It is appreciated that the antitumor drug HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) was found to prevent I/R-induced mitochondrial dysfunction and loss of mitochondrial membrane potential and reduce ROS production both pre-and post-myocardial ischemia. Moreover, these protective effects were counteracted in knocking out autophagy function-related genes Atg7 and Atg5, confirming that SAHA did reduce I/R myocardial damage by modulating autophagy flux; in addition, SAHA was found to be independent of the autophagy pathway, and can modulate mitochondrial function mediated by PGC-1 alpha to rescue I/R heart damage, exerting a protective effect, and also demonstrated that HDAC inhibitors have important clinical significance as autophagy modulators in ischemic heart disease. ( Min Xie, YIda Tang, joseph A.Hill, HDAC inhibition as a therapeutic strategy in myocardial ischemia/reperfusion injury, J Mol Cell cardiol.2019april;129:188-192 )

More surprisingly, both pre-operative administration and reperfusion administration of SAHA in model animals can reduce myocardial reperfusion injury in model animals, reduce myocardial infarction area, and partially save contractile function before reperfusion, with no significant difference in drug effect. Further studies have found that administration with SAHA during reperfusion is more beneficial in maintaining beneficial autophagy flow in the infarct border zone, protecting cardiomyocytes.

(Markus Wallner et al.HDAC inhibition improves cardiopulmonary function in a feline model of diastolic dysfunction.Science Translational Medicine,2020，January 08；12(525))

(Xie M,Hill JA.HDAC-dependent ventricular remodeling.Trends，Cardiovasc Med.2013；23:229–235)

(Targeting Autophagy for the Therapeutic Application of Histone Deacetylase Inhibitors in Ischemia/Reperfusion Heart Injury.Circulation March 11,2014Mar 11；129(10):1088-91)

Similar experimental results, also reflected in the patent description of the prior application (accession number 201810076352.3, application date: 2018.01.26), are presented with SAHA formulated as an emulsion at a concentration of 30mg/ml, and the pre-adapted group was pre-adapted to the administration of 50mg/kg at one time hour prior to surgery, and the post-reperfusion adapted group was administered with 25mg/kg of drug at 1 hour prior to surgery and during reperfusion procedures, respectively, both of which were effective in significantly reducing myocardial infarction dead space and heart function decline.

The research results lay a foundation for SAHA to be an adaptive drug for treating myocardial ischemia reperfusion injury. The ideal state of the post-adaptation medicine is to develop into injection, so that the injection can be used for subcutaneous injection, intravenous injection or direct PPCI operation for intracoronary administration of patients, is beneficial to patients (possibly in a coma state) in the perioperative period of cardiac intervention operation, and has clinical significance. Whereas SAHA is practically insoluble in water, readily soluble in organic solvents, its solubility in dimethyl sulfoxide (DMSO) is >15mg/ml.

Although SAHA was developed as an antitumor drug in the early days, it was designed as an intravenous solution (concentration 5mg/ml, pH 11.0-11.2). (W.K. Kelly et, phase I Clinical Trial of Histone Deacetylase Inhibitor: suberoylanilide Hydroxamic Acid Administered Intravenously Clinical Cancer Research, vol.9,3578-3588,September 1,2003) the SAHA intravenous injection has a pH of 11.2, is highly irritating to blood vessels, is prone to phlebitis, is not suitable for intravenous injection of patients with cardiovascular diseases, is not suitable for intracoronary injection in perioperative periods, and directly produces strong stimulation to the heart. Thus, prior applications (application number 201810076352.3, application day 2018.01.26) describe the preparation of SAHA as an emulsion.

As we have conducted intensive studies on the emulsion described in the above patent, it has unexpectedly been found that the drug metabolism of the emulsion is greatly changed relative to that of a conventional water-soluble injection using DMSO as a vehicle. The main variations are: 1) Compared with DMSO solvent injection and common water-soluble injection, the half-life of the emulsion is obviously prolonged; 2) The tissue distribution rule of the medicine is closely related to the formulation of the emulsion and the particle size of the emulsion.

In view of the use as a medicament for treating myocardial ischemia reperfusion injury, convenience of clinical use in myocardial infarction emergency surgery and safety should be considered. Therefore, the characteristic of prolonged half-life of the emulsion medicine is beneficial to reducing the dosage of the medicine, reducing the concentration of the medicine, changing small-capacity injection into large-capacity infusion, facilitating the establishment of a venous channel in the operation process, reducing the operation of medical staff, and facilitating clinical medication and timely control of the dosage of the medicine. This can further improve the safety of the medication, with self-evident benefits. In addition, the internal tissue distribution rules of the emulsions with different particle sizes and formulations are greatly different, and the optimized screening of the emulsions with better heart tissue distribution can further effectively reduce the dosage of the medicament, ensure the medicament effect and improve the safety of the medicament.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a pharmaceutical preparation for treating ischemic myocardial reperfusion injury and application thereof.

The pharmaceutical preparation of the invention has the advantages that: the pharmaceutical preparation prolongs the in vivo half-life of SAHA, prolongs the acting time of the medicine, improves the bioavailability, and improves the targeting property of tissue distribution of damaged heart parts, thereby reducing the actual clinical dosage and improving the safety of the medicine. In addition, the preparation is an infusion emulsion, has low medicine concentration, good tissue compatibility and small vascular irritation, is convenient for clinical use, reduces the operation and the mastering dosage of medical staff, and improves the convenience of clinical use. The preparation is more beneficial to the administration in the perioperative period of the heart operation (namely the period surrounding the operation, including preoperation, intraoperative and postoperative), can also be directly used for the intracoronary injection, and is particularly suitable for the clinical application of the medicine for treating ischemia reperfusion injury in the perioperative period of the myocardial infarction interventional therapy.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

the pharmaceutical preparation for treating ischemic myocardial reperfusion injury comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 30 parts of HDAC inhibitor medicine, 60 to 250 parts of oil phase, 8 to 30 parts of emulsifier, 10 to 30 parts of osmotic pressure regulator, 0 to 12 parts of stabilizer and 500 to 900 parts of water for injection.

As preferred pharmaceutical formulations of the invention: 0.5 to 15 parts of vorinostat, 80 to 150 parts of oil phase, 10 to 18 parts of emulsifying agent, 15 to 25 parts of osmotic pressure regulator, 0 to 10 parts of stabilizing agent and 600 to 800 parts of water for injection;

the oil phase auxiliary materials are as follows: one or more of soybean oil (containing injection grade), castor oil, tea oil, cottonseed oil, sesame oil, rapeseed oil, safflower oil, canola oil, olive oil, coconut oil, palm oil, cocoa butter, etc.; medium long chain fatty acid glycerides with chain lengths between C6 and C12, such as one or more of MCT medium chain triglycerides, arlacel 80, arlacel 86, capmul MCM, captex 200, captex 355, miglyol812, myvacet, myverol-92, glyceryl oleate, glyceryl linoleate, polyethylene glycol glyceryl laurate, ethyl oleate, ethyl linoleate, caprylic/capric triglyceride, and the like; one or more of the above mixtures of long chain fatty acid esters and medium chain fatty acid esters may also be used;

The auxiliary materials of the emulsifier are as follows: soy lecithin and its modifications (natural or synthetic), egg yolk lecithin and its modifications (natural or synthetic), PEG phospholipids, hydrogenated soy phospholipids HSPC, cultivated phospholipids DSPE-MPEG2000, ophase31, poloxamers, polyoxyethylene hydrogenated castor oil, water soluble VE (TPGS), solutol HS-15, PEG300, PEG400, PEG1750 and its monostearate, tween 80, span 20, phosphatidylcholine, distearoyl phosphatidylcholine, myristoyl lysolecithin, dimyristoyl lecithin, dilauroyl lecithin, dicapryl phosphatidylcholine, polyoxypropylene polyoxyethylene block copolymers;

the osmotic pressure regulator auxiliary materials are as follows: one or more of sodium chloride, glucose, sorbitol, xylitol, mannitol, and glycerol;

the stabilizer auxiliary materials are as follows: oleic acid, sodium oleate, sodium caprylate, cholesterol, cholic acid, deoxycholic acid and sodium salt thereof, vitamin A, vitamin C and vitamin E.

The invention is characterized in that the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of soybean oil, 10 to 15 parts of egg yolk lecithin (E80), 17 to 25 parts of glycerol, 0 to 10 parts of cholesterol, 0 to 10 parts of sodium oleate solution and 600 to 800 parts of water for injection.

As the preference of the pharmaceutical preparation, the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of soybean oil, 40 to 60 parts of MCT medium chain triglyceride, 10 to 15 parts of phosphatidylcholine, 17 to 25 parts of glycerol, 1 to 10 parts of sodium oleate solution and 600 to 800 parts of water for injection.

As the preference of the pharmaceutical preparation, the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of canola oil, 5 to 9 parts of hydrogenated soybean lecithin HSPC, 20001 to 3 parts of cultivated phospholipid DSPE-MPEG, 17 to 25 parts of mannitol, 1 to 10 parts of cholesterol and 600 to 800 parts of water for injection.

As the preference of the pharmaceutical preparation, the pharmaceutical preparation comprises the following components in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of soybean oil, 10 to 15 parts of PEG300 monostearate, 17 to 25 parts of glycerol, 1 to 10 parts of oleic acid and 600 to 800 parts of water for injection.

As the preference of the pharmaceutical preparation, the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of castor oil, 10 to 15 parts of poloxamer, 17 to 25 parts of glucose, 1 to 10 parts of deoxycholic acid and 600 to 800 parts of water for injection.

As a further preferred embodiment of the pharmaceutical preparation of the present invention, the pharmaceutical preparation comprises a pharmaceutically active ingredient and an auxiliary material in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of tea oil, 10 to 15 parts of soybean lecithin, 17 to 25 parts of sodium chloride, 1 to 10 parts of cholic acid and 600 to 800 parts of water for injection.

As a further preferred mode of the pharmaceutical preparation, the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of olive oil, 10 to 15 parts of egg yolk lecithin, 17 to 25 parts of sorbitol, 1 to 10 parts of sodium octoate and 600 to 800 parts of water for injection.

As a further preferred mode of the pharmaceutical preparation, the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 3 parts of vorinostat, 100 parts of soybean oil, 80 parts of egg yolk lecithin E, 22.5 parts of glycerol, 4 parts of cholesterol and 750 parts of water for injection.

As a further preferred mode of the pharmaceutical preparation, the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 3 parts of vorinostat, 50 parts of soybean oil, 50 parts of MCT medium chain triglyceride lecithin, 12 parts of phosphatidylcholine PL-100M, 22.5 parts of glycerin, 10 parts of 0.3% sodium oleate solution and 750 parts of water for injection.

As a further preferred mode of the pharmaceutical preparation, the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 3 parts of vorinostat, 100 parts of canola oil, 6 parts of HSPC (hydrogenated soybean phosphatidylcholine), 2000 parts of DSPE-mPEG, 25 parts of mannitol, 4 parts of cholesterol and 750 parts of water for injection.

As a further preferred mode of the pharmaceutical preparation, the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 3 parts of vorinostat, 100 parts of soybean oil, 12 parts of PEG300 monostearate, 25 parts of glycerin, 1 part of oleic acid and 750 parts of water for injection.

As a preferable mode of the pharmaceutical preparation, the pharmaceutical preparation is an intravenous emulsion preparation and an intracoronary injection.

The preparation formulation of the pharmaceutical preparation is injection, and can also be intravenous injection emulsion and intracoronary injection. Preferably an intravenous emulsion, the pH of which is in the range of 6.3 to 7.9.

The drug selected by the pharmaceutical preparation of the invention can be an HDAC inhibitor drug, and the specific drug is vorinostat, and the chemical structure is as follows:

SAHA (vorinostat)

Oil phase auxiliary materials are selected:

the oil phase is 0-50% by mass (W/W) in the emulsion according to the present invention, and it is required in the present invention that the prescribed amount of the drug can be dissolved in a smaller amount of the oil phase, and that the drug does not precipitate even under low-temperature storage conditions, and that stable emulsion can be formed with the water phase under the action of the emulsifier. The oil phase used in the invention is natural vegetable oil containing long-chain fatty acid ester or vegetable oil or fatty acid ester after structural modification and hydrolysis, such as one or more of soybean oil (containing injection grade), castor oil, tea oil, cotton seed oil, sesame oil, rapeseed oil, safflower oil, canola oil, olive oil, coconut oil, palm oil, cocoa butter and the like; or medium long chain fatty acid glycerides with chain lengths between C6 and C12, such as one or more of MCT medium chain triglycerides, arlacel 80, arlacel 86, capmul MCM, captex 200, captex 355, miglyol 812, myvacet, myverol-92, glyceryl oleate, glyceryl linoleate, polyethylene glycol glyceryl laurate, ethyl oleate, ethyl linoleate, caprylic/capric triglyceride, etc.; mixtures of the above long chain fatty acid esters and medium chain fatty acid esters are also possible.

Preferred oil phases are soybean oil, canola oil, MCT medium chain triglycerides, soybean oil: mixtures of MCT medium chain triglycerides (1:1).

Selection of an emulsifier:

the emulsifier used in the invention is one or more than one mixture of nonionic surfactant and ionic surfactant, and can use phospholipid with phosphatidylcholine PC content more than 70%, including soybean lecithin and its modifier (natural or synthetic), egg yolk lecithin and its modifier (natural or synthetic), PEG phospholipid, cultivated phospholipid DSPE-MPEG2000, ophase31, poloxamer 108, poloxamer 188, poloxamer 407, polyoxyethylene hydrogenated castor oil, water-soluble VE (TPGS), solvent HS-15, PEG300, PEG400, PEG1750 and its monostearate, tween 80, span 20, or a mixture of two or more. In the preparation of injection, a refined emulsifier with small hemolysis is selected.

Among the specific types of preferred emulsifiers of the present invention are: soybean lecithin PC80, hydrogenated soybean lecithin HSPC, egg yolk lecithin E80, cultivated lecithin DSPE-MPEG2000, phosphatidylcholine PL-100M, PC T, distearoyl phosphatidylcholine DSPC, myristoyl lysolecithin M-lysoPC, dimyristoyl lecithin DMPC, dilauryl lecithin DLPC, sinigyl phosphatidylcholine DEPC, polyoxypropylene polyoxyethylene block copolymer Poloxamer, most preferably egg yolk lecithin E80, cultivated lecithin DSPE-MPEG2000, phosphatidylcholine PL-100M.

Selection of osmotic pressure regulator:

preferably, the osmotic pressure regulator comprises one of sodium chloride, glucose, sorbitol, xylitol, mannitol and glycerin or a mixture of two or more of the substances, and the most preferred osmotic pressure regulator is glycerin and mannitol.

Selection of a stabilizer:

preferably, the stabilizer is oleic acid, sodium oleate, sodium octoate, cholesterol, cholic acid, deoxycholic acid and sodium salt thereof, vitamin A, vitamin C, vitamin E and the like or a mixture of the above substances in any proportion, and is a substance which can regulate the pH and reduce the hydrolysis speed of the emulsifier during high-temperature sterilization, can simultaneously lead the stomach of the emulsion to have negative charge and can improve the physical stability of the emulsion through electrostatic repulsion.

Preferably, the stabilizer is one or two of sodium oleate and cholesterol;

more preferably, the sodium oleate of the stabilizer is sodium oleate solution with the concentration of 0.1-0.6%; the cholesterol usage amount of the stabilizer is 20-50% of the phospholipid usage amount in the emulsifier; the stabilizer is one or two of the stabilizers.

The preparation process of the pharmaceutical preparation of the invention comprises the following steps:

the preparation method of the SAHA vorinostat venous emulsion comprises the steps of colostrum preparation, homogenization, sterilization, quality control and the like. The colostrum is prepared by ultrasonic method or high-speed shearing method (Y25 type laboratory high-shear dispersing emulsifying machine, shanghai wing electromechanical Co., ltd.). The homogenization is carried out by using a high-pressure homogenizer (AH-MINI 1, a general-purpose high-pressure homogenizer in ATS laboratory) or a microfluidics technology (Nanogenizer 20K, an experimental high-pressure microfluidizer). The sterilization adopts a wet heat sterilization method. The quality control mainly adopts particle size measurement. The particle size is controlled in the range of 50 to 300nm, preferably 80 to 250nm.

The preparation process of the pharmaceutical preparation comprises the following steps:

firstly, mixing SAHA (vorinostat) with an oil phase and an emulsifier, heating and dissolving the mixture on an ultrasonic oscillator (the temperature is 85-105 ℃) to prepare the oil phase, mixing pure water with a stabilizer, a permeation regulator and the like, and fully stirring the mixture under a magnetic stirrer to prepare a water phase; slowly adding the oil phase into the water phase under stirring of a high-speed shearing machine, controlling the temperature to be 60-80 ℃, shearing for 5-15min in the liquid high-speed shearing machine (the transmission speed is 5000-8000 RPM), repeating the high-speed shearing for 2-3 times, controlling the temperature to be not more than 60-80 ℃, and simultaneously performing nitrogen protection to prepare the colostrum.

Homogenizing the primary emulsion in a high pressure homogenizer or a high pressure microfluidizer for 6-8 times, controlling the temperature at 60-80deg.C, simultaneously introducing nitrogen for protection, cooling the emulsion to room temperature, collecting the emulsion, measuring the particle size of the emulsion within 100-250nm, and adjusting pH value to 6.3-7.9.

Packaging the emulsion, sealing with nitrogen-filled tank, and sterilizing with steam at 121deg.C for 10-15 min.

The SAHA emulsion of each formula prepared by the method has the particle size of 150-250nm, and other indexes meet the requirement of injection.

The pharmaceutical preparation has the beneficial effects that:

1. through a great deal of experimental researches, the invention can effectively prolong the half life of the medicament by adjusting the formula and the proportion. Compared with the prior application 201810076352.3, the formula is particularly selected and optimized for the emulsifier, the stabilizer, the osmotic pressure regulator and the like, sodium oleate or cholesterol and the like are particularly selected as the emulsion stabilizer, and phospholipid with the phosphatidylcholine PC content of more than 70% is used as the emulsifier, so that the affinity with the medicine is increased, the stability of the emulsion is improved, the release speed of the medicine from emulsion particles is slowed down, the in-vivo half-life of the medicine is prolonged, and the acting time of the medicine is prolonged.

2. Due to the adjustment of the formulation of the emulsion, the half life of the drug is prolonged, so that the administration dosage can be reduced. Further experimental results show that compared with the administration dose of 50mg/kg of the mice in the prior application patent 201810076352.3, the minimum effective dose is reduced to 6mg/kg, and under the condition of non-pretreatment administration, single dose administration during reperfusion can still effectively reduce myocardial infarction area and reduce myocardial cell apoptosis.

3. The pharmaceutical preparation of the invention improves the metabolic tissue distribution of the damaged heart part; through testing of drug metabolism tissue distribution, the emulsion for intravenous injection has targeting property of being easily distributed to heart damaged parts. Experiments show that the adjusted emulsion formula has good targeting of Heart tissue distribution by taking the relative uptake rate (re) as an index and Heart (re) =4.89 > 1.

4. According to the pharmaceutical preparation, the formulation auxiliary materials of the emulsion are optimized, sodium oleate is selected as a pH regulator, glycerin is selected as an emulsion osmotic regulator, the pH value and osmotic pressure are controlled in a proper range, and vascular irritation is reduced.

5. The SAHA injection emulsion of the invention is observed by an acceleration stability test for 6 months, the appearance of the SAHA injection emulsion is white emulsion, the average particle size of emulsion drops is 100-200nm, no layering phenomenon occurs, the Zeta potential of the emulsion after dilution is between-12 and-32, and the negative charge of the medicine emulsion is uniform and stable. The encapsulation rate of the pharmaceutical preparation is 89.6% -96.2%, the pH value of the pharmaceutical emulsion is 6.3-7.9, and the experimental result shows that the existing vorinostat emulsion has stable process and strong reproducibility.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention.

FIG. 1-a flow chart of a process for preparing a pharmaceutical formulation of the present invention;

FIG. 2-intravenous injection curves of formulations of the invention according to examples 1, 2, 3, 4, 5;

FIG. 3-comparison of the formulation of the pharmaceutical formulation of the invention example 1 between different doses versus the percentage (%) of myocardial infarction area/myocardial ischemia area of mice;

FIG. 4-comparison of the formulation of the pharmaceutical formulation of the invention 1 between the different doses versus the percentage (%) of myocardial infarction area/myocardial ischemia area of mice.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

1.1 preparation prescription and Process method of the pharmaceutical preparation of the invention

The invention carries out systematic study on the emulsion formula, the preparation process, the emulsion characterization and the stability study of the prior patent (201810076352.3) of the prescriptions 1, 2, 3 and 4 and the prescriptions 5.

1.1.1 prescribed doses of the pharmaceutical formulations of the invention

Table 1 the pharmaceutical formulation of the present invention relates to 5 prescribed proportioning doses

1.1.2 Process of the pharmaceutical preparation of the invention

Prescription 1: weighing 10g of soybean oil and 1.2g of egg yolk lecithin E, fully mixing by a magnetic stirrer, heating to 75-80 ℃, adding 0.3g of SAHA and 0.4g of cholesterol, mixing, maintaining the temperature on an ultrasonic oscillator at 75-80 ℃, and clarifying to obtain an oil phase; slowly adding preheated water phase (75 g of water and 2.25g of glycerol) into the oil phase under stirring of a high-speed shearing machine, controlling the temperature to be 60-80 ℃, shearing for 5-15min in the high-speed shearing machine (the transmission speed is 5000-8000 RPM), repeating the high-speed shearing for 2-3 times, and simultaneously carrying out nitrogen protection to prepare the colostrum. Homogenizing the primary emulsion in a micro-jet high pressure homogenizer for 6-8 times (pressure 1000-1200 bar), controlling temperature at 60-80deg.C, simultaneously introducing nitrogen for protection, cooling the emulsion to room temperature, collecting emulsion, measuring emulsion particle diameter at 100-250nm, and adjusting pH to 6.3-7.9.

Packaging the emulsion, sealing with nitrogen-filled tank, sterilizing with steam at 121deg.C for 15min under moist heat, and homogenizing all samples without layering.

Prescription 2: weighing 10g of soybean oil and 10g of MCT (1:1) mixture, fully mixing phosphatidylcholine PL-100M 1.2g by a magnetic stirrer, heating to 75-80 ℃, adding 0.3g of SAHA, mixing, maintaining the temperature at 75-80 ℃ by an ultrasonic oscillator, and clarifying the solution to obtain an oil phase; slowly adding water phase (75 g of water, 2.25g of glycerol, and 1.0g of 0.3% sodium oleate solution) into the oil phase under stirring of a high-speed shearing machine, shearing for 5-15min in the liquid high-speed shearing machine (transmission speed is 5000-8000 RPM) at 60-80 ℃, repeating the high-speed shearing for 2-3 times, and simultaneously introducing nitrogen for protection to prepare the colostrum. Homogenizing the primary emulsion in a high pressure homogenizer for 6-8 times (pressure 600-700 bar), controlling temperature at 60-80deg.C, introducing nitrogen for protection, cooling the emulsion to room temperature, collecting emulsion, measuring emulsion particle diameter at 100-250nm, and regulating pH value to 6.3-7.9.

Packaging the emulsion, sealing with nitrogen-filled tank, sterilizing with steam at 121deg.C for 15min under moist heat, and homogenizing all samples without layering.

Prescription 3: weighing 10g of canola oil, 0.6g of hydrogenated soybean phospholipid HSPC and 0.2g of cultivated phospholipid DSPE-MPEG2000, fully mixing by a magnetic stirrer, heating to 75-80 ℃, adding 0.3g of SAHA and 0.4g of cholesterol, mixing, maintaining the temperature at 75-80 ℃ by an ultrasonic oscillator, and clarifying the solution to obtain an oil phase; slowly adding water phase (75 g of water and 2.5g of mannitol) into the oil phase under stirring of a high-speed shearing machine, controlling the temperature to be 60-80 ℃, shearing for 5-15min in the high-speed shearing machine (the transmission speed is 5000-8000 RPM), repeating the high-speed shearing for 2-3 times, and simultaneously performing nitrogen protection to prepare the colostrum. Homogenizing the primary emulsion in a micro-jet high pressure homogenizer for 6-8 times (pressure 1000-1200 bar), controlling temperature at 60-80deg.C, simultaneously introducing nitrogen for protection, cooling the emulsion to room temperature, collecting emulsion, measuring emulsion particle diameter at 100-250nm, and adjusting pH to 6.3-7.9.

Packaging the emulsion, sealing with nitrogen-filled tank, sterilizing with steam at 121deg.C for 15min under moist heat, and homogenizing all samples without layering.

Prescription 4: weighing 10g of soybean oil, 1.2g of PEG300 monostearate and 0.1g of oleic acid, fully mixing by a magnetic stirrer, heating to 75-80 ℃, adding 0.3g of SAHA, maintaining the temperature at 75-80 ℃ in an ultrasonic oscillator after mixing, and obtaining an oil phase after the solution is clarified; slowly adding water phase (75 g of water and 2.5g of glycerin) into the oil phase under stirring of a high-speed shearing machine, controlling the temperature to be 60-80 ℃, shearing for 5-15min in the liquid high-speed shearing machine (the transmission speed is 5000-8000 RPM), repeating the high-speed shearing for 2-3 times, and simultaneously introducing nitrogen for protection to prepare the colostrum. Homogenizing the primary emulsion in a high pressure homogenizer for 6-8 times (pressure 600-700 bar), controlling temperature at 60-80deg.C, introducing nitrogen for protection, cooling the emulsion to room temperature, collecting emulsion, measuring emulsion particle diameter at 100-250nm, and regulating pH value to 6.3-7.9.

Packaging the emulsion, sealing with nitrogen-filled tank, sterilizing with steam at 121deg.C for 15min under moist heat, and homogenizing all samples without layering.

Recipe 5 (prior patent application 201810076352.3): weighing SAHA 3g, adding Tween 80 and glycerol, heating to 80deg.C, adding water about 100ml until the raw materials are dissolved, adding soybean phospholipid, shearing (6000 rpm), and mixing to obtain water phase; weighing oleic acid and soybean oil for injection, mixing uniformly, and preparing an oil phase; the oil-water phase was mixed under high shear (6000 rpm) at 70℃for 30 minutes to prepare a colostrum. Homogenizing the colostrum under 800bar pressure, circulating for 6 times, filling into ampoule, sealing, and sterilizing at 115deg.C for 30 min.

1.1.3 characterization of the emulsion of the pharmaceutical preparation of the invention after preparation of the emulsion, detection of the corresponding emulsion and quality detection of particle size, zeta potential encapsulation efficiency, pH value, etc. are carried out, and the detection results are shown in Table 2 below.

TABLE 2 results of quality detection indicators for pharmaceutical formulations of the invention

1.1.4 stability Studies of pharmaceutical formulations of the invention

The prescription intravenous emulsion formula is subjected to stability investigation (investigation indexes: average particle size, zeta potential, encapsulation efficiency content and pH value change) of a pharmaceutical preparation for 6 months, and under the room temperature condition, samples are subjected to investigation on appearance, average particle size, zeta potential, encapsulation efficiency content and pH value change respectively for 0, 1, 3 and 6 months. Wherein the average particle diameter and Zeta potential are measured by using a Zetasizer Lab nano-particle size potentiometer, the pH value is measured by using a pH meter, the encapsulation efficiency (content) is calculated by separating an oil phase, an emulsion layer and a water phase by using an ultracentrifugation method and measuring the SAHA content in water, the vorinostat content is measured by using a high performance liquid chromatography under the conditions of C18 column, (5 μm,50mm x 4.6 mm) and acetonitrile-0.1% phosphoric acid water (30:70) as a mobile phase (the pH is adjusted to 3.0 by using triethylamine); the detection wavelength is 241nm, the flow rate is 1.0ml/min, and the stability measurement results of the above pharmaceutical preparation are shown in Table 3.

TABLE 3 quality detection of various stability indicators for pharmaceutical formulations of the present invention

Example 2

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: 4g of vorinostat, 100g of soybean oil, 13g of egg yolk lecithin, 20g of glycerol, 6g of cholesterol and 700g of water for injection; the medicine intravenous emulsion is prepared.

Example 3

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: vorinostat 5g, soybean oil 120g, egg yolk lecithin 10g, glycerin 25g, cholesterol 10g and water for injection 800g; the medicine intravenous emulsion is prepared.

Example 4

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: 2g of vorinostat, 80g of soybean oil, 15g of egg yolk lecithin, 17g of glycerol, 1g of cholesterol and 600g of water for injection; the medicine intravenous emulsion is prepared.

Example 5

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: 4g of vorinostat, 50g of soybean oil, 50g of MCT medium chain triglyceride, 12g of phosphatidylcholine, 21g of glycerol, 5g of sodium oleate solution and 700g of water for injection; the medicine intravenous emulsion is prepared.

Example 6

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: 2g of vorinostat, 60g of soybean oil, 60g of MCT medium chain triglyceride, 10g of phosphatidylcholine, 25g of glycerol, 10g of sodium oleate solution and 800g of water for injection; the medicine intravenous emulsion is prepared.

Example 7

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: vorinostat 5g, soybean oil 40g, MCT medium chain triglyceride 40g, phosphatidylcholine 15g, glycerol 17g, sodium oleate solution 1g, and water for injection 600g; the medicine intravenous emulsion is prepared.

Example 8

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: vorinostat 4g, canola oil 100g, hydrogenated soybean lecithin 6g, cultivated phospholipid 2g, mannitol 21g, cholesterol 6g, water for injection 700g; the medicine intravenous emulsion is prepared.

Example 9

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: 2g of vorinostat, 80g of canola oil, 7g of hydrogenated soybean lecithin, 1g of cultivated lecithin, 25g of mannitol, 10g of cholesterol and 800g of water for injection; the medicine intravenous emulsion is prepared.

Example 10

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: vorinostat 5g, canola oil 120g, hydrogenated soybean lecithin 5g, cultivated phospholipid 3g, mannitol 17g, cholesterol 1g, water for injection 600g; the medicine intravenous emulsion is prepared.

Example 11

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: vorinostat 4g, soybean oil 90g, PEG300 monostearate 12g, glycerin 19g, oleic acid 6g, and water for injection 750g; the medicine intravenous emulsion is prepared.

Example 12

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: vorinostat 5g, soybean oil 80g, PEG300 monostearate 15g, glycerin 25g, oleic acid 10g, and water for injection 800g; the medicine intravenous emulsion is prepared.

Example 13

The pharmaceutical preparation of the invention is prepared according to the preparation method of the example 1, and the dosage proportion of the prescription is as follows: 2g of vorinostat, 120g of soybean oil, 10g of PEG300 monostearate, 17g of glycerol, 1g of oleic acid and 600g of water for injection; the medicine intravenous emulsion is prepared.

EXAMPLE 14 pharmacokinetic Studies of pharmaceutical formulations of the present invention

14.1 half-life and time of flight Curve determination of pharmaceutical formulations of the invention

14.1.1 test methods

The formulations 1, 2, 3, 4 and 5 of example 1 were administered intravenously to SD rats, respectively, and the blood concentration was measured. About 200g SD rats are selected, 15 SD rats are randomly grouped into 5 groups, 3 SD rats are respectively subjected to tail vein injection prescriptions 1, 2, 3, 4 and 5 (3 mg/mL) according to the dosage of 10mg/kg respectively, 0.5mL blood is obtained from eyeground venous plexus respectively at 0, 1, 5, 15, 30, 60, 120, 240, 360 and 720min, heparin is anticoagulated, 100 microliters of blood plasma is precisely sucked and mixed with 200 microliters of acetonitrile (0.15% formic acid and 300ng/mL benzyl benzamide are used as an internal standard). The samples were spun for 15s, incubated at room temperature for 10 minutes and the resulting plasma frozen at-20℃for use in a standard microcentrifuge at 13200 rpm. Then, 10. Mu.l of the supernatant was analyzed by liquid chromatography.

14.1.2 blood concentration measuring method

The test of the invention adopts liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to measure the concentration of the blood medicine. 100 microliters of plasma was mixed with 200 μl of acetonitrile (containing 0.15% formic acid, 300ng/mL benzyl benzamide as an internal standard). The samples were spun for 15s, incubated at room temperature for 10 minutes and spun at 13200rpm in a standard microcentrifuge. Then, 10. Mu.l of the supernatant was analyzed by liquid chromatography. The retention times of SAHA vorinostat (molecular ion peak 265.1/232.021) and internal standard (molecular ion peak 212.2/91.1) were 4.1 and 4.5min, respectively.

Mobile phase: buffer a: water +0.1% formic acid; buffer B: methanol +0.1% formic acid; flow, 1.5mL/min; chromatographic column: agilent C18 XDB column, (5 μm,50 mm. Times.4.6 mm); liquid phase conditions: 0-1 min 95% a,1-1.5 min gradient to 95% b; 100% b for 1.5 to 2.5 minutes, gradient to 95% a for 2.5 to 2.6 minutes; 95% A for 2.6 to 3.5 minutes.

And adopting an electrospray ion source and detecting a positive ion multi-reaction ion monitoring mode. The peak areas of vorinostat were calculated from the internal standard. And measuring the concentration of the drug in the blood plasma at different time points, and deducting the background concentration of the drug in the blood plasma to obtain the blood drug concentration. The intravenous drug curves for prescriptions 1, 2, 3, 4, 5 were obtained and can be seen in figure 2.

14.1.3 pharmacokinetic parameters of the pharmaceutical formulations of the invention

The results of the atrioventricular model fitting of the blood concentration-time data using DAS2.2.2.1 pharmacokinetic software show that the pharmacokinetic process of the SAHA formulation for rat tail intravenous administration is consistent with the two-compartment model, and the results are shown in the table. As can be seen from the results in Table 4, the AUC of formulas 1, 2, 3, 4, and 5 vary widely from one formula to another. Wherein prescription 1 effectively prolongs half-life and AUC area suggests good bioavailability.

TABLE 4 pharmacokinetic study results for different prescriptions

14.1.4 tissue distribution of the medicament of the invention

1.1.4.1 test method

Fasted 12h mice 135 were randomly divided into 5 groups, each with a dose of 10mg/kg given by tail intravenous injection prescription 1, 2, 3, 4, 5, 3 mice per time point, and blood was collected from the eyes of 1, 5, 15, 30, 60, 120, 240, 360, 720min after administration in heparin anticoagulation tube, plasma was centrifuged at 13200r/min, animals were rapidly sacrificed, and heart, liver, brain, spleen, lung, kidney were removed, washed with ice physiological saline, and filter paper was dipped to dryness, and 0.5g was weighed. The obtained tissue is frozen and preserved at-20deg.C for use.

Treatment of 14.1.4.2 plasma samples:

0.1ml of mouse plasma is taken in a 1.5ml centrifuge tube, 0.2ml of methanol and 0.4ml of normal hexane are added, the mixture is centrifuged for 10min at 10000r/min at 4 ℃,20 mu L of supernatant is taken for sample injection measurement, and the operation is carried out in a dark place.

14.1.4.3 treatment and detection of tissue samples:

precisely weighing 0.1g of tissue, homogenizing, placing the homogenate in 0.2ml of physiological saline at a homogenate medium position in a 1.5ml centrifuge tube, adding 0.2ml of methanol, 0.4ml of normal hexane, swirling for 3min, centrifuging for 10min at 10000r/min at 4 ℃, transferring normal hexane into a 1.5ml centrifuge tube, repeating the operation for 2 times, combining normal hexane layers, centrifuging, concentrating, volatilizing normal hexane, dissolving 200 mu L of mobile phase, swirling for 3min, centrifuging for 10min at 10000r/min, taking 20 mu L of supernatant, performing sample introduction measurement, and performing light-shielding operation.

Mobile phase: buffer a: water +0.1% formic acid; buffer B: methanol +0.1% formic acid; flow, 1.5mL/min; chromatographic column: agilent C18 XDB column, (5 μm,50 mm. Times.4.6 mm); liquid phase conditions: 0-1 min 95% a,1-1.5 min gradient to 95% b; 100% b for 1.5 to 2.5 minutes, gradient to 95% a for 2.5 to 2.6 minutes; 95% A for 2.6 to 3.5 minutes. And adopting an electrospray ion source and detecting a positive ion multi-reaction ion monitoring mode. The peak areas of vorinostat were calculated from the internal standard.

And measuring the drug concentration in the tissues at different time points, subtracting the background drug concentration in the tissues to obtain the blood drug concentration in the obtained tissues, and obtaining the area under the curve AUC value of SAHA in different tissues by calculating the drug time change in the different tissues with time, wherein the result is shown in Table 5.

TABLE 5 AUC values of SAHA in different tissues

14.1.4.4 Targeted preliminary evaluation

At a relative uptake rate (r _e ) As an indicator, the relative targeting of different SAHA emulsion formulations in vivo was evaluated. The calculation formula is as follows: r is (r) _e ＝(AUC) _p /(AUC) _s AUC is the area under the drug curve, p and s represent formulation prescriptions 1-4 and prior patent prescription 5, respectively. r is (r) _e When the ratio is greater than 1, the medicine preparation has targeting effect on the tissue, and the targeting effect is better as the medicine preparation is greater than that of the prior patent; r is (r) _e Equal to or less than 1, represents a non-targeting advantage over the prior patent, r _e The results are shown in Table 6.

TABLE 6 targeting of SAHA in different tissues

The relative uptake rate results show that, during the investigation time, the content of prescription 1 in the heart and spleen is 5.75 and 3.6 times that of the group 5 of the prior patent, the content of prescription 2 in the heart and spleen is 2.75 and 2.32 times that of the group 5 of the prior patent, the content of prescription 3 and prescription 4 in the lung is 2.4 and 2.17 times that of the prescription 5 respectively, the emulsion particle size of prescription 4 is the smallest, and the content of the emulsion in the brain is 3.17 times that of the prescription 5, which also shows that the optimized intravenous emulsion formula of the invention can be adjusted, and the tissue distribution of the medicine can be effectively adjusted through controlling the particle size.

EXAMPLE 15 in vitro hemolysis experiments of pharmaceutical formulations of the invention

Taking 5ml of rabbit blood, placing into an anticoagulation centrifuge tube, shaking for 10min in an conical flask loaded with small glass beads, removing fibrinogen, centrifuging to obtain supernatant, flushing a red blood cell layer with 0.9% sodium chloride injection for 4 times, 10ml each time, centrifuging to obtain supernatant until the supernatant is red, then sucking a certain amount of red blood cells, diluting with 0.9% sodium chloride injection according to a volume ratio to prepare 2% red blood cell suspension, and refrigerating in a refrigerator at 4 ℃ for later use.

7 test tubes are taken, the serial numbers of the test tubes are 1-7, the design of a hemolysis test is shown in Table 6, a proper amount of 2% erythrocyte suspension and 0.9% sodium chloride injection (distilled water is added into the No. 7 tube to serve as positive control) are respectively added into each tube, the test tubes are placed into a constant-temperature water bath at 37 ℃ for 30min, different amounts of prescriptions 1-5 (No. 6 tube is used as negative blank control) are respectively added, the test tubes are uniformly shaken, and the hemolysis conditions of the erythrocyte in the test tubes are respectively observed at 0.5, 1, 2, 3 and 4 h. The hemolysis test is designed into a sample adding table.

Test result observation judgment standard: grading: test tube condition; hemolysis: the solution is clear red, and the bottom of the tube has no cell residue or little red cell residue; no hemolysis: the red blood cells are all submerged, and the upper liquid is colorless and clear; condensing: the solution had a brownish red or reddish brown flocculent precipitate which could not be dispersed after shaking.

TABLE 7 design of intravenous emulsion hemolysis experiments (ml) for different prescriptions

Sample of	1	2	3	4	5	6	7
								2% erythrocyte suspension	2.5	2.5	2.5	2.5	2.5	2.5	2.5
Sodium chloride injection with concentration of 0.9%	2.0	2.1	2.2	2.3	2.4	2.5	0
								Distilled water	0	0	0	0	0	0	2.5
Prescription 1	0.5	0.4	0.3	0.2	0.1	0	0
								Prescription 2	0.5	0.4	0.3	0.2	0.1	0	0
Prescription 3	0.5	0.4	0.3	0.2	0.1	0	0
								Prescription 4	0.5	0.4	0.3	0.2	0.1	0	0
Prescription 5	0.5	0.4	0.3	0.2	0.1	0	0

TABLE 8 observations of pharmaceutical formulations of the invention

Sample of	Test tube condition
		Prescription 1	None of them has hemolysis
Prescription 2	None of them has hemolysis
		Prescription 3	None of them has hemolysis
Prescription 4	None of them has hemolysis
		Prescription 5	If the time is more than 4 hours, the tube has little condensation except 0.5ml tube, and all the tubes have no hemolysis in 3 hours

EXAMPLE 16 Effect of the pharmaceutical formulation of the invention on vascular irritation in animals

Vascular irritation test 21 healthy New Zealand white rabbits, male and female, were randomly divided into 7 groups of 3. Intravenous drip (10 ml/kg,1ml/min,1 time/Day) is performed on the edge of the ear according to a sterile operation method, the first group of right ear instils the solvent physiological saline solution, and the left ear instils the physiological saline solution; the second group of left and right ears instilled with a 5% dextrose solution of the solvent; the third group to the seventh group are instilled with the prescription of 1-5 respectively, the volume is 10ml/kg,1 time/day, and 5 days are continuous, and the irritation phenomena such as red swelling, congestion and the like at the injection part are observed with naked eyes before and after the last administration of each day for 72 hours. Animals are sacrificed, 3 sections of blood vessels and tissues are continuously taken from the ear edge intravenous injection point to the centripetal direction of 2cm, pathological examination is carried out, and whether inflammatory reaction exists on the venous blood vessel tissues or not, and even obvious stimulation reaction such as denaturation necrosis and the like are observed. The other side was treated in the same manner as the control vessel.

The results of pathological observation and histological examination of each section of vein and tissue after 5 days of administration and 72 hours of the last administration are shown in Table 9. No irritation such as reddening, swelling and congestion is seen at the intravenous injection sites of 3 rabbits in each administration group. As a result of pathological histology examination, normal saline and 5% glucose left ear control group, the arrangement of vascular endothelial cells of the auricular veins is normal, no change of hierarchy increase and arrangement disorder is seen, and no wall-attached thrombus and inflammatory cell infiltration exist in the vascular cavity. The inner, middle and outer layers of the tube wall have clear structures, the inner elastic plate is visible, and fibrous tissue hyperplasia is not visible. The wall of the tube has no morphological changes such as thickening, necrosis, inflammation and the like, and is a normal tissue structure; the vascular tissue structures of the solvent and each prescription administration group are normal, and compared with the blood vessels of the left ear control group, the vascular tissue structures of the solvent and each prescription administration group are not abnormally changed.

Table 9 comparison of pathological lesions of the Rabbit ear margin vein

The test results show that each prescription of the invention has good vascular tolerance, small vascular irritation and good safety, and provides further safety guarantee for injection used as intracoronary injection. EXAMPLE 17 Effect of the pharmaceutical formulation of the invention on myocardial infarction/ischemia area in model animals

17.1 establishing a model of myocardial ischemia reperfusion in mice

Model group (I/R) mice received 2% isoflurane inhalation anesthesia, and the operator made a skin incision of about 1.2cm in the left anterior chest wall, and exposed the left chest fourth rib space obliquely, gently and rapidly expanding the pleural and pericardial cavities, squeezing the heart. A6-gauge needle suture is used for ligating the slipknot at the anterior descending branch of the left coronary artery of the anterior wall of the heart, and the color of myocardial tissue of the left ventricle is visually observed to turn into gray, so that the success of ligature is confirmed. Thereafter, the heart is quickly placed in the chest cavity, the air in the cavity is manually vented, the chest cavity is closed, the skin wound is sutured, and the end of the knot ligating the anterior descending branch of the left coronary artery of the heart is left outside the chest. Mice were placed in an air-ventilated environment and monitored for resuscitation. 45 minutes after ischemia, the sliding knot of the anterior descending branch of the ligation was loosened by gently and gently dragging the wire head left outside the chest, and the myocardial tissue was subjected to blood reperfusion (perfusion time was 24 h). The sham group (sham) extruded the heart following the same procedure as described above for model group (I/R), 6 gauge needle suture passed through the anterior descending left coronary artery of the anterior wall of the heart but not knotted, and the thoracic air was removed in the same manner, the thoracic was closed, and the wound was sutured.

17.2 mice were dosed with prescription 1 at myocardial ischemia reperfusion, specific groupings and dosing are shown in Table 9.

Table 10 different dosage groupings and dosing amounts for the inventive pharmaceutical formulation of prescription 1

Animals were randomly grouped by the on-line grouping tool Quickcalcs (http:// www.graphpad.com/quick calcs /), and were grouped into I/R, I/R+4.5mg/kg, I/R+6mg/kg, I/R+9mg/kgI/R+13.5mg/kg groups. Prescription 1 or solvent was administered to I/R mice by tail vein injection, and all drugs were administered once according to body weight at the time of myocardial reperfusion procedure.

17.3 Evans Blue & TTC double staining method for detecting heart infarction/ischemia area of mice

The mice of the myocardial ischemia reperfusion group were reinfused with heart vessels 24h after reperfusion, and femoral artery or aorta were injected with 1% Evans Blue 200 μl, showing ischemic myocardium; the sham group did not ligate the heart vessels and the femoral artery or aorta were injected with 200 μl of 1% Evans Blue. Taking heart, washing blood in PBS until PBS is not discolored, wiping off water, freezing at-40deg.C for 60 min, placing into a mold, slicing at equal distance (5 slices of coronal slices) perpendicular to the longitudinal axis of heart, placing into 2% TTC phosphate buffer solution PH 7.4, and incubating at 37deg.C for 20 min. And after the Image acquisition system scans, calculating and counting myocardial ischemia range and infarction range through Image J.

The statistical results of the test results are shown in Table 10.

Table 11 comparison between pharmaceutical formulations of different doses administered versus mice myocardial infarction area (%) group

Table 11 comparison between different doses of pharmaceutical formulation versus percent myocardial infarction area/myocardial ischemia area%

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One-way analysis of variance for Tukey test of C57BL/6 male mice acute myocardial ischemia/reperfusion injury index was performed in prescription 1 (4.5 mg/kg,6mg/kg,9mg/kg,13.5 mg/kg). Expressed as mean±sd, n=10 per group. * p <0.05, <0.01vs. I/R group.

Conclusion: compared with a model group, the drug preparation prescription 1 dose of 9mg/kg group effectively reduces the myocardial infarction area and the myocardial infarction/ischemia area percentage (p is less than 0.05); in addition, the 13.5mg/kg dose group effectively reduced myocardial infarction area than the model group. Therefore, the drug preparation formula 1 with the optimal heart tissue distribution screened by the emulsion formula optimization is administered at a low dose of 9mg/kg and only in a single dose during reperfusion, and can also remarkably reduce the myocardial infarction area by 40 percent and reduce the myocardial infarction/ischemia area percentage by 35 percent. While the emulsion of the prior patent application needs to be respectively administered with 25mg/kg before and after reperfusion before operation or 50mg/kg once in one hour before operation to achieve significant reduction of myocardial infarction area. Therefore, the intravenous emulsion can effectively reduce the dosage of clinical medicines and provide further guarantee for the safety of the medicines.

The invention is not limited to the specific embodiments described above, which are intended to be illustrative, instructive, and not limiting. Those skilled in the art should, in light of the present disclosure, make any changes, equivalents, and modifications that are within the spirit and scope of the present invention.

Claims (10)

1. The pharmaceutical preparation for treating ischemic myocardial reperfusion injury is characterized by comprising the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 30 parts of HDAC inhibitor medicine, 60 to 250 parts of oil phase, 8 to 30 parts of emulsifier, 10 to 30 parts of osmotic pressure regulator, 0 to 12 parts of stabilizer and 500 to 900 parts of water for injection.

2. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation comprises the following components in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of oil phase, 10 to 18 parts of emulsifying agent, 13 to 27 parts of osmotic pressure regulator, 0 to 10 parts of stabilizing agent and 600 to 800 parts of water for injection.

3. The pharmaceutical formulation according to claim 1 or 2, wherein the pharmaceutical formulation excipients are:

The oil phase is as follows: one or more of soybean oil (containing injection grade), castor oil, tea oil, cottonseed oil, sesame oil, rapeseed oil, safflower oil, canola oil, olive oil, coconut oil, palm oil, cocoa butter, etc.; medium long chain fatty acid glycerides with chain lengths between C6 and C12, such as one or more of MCT medium chain triglycerides, arlacel 80, arlacel 86, capmul MCM, captex 200, captex 355, miglyol 812, myvacet, myverol-92, glyceryl oleate, glyceryl linoleate, polyethylene glycol glyceryl laurate, ethyl oleate, ethyl linoleate, caprylic/capric triglyceride, and the like; one or more of the above mixtures of long chain fatty acid esters and medium chain fatty acid esters may also be used;

the emulsifying agent is as follows: soy lecithin and its modifications (natural or synthetic), egg yolk lecithin and its modifications (natural or synthetic), PEG phospholipids, hydrogenated soy phospholipids HSPC, cultivated phospholipids DSPE-MPEG2000, ophase31, poloxamers, polyoxyethylene hydrogenated castor oil, water soluble VE (TPGS), solutol HS-15, PEG300, PEG400, PEG1750 and its monostearate, tween 80, span 20, phosphatidylcholine, distearoyl phosphatidylcholine, myristoyl lysolecithin, dimyristoyl lecithin, dilauroyl lecithin, dicapryl phosphatidylcholine, polyoxypropylene polyoxyethylene block copolymers;

The osmotic pressure regulator is as follows: one or more of sodium chloride, glucose, sorbitol, xylitol, mannitol, and glycerol;

the stabilizer is as follows: oleic acid, sodium oleate, sodium caprylate, cholesterol, cholic acid, deoxycholic acid and sodium salt thereof, vitamin A, vitamin C and vitamin E.

4. The pharmaceutical preparation according to claim 1 or 2, wherein the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of soybean oil, 10 to 15 parts of egg yolk lecithin (E80), 17 to 25 parts of glycerol, 0 to 10 parts of cholesterol, 0 to 10 parts of sodium oleate solution and 600 to 800 parts of water for injection.

5. The pharmaceutical preparation according to claim 1 or 2, wherein the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of soybean oil, 40 to 60 parts of MCT medium chain triglyceride, 10 to 15 parts of phosphatidylcholine, 17 to 25 parts of glycerol, 1 to 10 parts of sodium oleate solution and 600 to 800 parts of water for injection.

6. The pharmaceutical preparation according to claim 1 or 2, wherein the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 0.5 to 15 parts of vorinostat, 80 to 120 parts of canola oil, 5 to 9 parts of hydrogenated soybean lecithin HSPC, 20001 to 3 parts of cultivated phospholipid DSPE-MPEG, 17 to 25 parts of mannitol, 1 to 10 parts of cholesterol and 600 to 800 parts of water for injection.

7. The pharmaceutical preparation according to claim 1 or 2, wherein the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: : 0.5 to 15 parts of vorinostat, 80 to 120 parts of soybean oil, 10 to 15 parts of PEG300 monostearate, 17 to 25 parts of glycerol, 1 to 10 parts of oleic acid and 600 to 800 parts of water for injection.

8. The pharmaceutical preparation according to claim 1 or 2, wherein the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 3 parts of vorinostat, 100 parts of soybean oil, 80 parts of egg yolk lecithin E, 22.5 parts of glycerol, 4 parts of cholesterol and 750 parts of water for injection.

9. The pharmaceutical preparation according to claim 1 or 2, wherein the pharmaceutical preparation comprises the following pharmaceutical active ingredients and auxiliary materials in parts by weight: 3 parts of vorinostat, 50 parts of soybean oil, 50 parts of MCT medium chain triglyceride lecithin, 12 parts of phosphatidylcholine PL-100M, 22.5 parts of glycerin, 10 parts of 0.3% sodium oleate solution and 750 parts of water for injection.

10. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation is an intravenous emulsion formulation or an intracoronary injection.