CN117243933B - Coenzyme Q-containing 10 Cardioplegic fluid composition and use thereof - Google Patents
Coenzyme Q-containing 10 Cardioplegic fluid composition and use thereof Download PDFInfo
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- CN117243933B CN117243933B CN202311543507.7A CN202311543507A CN117243933B CN 117243933 B CN117243933 B CN 117243933B CN 202311543507 A CN202311543507 A CN 202311543507A CN 117243933 B CN117243933 B CN 117243933B
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- coenzyme
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- polyoxyethylene
- asystole
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- QUANRIQJNFHVEU-UHFFFAOYSA-N oxirane;propane-1,2,3-triol Chemical compound C1CO1.OCC(O)CO QUANRIQJNFHVEU-UHFFFAOYSA-N 0.000 description 1
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
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- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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Abstract
The invention relates to the technical field of biological medicine, in particular to a coenzyme Q-containing medicine 10 Is of the heart of (a)Zygosaccharide composition and application thereof, and coenzyme Q-containing composition 10 Comprises coenzyme Q 10 Solution and cardioplegic solution, wherein the coenzyme Q 10 In the solution, the dissolvent comprises 33-60 parts by weight of polyoxyethylene nonionic surfactant and 40-67 parts by weight of water-soluble polyhydroxy compound; coenzyme Q 10 The concentration is 14.8-58.8 mug/mg, and the obtained solution is a clear solution with HLB value of 9-29. Containing coenzyme Q 10 The use of cardioplegic composition for ex vivo donor heart preservation in cardiac transplantation and cardioplegic drug for perfusion in establishing extracorporeal circulation.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a coenzyme Q-containing medicine 10 Cardioplegia composition and its application.
Background
Coenzyme Q 10 The fat-soluble quinone compound has the common characteristics of vitamins, has a chemical structure similar to vitamin K, has multiple biological functions, and particularly has the advantages of small dosage and low toxicity when used, and can be used for assisting in treating multiple diseases, thereby being widely applied clinically. Coenzyme Q 10 The medicine is a good biochemical medicine, has the functions of natural antioxidation and cell metabolism activation, can obviously improve the immunity of human bodies, is clinically mainly used for treating cardiovascular diseases, scurvy, aplastic anemia, duodenal ulcer, acute and chronic viral hepatitis, subacute liver necrosis, congestive heart disease, emphysema and other diseases and assisting treatment of cancer patients, and is widely used for nutritional health care products and food additives at home and abroad.
But coenzyme Q 10 In terms of solubility, the water-soluble organic solvent has the advantages of being very soluble in chloroform and benzene, being very soluble in acetone and diethyl ether and being very insoluble in ethanolIs insoluble in water and methanol, and due to coenzyme Q 10 The quinone group exists in the molecular structure of the catalyst, and is easy to decompose in the presence of oxygen and light. To increase coenzyme Q 10 The solubility requires more auxiliary materials such as surfactants, organic solvents or long chain fatty acids (oils) or lipids. Coenzyme Q disclosed in Chinese patent application No. 200410022007.X 10 Intravenous infusion and method for preparing same, which provides a method for preparing coenzyme Q 10 The method for preparing intravenous transfusion mainly comprises adding high concentration of surfactant polysorbate 80 (Tween 80) to coenzyme Q 10 Solubilization is performed to successfully prepare the medicine into intravenous transfusion. Polysorbate 80 (Tween 80) is a commonly used coenzyme Q 10 Solubilizing adjuvant, and dissolving coenzyme Q alone 10 The solution can be kept clear for a short period of time, but after a few days, the coenzyme Q is 10 Will re-precipitate. Coenzyme Q disclosed in Chinese patent application No. 200610086747.9 10 Intravenous infusion injection "which provides another alternative to coenzyme Q 10 The key point of the method is to use a compound solubilizer which is composed of polysorbate 80 (Tween 80) and polyoxyethylene fatty acid ester (polyoxyethylene fatty acid 40 ester) according to a certain proportion. Coenzyme Q in patent CN201510565360.0 10 The injection adopts a combination mode of a solubilizing emulsifier and a surfactant polysorbate to increase coenzyme Q 10 Is a solvent for the polymer. Patent CN200810069760.2 coenzyme Q 10 The injection adopts a mode of mixing polyethylene glycol 15-hydroxystearate (Solutol HS 15) with polysorbate 80. These patent techniques are all combinations of various pro-solvents, and the greater amounts of these components, the safety and impact on cardiomyocytes are also considered. In addition, excessive addition of polysorbate 80 may cause safety problems such as allergy and the like.
In another example, solubilization with micelles, e.g. coenzyme Q 10 Encapsulated in a micelle comprising glycyrrhizic acid or glycyrrhizic acid salt, bile acid and unsaturated salt (coenzyme Q 10 Solubilizing composition and method for preparing the same, CN 201780024843.7), or adding vegetable oil, and preparing into emulsion. Also researchers have performed the treatment of coenzyme Q 10 And gatherMixing ethylene glycol dodecahydroxystearate solubilizer, heating to melt, adding injectable solvent to give clear solution (coenzyme Q) 10 Intravenous infusion and methods of making the same, CN101480375 a).
However, the cardioplegia has complex formulation, and each 1000mL of the solution contains 0.8766g of sodium chloride, 0.6710g of potassium chloride, 0.1842g of 2-ketoglutarate-hydrogen-potassium, 0.8132g of magnesium chloride hexahydrate, 3.7733g of histidine monohydrochloride, 27.9289g of histidine, 0.4085g of tryptophan, 5.4651g of mannitol and 0.0022g of calcium chloride dihydrate. Thus, unlike the intravenous solutions described in the above-mentioned patents (which typically employ 0.9% sodium chloride solution or 5% dextrose solution), the coenzyme Q10 solubilizing formulation may aggregate upon addition to cardioplegia due to ionic or molecular interactions with cardioplegia, resulting in encapsulated coenzyme Q 10 Leakage of (1) and precipitation; meanwhile, the types or the amounts of auxiliary materials used in the preparations cannot meet the safety requirements of heart protection.
The complexity of the components of the asystole formulation also ensures that the variety and the content of the newly added components are reduced as much as possible while the original component content is unchanged when the formulation is improved, so as to reduce the disturbance on the ion components in the asystole and further influence the asystole effect of the asystole.
In terms of stability, due to coenzyme Q 10 Is easy to oxidize, and the prior art mainly maintains coenzyme Q by adding sodium bisulphite and sodium calcium edetate as reducing agents 10 Is stable. The HTK cardioplegic solution is mainly prepared by inhibiting action potential of myocardial cells through high-concentration potassium ions and low-concentration sodium and calcium ions in the solution, so as to achieve the cardioplegic effect. Therefore, the direct addition of these two ionic reductants to the asystole will result in a change in the concentration of ions in the asystole, affecting the asystole effect. If the concentration of sodium ions in asystole is increased, the consumption of sodium-potassium pump energy increases due to the tendency of extracellular sodium ions to diffuse into the cells, and the reduced ATP will further increase the risk of cellular oedema during asystole.
Disclosure of Invention
The invention aims to provide a deviceCoenzyme Q-containing species 10 The cardioplegic solution composition has high drug loading capacity and stability.
It is a further object of the present invention to provide the use of the above composition.
The invention aims at realizing the following scheme: coenzyme Q-containing 10 Cardioplegia composition comprising coenzyme Q 10 A solution and cardioplegia solution, wherein,
said coenzyme Q 10 The solution comprises 33-60 parts by weight of polyoxyethylene nonionic surfactant, 40-67 parts by weight of water-soluble polyhydroxy compound and coenzyme Q 10 The concentration is 14.8-58.8 mug/mg, the obtained solution is clear solution with HLB value of 9-29, and the solution passes through coenzyme Q 10 Self-assembling with a surface active substance to form micelle-like structure particles, so that the loading process of the drug is always carried out in a micelle-like structure with nano scale; the concentration of the dissolving agent in the cardioplegia is not more than 10mg/mL, and the coenzyme Q 10 The concentration in the cardioplegia solution is 15-60 mg/L, e.g. the coenzyme Q 10 15mg/L, 17 mg/L, 20 mg/L, 23 mg/L, 25mg/L, 27 mg/L, 30 mg/L, 35 mg/L, 40 mg/L, 45 mg/L, 50 mg/L, 55 mg/L, and 60mg/L;
said coenzyme Q 10 In the solution, the water is mixed with the water,
the polyoxyethylene nonionic surfactant is used as a solubilizer and comprises one or more of polyoxyethylene hydrogenated castor oil, polyoxyethylenated 12-hydroxystearic acid, caprylic/capric acid polyethylene glycol glyceride, poloxamer, lauroyl polyoxyethylene-6 glyceride, polyoxyethylene castor oil, polyoxyethylene-8 glyceryl behenate, lauroyl polyoxyethylene-32 glyceride, stearoyl polyoxyethylene glyceride, oleoyl polyoxyethylene glyceride and linoleoyl polyoxyethylene-6 glyceride;
the water-soluble polyhydroxy compound comprises one or more of glycerol, polyethylene glycol, polyglycerol, copolymer of polyethylene glycol and polypropylene glycol.
The obtained coenzyme Q 10 Adding the solution into water or aqueous solution, and passing through coenzyme Q 10 Self-assembling with surface active substances to form micelle-like structure particles, so that the drug loading process is always carried out in a micelle-like structure with nanometer scale, and therefore, coenzyme Q in the micelle-like structure can be greatly increased by only a small amount of surface active substances 10 Is a load of (a) in the vehicle. Is beneficial to reducing the dosage of auxiliary materials in the solution and improving the coenzyme Q 10 Stability of the solution.
Further, the solubilizer and the cosolvent in the dissolvent are mixed with coenzyme Q at the temperature of 40-50 DEG C 10 Uniformly mixing to obtain coenzyme Q 10 Solution, coenzyme Q obtained 10 The solution exists in semisolid form at low temperature, after heating in water bath, it is recovered to clear solution, and added into heart protecting solution, and self-assembled with surfactant to form coenzyme Q 10 Micelle-like self-assembled structure of (a) to make coenzyme Q 10 Stably dispersed in cardioprotective solution, the coenzyme Q 10 Solution to coenzyme Q 10 Insoluble in water and easily oxidized.
Coenzyme Q 10 Comprises two parts of a solubilizer and a cosolvent. Wherein, the solubilizer is a nonionic surfactant, the polyethylene glycol structure is used as hydrophilic group, and the HLB value is between 9 and 29; the cosolvent is polyhydroxy hydrophilic component such as polyethylene glycol 300 and 400, and glycerol (glycerol).
Based on the scheme, the weight average molecular weight of the polyethylene glycol is 200-1000.
The invention takes single and small amount of cosolvent as main material to improve coenzyme Q 10 Solubility in water, which is capable of dissolving coenzyme Q 10 Reducing the influence on the components of the cardioplegia.
Preferably, the concentration of the dissolving agent in the cardioplegia solution is 1.1 mg/mL-10 mg/mL.
Preferably, the coenzyme Q 10 The concentration of (2) is 15-30 mg/L.
Furthermore, the composition also contains thiourea to further improve coenzyme Q 10 Stability in the composition.
Preferably, the concentration of the thiourea is 0.06 mg/mL-0.54 mg/mL.
Further, the cardioplegia is an intracellular fluid type cardioplegia, such as: HTK asystole, and the like. Q prepared by dissolving agent 10 The solution can be well dissolved in HTK stopping liquid, and aggregation and precipitation do not occur.
The invention adopts the non-ionic reducing agent thiourea to maintain the coenzyme Q on the premise of not affecting the cardiac arrest effect of the arrest liquid 10 Is stable. Said coenzyme Q 10 Thiourea can be further stabilized in HTK liquid, and oxidation deterioration does not occur.
The invention also provides application of the composition in the preservation of isolated donor hearts in heart transplantation.
The invention also provides application of the composition in preparing a medicine for preparing cardioplegia which is infused when the extracorporeal circulation is established. Containing coenzyme Q 10 The heart arrest liquid has a protective effect on myocardial cells after ischemia reperfusion.
The mechanism of the invention is as follows: polyoxyethylene surfactant is used as solubilizer, and coenzyme Q is improved through micelle solubilization 10 Solubility in aqueous solutions, wherein the hydrophilic structure of the polyoxyethylene surfactant is polymerized from ethylene glycol; hydroxyl groups of polyhydroxy polymers such as glycerin, polyethylene glycol, and the like are capable of forming hydrogen bonds with polyoxyethylene groups (formula 1). The polyhydroxy polymer stabilizes coenzyme Q through hydrogen bonding 10 Micelle structure, so that coenzyme Q can be reduced in amount by using less surfactant 10 Stably solubilizes in micelles.
The coenzyme Q of the invention is 10 Reaction mechanism of the solution:
。
in formula 1, the polyhydroxy polymer (glycerol for example) forms intermolecular hydrogen bonds with polyoxyethylene-based surfactants such as polyoxyethylene hydrogenated castor oil (Cremophor RH 40), pluronic F68, etc., containing polyoxyethylene groups. Hydrophilic structures of such surfactants are within the dashed box.
The invention takes single solubilizer with small dosage as main material to improve coenzyme Q 10 Solubility in water, solubility in coenzyme Q 10 Reducing the influence on the components of the cardioplegia. Considering the ion type and content in asystole and the safety requirement of asystole heart protection, the invention selects the polyoxyethylene type nonionic surfactant with better safety as the solubilizer to reduce the ion pair coenzyme Q 10 The effect of solubilising formulation stability; further, a water-soluble polyhydroxy compound is added as a cosolvent, and the hydrogen bonding effect (formula 1) between the polyhydroxy compound and hydrophilic groups of the polyoxyethylene surfactant is utilized, so that the consumption of the surfactant is reduced, and meanwhile, the coenzyme Q is ensured 10 Can be solubilized in cardioplegia for a long time.
The invention has the advantages that: solves the problem of coenzyme Q 10 Solubility in cardioplegic solution, stability and efficacy of its action. Wherein,
(1) The invention solves the problems of coenzyme Q 10 Solubility problem in cardioplegia:
by preparing coenzyme Q with high drug loading 10 A solution in a semi-solid form at low temperature; the coenzyme Q is treated 10 Heating the solution in water bath, recovering to obtain clear solution, adding into cardioprotective solution or cardioplegic solution according to required concentration, and adding coenzyme Q 10 Self-assembling with surfactant to form coenzyme Q with internal package 10 Micelle-like self-assembled structure of (2) to thereby give coenzyme Q 10 Can be stably dispersed in cardioprotective solution. This coenzyme Q 10 The solution of (2) solves the problems that coenzyme Q10 is insoluble in water and is easy to oxidize;
(2) The invention improves the coenzyme Q 10 Compatibility in cardioplegic fluid:
the invention uses coenzyme Q 10 And/or the analogues thereof and the surface active substances are self-assembled to form micelle-like structure particles, so that the loading process of the medicine is always carried out in the micelle-like structure with nanometer scale, and therefore, only a small amount of surface active substances are neededCoenzyme Q in micelle-like structure can be greatly increased 10 Is a load of (a) in the vehicle. The dosage of auxiliary materials in the solution is reduced, so that the compatibility of the solution with cardioprotective solution or cardioplegic solution is improved when the solution is used for myocardial protection;
(3) The invention improves the drug carrying stability:
coenzyme Q Using the invention 10 Upon loading, the micelle-like structure dispersed in water has a limiting effect on particle growth, so that coenzyme Q dissolved in the cosolvent 10 It has not been possible to aggregate large particles, i.e., to effectively load the particles into micelle-like structures in the form of molecules or crystallites. The microcrystalline medicine not only greatly improves the medicine carrying quantity, but also further improves the medicine carrying stability, and can avoid coenzyme Q 10 Precipitation in cardioprotective or cardioplegic fluids. In addition, the present invention provides the above coenzyme Q 10 The solution avoids the use of large amount of grease and lipid components, and has good stability and safety. And coenzyme Q 10 The solution preparation process is simple and easy to expand production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the coenzyme Q provided in example 1 10 Dissolving agents (A and B, respectively, formula 1 and formula 2 in example 1) and diluted with cardioplegic solution (C, 1,2,3,7 respectively correspond to the number of each formula in example 1);
FIG. 2 shows coenzyme Q prepared by several sets of formulations provided in example 1 10 Particle size distribution of particles in the solution obtained after the dissolving agent is diluted by cardioplegic solution; a to C are samples prepared in formulation examples 1,2 and 7 in example 1, respectively;
In FIG. 3, A-E are flow assays of human induced pluripotent stem cell-derived cardiomyocytes; wherein, A selects living cell group P1 according to forward scattered light and side scattered light, and removes dead cells; b, selecting a cell group P2 positioned on a diagonal line according to the forward scattered light height and the area, and removing a viscous cell group; c circling a control cell population unlabeled with cardiac troponin T antibody out of the door; D. e, determining a cardiac troponin T positive cardiac myocyte group P3 according to the middle gate of C, wherein the cardiac troponin T positive cardiac myocytes obtained by flow cytometry detection account for 85.9%; f is the action potential of the cardiomyocytes derived from the induced pluripotent stem cells;
in FIG. 4, A is the Western blot detection result of apoptosis signal pathway proteins (Caspase-3 and cleaned Caspase-3) after culturing cardiomyocytes in HTK asystole containing 10mg/mL, 3.3mg/mL and 1.1mg/mL of lytic agent, respectively; b is a histogram of activation level of apoptosis channel detected by Western blot; c is TUNEL staining pattern of myocardial cells, wherein C1-C4, C5-C8, C9-C12 and C13-C16 are respectively TUNEL staining pattern, DAPI (cell nucleus), TUNEL (apoptotic cells), actinin (cytoskeleton) and Merge (superposition pattern of DAPI, TUNEL and Actinin staining) of myocardial cells after culturing in HTK asystole containing 10mg/mL, 3.3mg/mL and 1.1mg/mL of dissolving agent, and the scale is 50 mu m; d is a histogram of TUNEL staining for detecting the rate of myocardial apoptosis;
In FIG. 5, A is the Western blot detection result of apoptosis signal pathway proteins (Caspase-3 and cleaned Caspase-3) of cardiomyocytes cultured in HTK asystole containing 0.54mg/mL, 0.18mg/mL and 0.06mg/mL thiourea; b is a histogram of activation level of apoptosis channel detected by Western blot; c is TUNEL staining pattern of myocardial cells, wherein C1-C4, C5-C8, C9-C12 and C13-C16 are respectively TUNEL staining pattern, DAPI (cell nucleus), TUNEL (apoptotic cells), actinin (cytoskeleton) and Merge (superposition pattern of DAPI, TUNEL and Actinin staining) of myocardial cells after being cultured in HTK asystole containing 0.54mg/mL, 0.18mg/mL and 0.06mg/mL thiourea, and the scale is 50 mu m; d is a histogram of TUNEL staining for detecting the rate of myocardial apoptosis;
fig. 6: A. b, C (V),D is coenzyme Q in HTK asystole containing 0.54mg/mL, 0.18mg/mL, 0.06mg/mL thiourea and no thiourea respectively 10 A time-dependent content profile of (c); E. f, G is the time-dependent change curve of the contents of alpha-ketoglutarate, tryptophan and histidine in HTK asystole containing 0.54mg/mL thiourea;
fig. 7: A. b, C, D the myocardial cells are perfused with HTK asystole (A), HTK asystole (B) containing 0.54mg/mL thiourea, and HTK asystole containing 60mg/L coenzyme Q 10 An action potential after HTK asystole (C) and HTK asystole (D) containing 2.5mg/mL sodium bisulphite, wherein an arrow indicates to the lower left to represent myocardial cells to start stopping electrophysiological activity, and an arrow indicates to the lower right to represent myocardial cells to restart action potential;
fig. 8: a is cardiomyocyte and contains 15mg/L, 30mg/L, 45mg/L and 60mg/L coenzyme Q in HTK asystole 10 Western blot detection results of apoptosis signal pathway proteins (Caspase-3 and clean Caspase-3) in the HTK asystole composition after ischemia reperfusion process; b is a histogram of activation level of apoptosis channel detected by Western blot; TUNEL staining pattern of cardiomyocytes with C1-C4, C5-C8, C9-C12, C13-C16, and C17-C20 respectively, containing 15mg/L, 30mg/L, 45mg/L and 60mg/L coenzyme Q in HTK asystole 10 TUNEL staining patterns after ischemia reperfusion process in HTK asystole, DAPI (cell nucleus), TUNEL (apoptotic cells), actinin (cell skeleton) and Merge (superimposed patterns of DAPI, TUNEL and Actinin staining), with a scale of 50 μm; d is a histogram of TUNEL staining for detecting the rate of myocardial apoptosis; *P<0.05, vs HTK；
Fig. 9: a is cardiomyocyte and contains 15mg/L, 30mg/L, 45mg/L and 60mg/L coenzyme Q in HTK asystole 10 Western blot detection results of apoptosis signal pathway proteins (Caspase-3 and clean Caspase-3) in the HTK asystole composition after ischemia reperfusion process; b is a histogram of activation level of apoptosis channel detected by Western blot; TUNEL staining pattern of cardiomyocytes with C1-C4, C5-C8, C9-C12, C13-C16, and C17-C20, respectively, of cardiomyocytes in HTK asystole at 15mg/L, 30mg/L, 45mg/L, and 60mgL coenzyme Q 10 TUNEL staining patterns after ischemia reperfusion process in HTK asystole, DAPI (cell nucleus), TUNEL (apoptotic cells), actinin (cell skeleton) and Merge (superimposed patterns of DAPI, TUNEL and Actinin staining), with a scale of 50 μm; d is a histogram of TUNEL staining for detecting the rate of myocardial apoptosis; *P<0.05, vs HTK。
Description of the embodiments
Each of the following examples is intended to illustrate and not limit the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).
The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.
Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated. Molecular weight is referred to, allowing its meaning to include fluctuations of + -10%. In the present invention, the term "about" is used as a modifier for an amount, and is intended to encompass +or-5% of the amount modified.
In the present invention, the descriptions of "plural", and the like are referred to, and the number of the terms "plural", and the like is not particularly limited, and is 2 or more.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.
The invention relates to a coenzyme Q 10 The solution contains 33-60 parts by weight of polyoxyethylene nonionic surfactant and 40-67 parts by weight of water-soluble polyhydroxy compound.
Adding the solution to water or an aqueous solution, and passing the solution through coenzyme Q 10 Self-assembling with surface active substances to form micelle-like structure particles, so that the drug loading process is always carried out in a micelle-like structure with nanometer scale, and therefore, coenzyme Q in the micelle-like structure can be greatly increased by only a small amount of surface active substances 10 Is a load of (a) in the vehicle. Is beneficial to reducing the dosage of auxiliary materials in the solution so as to improve the compatibility of the solution with the heart when the solution is used for protecting the cardiac muscle. Meanwhile, coenzyme Q is carried out by adopting the preparation method 10 The micelle-like structure dispersed in water also has a limiting effect on particle growth during loading, so that coenzyme Q dissolved in the lytic reagent 10 It has not been possible to aggregate large particles, i.e., to effectively load the particles into micelle-like structures in the form of molecules or crystallites. The microcrystalline medicine not only greatly improves the medicine carrying quantity, but also further improves the medicine carrying stability, and can avoid coenzyme Q 10 Precipitation in cardioprotective fluids. In addition, the present invention provides the above coenzyme Q 10 The solution avoids the use of large amount of grease and lipid components, and has good stability and safety. And has the advantages of simple process and easy amplification. "coenzyme Q" in the present invention 10 "is understood to include pharmaceutically analogues or derivatives thereof.
The term "micelle-like self-assembled structure", "micelle-like particle", "micelle-like structure", "micelle-like drug" or "self-assembled structure" as used herein refers to a dispersion formed by the spontaneous directional arrangement of a surface active substance, and optionally a binding drug molecule, at an interface in a medium. The surface activity of the surface active substance is derived from the amphiphilic structure of the molecule, the hydrophilic group makes the molecule have a tendency to enter the water phase, the hydrophobic group strives to prevent the dissolution in water and the migration from the inside to the outside of the water, the water phase is escaped, and the balance of the two tendencies is that the surface active agent is enriched at the interface. When the surface adsorption reaches saturation, under the action of hydrophobic groups, the surface active substance molecules are self-polymerized in the solution, namely the hydrophobic groups are together to form an inner core, and hydrophilic groups are outwards contacted with water to form the simplest micelle sample self-assembled structure.
Micelles may form when amphiphilic molecules (including surfactants) encounter selective solvents (i.e., only one of the hydrophilic or hydrophobic segments is soluble, and the other is insoluble). There are a wide variety of selective solvents that can be generally used, including, for example, water. In the present invention, the terms "loaded in micelle-like self-assembled structure" and "self-assembled with a surface active substance to form micelle-like self-assembled structure" are used interchangeably to refer to coenzyme Q 10 Interact with the surface active substances, wherein the drug molecules are wrapped in particles formed by the surface active substances and form a stable drug-carrying micelle-like structure.
The polyoxyethylene nonionic surfactant suitable for the present invention means a surfactant which is mainly composed of oxyethylene (EO) groups combined with a hydrophobic compound containing an active hydrogen atom and is combined into an arbitrary length as required, and which mainly plays a role of solubilization. The content thereof may be 33 to 60 parts, for example 40, 45, 50, 55 parts. Surfactants suitable for use in the present invention include Peregal (Peregal), alkylphenol ethoxylates, fatty acid ethoxylates, polyol surfactants, span and polyethers, and the like. In some preferred embodiments, the polyoxyethylene nonionic surfactant comprises one or more of polyoxyethylene hydrogenated castor oil, polyoxyethylenated 12-hydroxystearic acid, caprylic capric polyethylene glycol glyceride, poloxamer, lauroyl polyoxyethylene-6 glyceride, polyoxyethylene castor oil, polyoxyethylene-8 glyceryl behenate, lauroyl polyoxyethylene-32 glyceride, stearoyl polyoxyethylene glyceride, oleoyl polyoxyethylene glyceride, and linoleoyl polyoxyethylene-6 glyceride.
The water-soluble polyol content may be 40 to 67 parts, for example 45, 55, 60 parts. In some embodiments, the water-soluble polyhydroxy compound comprises one or more of glycerin, polyethylene glycol, polyglycerol, a copolymer of polyethylene glycol and polypropylene glycol, polypropylene glycol.
In some embodiments, the polyethylene glycol has a weight average molecular weight of 200 to 1000, such as 300, 400, 500, 600, 700, 800, 900.
In some embodiments, the solvent has an HLB value of 9 to 29, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29.
In some embodiments, the coenzyme Q 10 The solution was a clear solution. Clear solutions are used to distinguish liquid formulations such as suspensions or emulsions.
The invention also relates to a liquid composition comprising a coenzyme Q as described above 10 A solution and cardioplegia, wherein the concentration of the lytic agent in the cardioplegia is 10mg/mL or less, for example 9mg/mL, 8mg/mL, 7mg/mL, 6mg/mL, 5mg/mL, 4mg/mL, 3mg/mL, 2mg/mL, 1mg/mL; preferably 1.1mg/mL to 10mg/mL.
In some embodiments, the composition further comprises thiourea, preferably at a concentration of 0.06mg/mL to 0.54mg/mL, such as 0.10mg/mL, 0.15mg/mL, 0.20mg/mL, 0.25mg/mL, 0.30mg/mL, 0.35mg/mL, 0.40mg/mL, 0.45mg/mL, 0.50mg/mL.
In some embodiments, the coenzyme Q in the composition 10 The concentration is 15 mg/L to 60mg/L, for example 17 mg/L, 20 mg/L, 23 mg/L, 25mg/L, 27 mg/L, 30 mg/L, 35 mg/L, 40 mg/L, 45 mg/L, 50 mg/L and 55 mg/L.
Cardioplegia (abbreviated as "asystole") is a liquid that perfuses the heart through the coronary ostium or coronary sinus ostium during open-heart surgery, and that chemically induces, causing the heart to rapidly stop beating. Cardioplegia suitable for use in the present invention is preferably an intracellular fluid type cardioplegia, more preferably a low sodium microcalcium cardioplegia, for example having a sodium ion concentration below 15mEq/L and a calcium ion concentration below 0.015mEq/L; more preferably, the asystole comprises tryptophane and ketoglutarate ions, and histidine is used as a buffer solution. In some embodiments, the cardioplegia is HTK cardioplegia (the histoptine-trytophan-ke-toglutarate solution; custodiol, also known as Kang Site protective fluid).
The liquid composition of the present invention may contain other components as required. In some cases, it may be beneficial for the liquid composition to contain thrombolytic agents, such as tissue plasminogen activator, as well as other free radical scavengers or drugs that prevent free radical generation. Examples of free radical scavengers are superoxide dismutase, a protein that perfuses after an ischemic event; or substances having a smaller efficacy such as catalase, acetylcysteine, vitamin E, glutathione and selenium.
According to a further aspect of the invention, it also relates to the use of a composition as described above for ex vivo donor heart preservation in heart transplantation.
Preferably, the temperature of storage is from about 0deg.C to about 35deg.C, preferably from about 0deg.C to about 16deg.C, more preferably from about 0deg.C to about 4deg.C. In general, the time for an isolated organ to withstand ischemia at low temperatures can be prolonged by a factor of 10 compared to normal temperature.
According to a further aspect of the invention, it also relates to the use of a composition as described above for the preparation of a medicament for cardioplegia perfused when establishing extracorporeal circulation.
According to a further aspect of the present invention, it also relates to a method of inducing temporary paralysis of the heart at cardiac surgery/a method of establishing extracorporeal circulation comprising the step of administering an effective amount of a liquid composition as described above to perfuse the heart of a patient.
In the present invention, the term "effective amount" or "therapeutically effective amount" of a drug or pharmaceutical active ingredient is used to refer to an amount of the drug or active agent that is non-toxic but sufficient to provide the desired therapeutic effect. The "effective" amount will vary from individual to individual, depending on the age and general condition of the individual, the particular active agent, and the like. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.
In some embodiments, wherein the liquid composition is administered only once during surgery.
In some embodiments, wherein the liquid composition is administered more than once during surgery.
In some embodiments, the liquid composition is infused into the root of a blocked aorta (cross-clamped aorta), and/or directly into the coronary sinus (coronary sinus). A balloon catheter (balloon catheter) may be used as the fluid composition catheter for introducing the coronary sinus through the right atrium and infusing the fluid composition into the coronary vascular circulation (coronary circulation) through the venous circulation (venous circulation). The method has the advantages of being more uniformly distributed when used for patients with diffuse coronary artery disease (diffuse coronary artery disease), and delivery of the method is independent of an arterial valve (aortic valve) with perfect functions.
In some embodiments, a method of using the liquid composition of the invention in cardiac surgery to induce cardioplegia comprises perfusing a patient's heart with a liquid composition of the invention at about 4 ℃ to about 35 ℃, more preferably at about 10 ℃ to about 21 ℃, and most preferably at about 13 ℃. It is known to those skilled in the art that the use of the present invention to induce cardioplegia in a patient to preserve cardiac function involves perfusing the heart with the liquid composition at moderate low temperature (hypothermia). The administration process of the present invention further comprises perfusing the heart with preferably about 20 to about 30mL/kg of the liquid composition. In some embodiments, comprising perfusing a patient's heart with a liquid composition of the invention at moderate to low temperature every 20 to 40 minutes, cardioplegia is induced to preserve cardiac function. The methods of use of the present invention may comprise perfusing the heart with the liquid composition of the present invention for a cycle of about 20 to 40 minutes, at moderate low temperatures for at least about 24 cycles. The methods of use of the present invention comprise the sustained administration of a liquid composition.
Myocardial ischemia reperfusion injury after extracorporeal circulation of a heart operation patient can seriously damage myocardial tissue activity and influence the postoperative cardiac function of the patient. Our previous studies have shown that additional coenzyme Q supplementation in cardioplegia 10 Can enhance myocardial protection effect in extracorporeal circulation operation and relieve postoperative ischemia reperfusion injury.
Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
Example 1
Coenzyme Q 10 Preparation of the solution:
formula 1. Weighing 400mg of polyoxyethylene hydrogenated castor oil (Cremophor RH 40), heating 600mg of glycerol to 40deg.C, adding coenzyme Q 10 20mg, and uniformly mixing by vortex oscillation to obtain a clear solution with an HLB value of 14-16.
Formula 2. Weighing 600mg polyoxyethylenated 12-hydroxystearic acid (Kolliphor HS 15), 400mg polyethylene glycol 400, heating to 40deg.C, adding coenzyme Q 10 40mg, vortex shaking and mixing uniformly to obtain clear solution with HLB value of 16.
Formulation 3. Weighing 200mg Labrafil M1944 (Oleoyl Polyoxy-6 glycerides), 400mg glycerol, adding coenzyme Q 10 15mg, heated in a 50℃water bath for about 5min, and mixed well to give a clear solution with HLB value of 9.
Formula 4. Weighing 400mg Labrafil M2130 (Lauroyl polyoxyethylene-6 glycerides), 600mg glycerol, and heating to 50deg.C; addition of coenzyme Q 10 15mg, and mixed to obtain a clear solution having HLB value of 9.
Formula 5 weighing 400mg of polyoxyethylated castor oil (Cremophor EL), 400mg of glycerol, adding coenzyme Q 10 50mg, heating in 50deg.C water bath for about 5min, and mixing to obtain clear solution with HLB value of 13.5.
Formulation 6. 200mg Labrosol (caprylic capric acid polyethylene glycol glyceride l (Caprylocaproyl Polyoxyl-8 glycerides)) were weighed,400mg of polyethylene glycol 300 and coenzyme Q are added 10 15mg, heated in a 50℃water bath for about 5min, and mixed well to give a clear solution with HLB value of 12.
Formula 7. Weighing 180mg of pluronic F68 and 520mg of polyethylene glycol 300, and heating and melting; addition of coenzyme Q 10 20mg, and mixed to obtain a translucent clear solution having an HLB value of 29.
The main property characterization of formulations 1 to 7 is shown in table 1:
。
as shown in A and B of FIG. 1, coenzyme Q prepared in formulas 1 and 2 10 The solution was a yellow transparent viscous solution. After diluting this solution to about 60. Mu.g/mL with cardioplegia, a clear pale yellow solution was obtained (formulas 1 and 2 in FIG. 1C). In addition, coenzyme Q prepared in formula 3 10 The solution obtained after dilution was also clear pale yellow. Whereas diluted coenzyme Q prepared in formulation 7 10 The solution was translucent in appearance.
Example 2
Containing coenzyme Q 10 Particle size measurement of cardioplegic solution
The coenzyme Q of formulations 1, 2 and 7 of example 1 was taken 10 The solution was diluted to 60ug/mL with cardioplegic solution. The mean particle size and particle size distribution were determined by dynamic light scattering (Zetasizer Nano ZS, markov UK). The laser wavelength was 633nm, the temperature was 25℃and the equilibration time 120sec. The determination is made in terms of viscosity and refractive index of water. Results are shown in Table 2 coenzyme Q 10 The results of the particle size measurement of the solution in cardioplegia are shown in table 2:
。
The volume average particle size distribution of the three samples is shown in fig. 2. FIGS. 2A-C are samples prepared according to formulation 1, formulation 2 and formulation 7, respectively, of example 1.
The particle size measurement result shows that during the dilution process of the asystole, the coenzyme Q 10 Self-assembling with polyoxyethylene surfactant to form micelle, and adding coenzyme Q 10 Effectively solubilizes in the micelle, and avoids precipitation of the medicine. The micelle size distribution is relatively uniform. The Z-average particle diameter of formulation 7 was 26 times or more larger than that of the other formulations, which was also the reason why the clarity of formulation 7 was not high.
Example 3
The lytic reagent in this example is coenzyme Q 10 Comprises a nonionic surfactant and a water-soluble polyhydroxy compound
1. Acquisition of cardiomyocytes
Taking human induced pluripotent stem cells for in vitro culture and inducing and differentiating the human induced pluripotent stem cells into myocardial cells. The differentiation efficiency was examined by flow cytometry, and the electrophysiological activity was examined by cell patch clamp technique to verify whether it differentiated into cardiomyocytes.
In fig. 3, fig. 3A, a population of living cells P1 is selected based on forward scattered light and side scattered light, and dead cells are removed; FIG. 3B selects the diagonally positioned cell population P2 based on forward scattered light height and area to remove the viscous cell population; FIG. 3C circles a control cell population unlabeled cardiac troponin T antibody out of the door; FIGS. 3D and E determine a cardiac troponin T-positive cardiomyocyte population P3 from C, with 85.9% of the cardiac troponin T-positive cardiomyocytes detected by flow cytometry; FIG. 3F shows that the action potential of human induced pluripotent stem cells-derived cardiomyocytes can always maintain a strong and stable spontaneous action potential.
As shown in fig. 3A-E for flow assays of human induced pluripotent stem cell-derived cardiomyocytes: the positive rate of troponin T, a cardiomyocyte-specific marker, was 81.28% ± 2.41% (n=5), indicating that a large number of high purity cardiomyocytes could be obtained in this protocol. The results in fig. 3F show that cardiomyocytes were able to maintain a strong and stable spontaneous action potential at all times, demonstrating that cardiomyocytes obtained by this protocol can function normally.
2. Assessment and preference of the myocardial cytotoxicity of lytic agents
400mg of polyoxyethylene hydrogenated castor oil (Cremophor RH 40) was weighed, 600mg of glycerin was heated to 40℃and mixed well by vortexing to obtain a clear mixed solution. And adding 1000 mg,330 mg,110 mg mixed solution into 100 mL HTK asystole to prepare three concentrations of HTK asystole containing a dissolving agent of 10mg/mL, 3.3 mg/mL and 1.1 mg/mL respectively, culturing myocardial cells in asystole containing the dissolving agent of different concentrations for 30 minutes, detecting the activation level of an apoptosis pathway of the myocardial cells through Western blot and detecting the apoptosis rate of the myocardial cells through TUNEL staining, and evaluating the toxicity of the dissolving agent of different concentrations to the myocardial cells so as to explore the safe concentration range of the dissolving agent.
In FIG. 4, FIG. 4A shows the Western blot detection results of apoptosis signal pathway proteins (Caspase-3 and clear Caspase-3) cultured in HTK asystole containing 10mg/mL, 3.3mg/mL and 1.1mg/mL of lytic agent in HTK asystole, and FIG. 4B shows a histogram of Western blot detection of activation levels of apoptosis pathway. The results showed that there was no statistical difference in myocardial apoptosis levels after the addition of three concentrations of lytic agent at asystole of 10mg/mL, 3.3. 3.3mg/mL and 1.1mg/mL compared to the negative control group (HTK asystole without any additional ingredients). FIG. 4C is a TUNEL staining pattern of cardiomyocytes in asystole with different concentrations of lytic agent, wherein C1-C4, C5-C8, C9-C12, C13-C16 are respectively TUNEL staining patterns of cardiomyocytes after incubation in HTK asystole with 10mg/mL, 3.3mg/mL and 1.1mg/mL lytic agent, DAPI (nucleus), TUNEL (apoptotic cells), actinin (cytoskeleton) in a scale of 50 μm; fig. 4D is a histogram of TUNEL staining for detection of myocardial apoptosis rate. The results showed that after three concentrations of dissolution agent, 10mg/mL, 3.3. 3.3mg/mL and 1.1. 1.1mg/mL, were added to the asystole, the rate of myocardial apoptosis was very low, and there was no statistical difference between the groups, as compared to the negative control group.
From the results of fig. 4, it can be seen that: the concentration of the dissolving agent below 10mg/mL has no obvious effect on the myocardial apoptosis level, i.e. the concentration of the dissolving agent below 10mg/mL has no myocardial cytotoxicity.
3. Assessment and preference of reducing agent for myocardial cytotoxicity
Respectively weighing 54mg, 18mg and 6mg of thiourea, uniformly dissolving in 100mL of HTK stopping liquid, preparing three concentrations of stopping liquid containing reducing agent thiourea, namely 0.54mg/mL, 0.18mg/mL and 0.06mg/mL, culturing myocardial cells in stopping liquid containing reducing agents with different concentrations for 30 minutes, detecting the activation level of myocardial apoptosis channels through Western blot and detecting the myocardial apoptosis rate through TUNEL staining, and evaluating the toxicity of the reducing agents with different concentrations on the myocardial cells so as to explore the safe concentration range of the reducing agents.
In FIG. 5, FIG. 5A shows the Western blot detection results of apoptosis signal pathway proteins (Caspase-3 and cleaned Caspase-3) after culturing cardiomyocytes in HTK asystole, asystole containing 0.54mg/mL, 0.18mg/mL and 0.06mg/mL of thiourea as reducing agents at different concentrations, and FIG. 5B shows a histogram of Western blot detection of activation levels of apoptosis pathway. The results showed that there was no statistical difference in myocardial apoptosis levels after the addition of three concentrations of reducing agent, 0.54mg/mL, 0.18mg/mL, and 0.06mg/mL, to the asystole compared to the negative control group (HTK asystole without any additional ingredients). FIG. 5C is a TUNEL staining pattern of cardiomyocytes in asystole with varying concentrations of reducing agent, wherein C1-C4, C5-C8, C9-C12, C13-C16 are respectively TUNEL staining patterns of cardiomyocytes after incubation in HTK asystole with 0.54mg/mL, 0.18mg/mL and 0.06mg/mL thiourea, DAPI (nuclei), TUNEL (apoptotic cells), actinin (cytoskeleton) at a scale of 50 μm; fig. 5D is a histogram of TUNEL staining for detection of myocardial apoptosis rate. The results show that after three concentrations of reducing agents of 0.54mg/mL, 0.18mg/mL and 0.06mg/mL are added to the asystole, the myocardial apoptosis rate is very low compared with the negative control group, and no statistical difference exists between the groups.
From the results of fig. 5, it can be seen that: the thiourea concentration is below 0.54mg/mL, and has no obvious effect on the myocardial apoptosis rate, namely the thiourea concentration is below 0.54mg/mL, and has no myocardial cytotoxicity.
Example 4
Coenzyme Q 10 Solution stability assessment
400mg of polyoxyethylene hydrogenated castor oil (Cremophor RH 40) were weighed according to formula 1 of example 1, 600mg of glycerol was heated to 40℃and coenzyme Q was added 10 20mg, vortexMixing uniformly by rotary oscillation to obtain clear coenzyme Q 10 A solution; weighing 306mg of coenzyme Q 10 Adding the solution into 100ml HTK asystole, stirring to mix uniformly to prepare 60 mg/L coenzyme Q-containing solution 10 Asystole of (a);
then, 54mg, 18mg and 6mg of thiourea were weighed and dissolved in 100mL of the above coenzyme Q-containing solution, respectively 10 Preparing three types of novel asystole with different concentrations of thiourea of 0.54mg/mL, 0.18mg/mL and 0.06 mg/mL;
finally, the prepared solution is placed at 4 ℃ to be protected from light and stored in a sealed manner. After standing for 0, 12, 15, 18, 21, 24, 27 and 30 days, detecting coenzyme Q in three novel asystole solutions containing thiourea with different concentrations and novel asystole solution without thiourea respectively by high performance liquid chromatography 10 Simultaneously, the contents of alpha-ketoglutaric acid, tryptophan and histidine are respectively detected by a colorimetric ketoglutaric acid quantitative kit and an ultra-high performance liquid chromatography tandem mass spectrometry respectively after standing for 0,3,6,9, 12, 15, 18, 21, 24, 27 and 30 days, and the contents of the dissolving agent and the coenzyme Q are analyzed 10 Whether the addition of the reducing agent has an influence on the stability of the HTK asystole or not and evaluate the coenzyme Q by using thiourea with different concentrations as the reducing agent 10 Protection effect of stability.
In fig. 6: FIG. 6A, B, C, D shows coenzyme Q in novel asystole solutions containing 0.54mg/mL, 0.18mg/mL, 0.06mg/mL and no thiourea, respectively 10 A time-dependent content profile of (c); FIG. 6 is a graph E, F, G showing the time course of the content of alpha-ketoglutarate, tryptophan and histidine in a novel asystole containing 0.54mg/mL thiourea.
FIG. 6A, B, C, D shows the results of coenzyme Q in three novel asystole solutions of different thiourea concentrations 10 No obvious decrease in concentration within 30 days, but no thiourea in the novel asystole 10 The concentration was significantly decreased within 30 days, indicating coenzyme Q 10 The stability is poor without the protection of the reducing agent, and 0.54mg/mL, 0.18mg/mL and 0.06mg/mL of thiourea can play a good role in coenzyme Q 10 Protecting effect. Thus ensuring coenzyme Q 10 While at the same time reducing the stabilityThe optimal concentration of thiourea in the asystole is 0.06mg/L.
FIG. 6E, F, G shows no significant decrease in the levels of alpha-ketoglutarate, tryptophan and histidine within 30 days, indicating that the novel asystole solubilizes and coenzyme Q 10 And the addition of the reducing agent can not disturb the main components of the HTK asystole, and the solution stability is high.
Example 5
Containing coenzyme Q 10 Is used for assessing the asystole effect of the cardioplegic liquid composition
The 54-mg thiourea is weighed and evenly dissolved in 100mL of HTK asystole, and 0.54-mg/mL of HTK asystole containing thiourea is prepared.
400mg of polyoxyethylene hydrogenated castor oil (Cremophor RH 40) were weighed according to formula 1 of example 1, 600mg of glycerol was heated to 40℃and coenzyme Q was added 10 20mg, vortex oscillation and uniform mixing to obtain clear coenzyme Q 10 The solution was then weighed 306mg of coenzyme Q 10 Adding the solution into 100ml HTK asystole, stirring to mix uniformly to prepare 60mg/L coenzyme Q-containing solution 10 HTK asystole compositions of (a).
Weighing 250 mg sodium bisulphite, uniformly dissolving in 100mL of HTK asystole, and preparing 2.5 mg/mL of HTK asystole containing sodium bisulphite.
The HTK asystole containing thiourea and the coenzyme Q are mixed 10 The HTK asystole composition, the HTK asystole containing sodium bisulphite and the HTK asystole without any other components are respectively perfused into myocardial cells, the action potential of the myocardial cells is detected by adopting a cell patch clamp technology, and the difference of the asystole effects among 4 groups is compared.
FIG. 7 shows that FIG. 7A, B, C, D shows cardiomyocytes perfusing HTK asystole with 0.54. 0.54 mg/mL thiourea, with 60mg/L coenzyme Q 10 HTK asystole of (c) and HTK asystole containing 2.5mg/mL sodium bisulfite, red arrows indicate the onset of stopping electrophysiological activity by cardiomyocytes, and blue arrows indicate the restart of action potential by cardiomyocytes.
FIG. 7 shows the results of the almost simultaneous cessation of action potential by cardiomyocytes after perfusion with 4 groups of asystole, a graphGroup 7A, B, C restored the action potential within 11 minutes, while group 7D did not yet restore the action potential within 45 minutes. Description of thiourea, coenzyme Q 10 The addition of the dissolving agent has no obvious influence on the stopping effect of the stopping liquid, but the rebound time of the myocardial cells is obviously prolonged after the sodium bisulphite is added.
Example 6
Containing coenzyme Q 10 Is used for evaluating myocardial protection effect of cardioplegic liquid composition
1. 400mg of polyoxyethylene hydrogenated castor oil (Cremophor RH 40) were weighed according to formula 1 of example 1, 600mg of glycerol was heated to 40℃and coenzyme Q was added 10 20mg, vortex oscillation and uniform mixing to obtain clear coenzyme Q 10 A solution; then, 76.5mg, 153mg, 229.5mg and 306mg of the obtained coenzyme Q were weighed respectively 10 Adding the solution into 100ml HTK asystole, stirring to mix uniformly, and respectively preparing into four different concentrations of coenzyme Q containing 15mg/L, 30mg/L, 45mg/L and 60mg/L 10 Asystole composition of (a).
Firstly, the myocardial cells are placed in a low-oxygen environment and contain coenzyme Q with different concentrations 10 The HTK asystole composition of (2) and HTK asystole without any other components added were cultured for 45min to simulate myocardial ischemia process, and then the cardiomyocytes were placed in a high-oxygen environment and cultured in a common cell culture medium for 3 h to simulate myocardial reperfusion process. The activation level of myocardial apoptosis pathway is detected by Western blot, and the apoptosis rate of myocardial cells is detected by TUNEL staining, so that the coenzyme Q with different concentrations is estimated 10 Protective action on myocardial cells to explore coenzyme Q 10 Is used in the composition according to the optimum amount.
FIG. 8A shows that cardiomyocytes contained 15mg/L, 30mg/L, 45mg/L and 60mg/L of coenzyme Q in HTK asystole 10 In the HTK asystole, apoptosis signal pathway proteins (Caspase-3 and clean Caspase-3) after ischemia reperfusion process, and FIG. 8B is a histogram of Western blot detection of activation level of apoptosis pathway. FIG. 8C is a TUNEL staining pattern of cardiomyocytes wherein C1-C4, C5-C8, C9-C12, C13-C16, C17-C20 are cardiomyocytes in HTK asystole with 15mg/L, 30mg/L, 45mg/L and 60mg/L coenzyme Q 10 HTK of (c)TUNEL staining patterns, DAPI (cell nucleus), TUNEL (apoptotic cells) and Actinin (cell skeleton) of the asystole after ischemia reperfusion process, with a scale of 50 mu m; fig. 8D is a histogram of TUNEL staining for detection of cardiomyocyte apoptosis rate.
FIGS. 8A-B show that activation of the myocardial apoptosis signaling pathway is at the level of coenzyme Q compared to HTK asystole without any additional components 10 The concentration began to drop at 15mg/L and was significantly lower at 30 mg/L. While when coenzyme Q 10 The level of apoptosis did not change significantly as the concentration continued to increase. FIGS. 8C-D show that the rate of myocardial apoptosis is at coenzyme Q compared to HTK asystole without any additional ingredients 10 The concentration of the coenzyme Q begins to fall at 15mg/L 10 The concentration was significantly decreased at 30 mg/L. While when coenzyme Q 10 The rate of apoptosis of cardiomyocytes did not significantly decrease as the concentration continued to increase.
2. 600mg of polyoxyethylenated 12-hydroxystearic acid (Kolliphor HS 15) were weighed according to formula 2 of example 1, 400mg of polyethylene glycol 400 were heated to 40℃and 40mg of coenzyme Q were added 10 Vortex oscillation and even mixing to obtain clear coenzyme Q 10 Mixing the solutions; then 39 mg, 78mg, 117mg and 156mg of coenzyme Q are weighed respectively 10 Adding the mixed solution into 100ml HTK asystole, stirring to mix uniformly, and respectively preparing into 15mg/L, 30mg/L, 45mg/L and 60mg/L four kinds of coenzyme Q-containing solutions with different concentrations 10 HTK asystole compositions of (a).
Firstly, the myocardial cells are placed in a low-oxygen environment and contain coenzyme Q with different concentrations 10 The HTK asystole composition of (2) and HTK asystole without any other components added were cultured for 45min to simulate myocardial ischemia process, and then the cardiomyocytes were placed in a high-oxygen environment and cultured in a common cell culture medium for 3 h to simulate myocardial reperfusion process. The activation level of myocardial apoptosis pathway is detected by Western blot, and the apoptosis rate of myocardial cells is detected by TUNEL staining, so that the coenzyme Q with different concentrations is estimated 10 Protective action on myocardial cells to explore coenzyme Q 10 Is used in the composition according to the optimum amount.
In FIG. 9, FIG. 9A shows that cardiomyocytes contained 15mg/L, 30mg/L, in HTK asystole,45mg/L and 60mg/L coenzyme Q 10 In the HTK asystole, apoptosis signal pathway proteins (Caspase-3 and clean Caspase-3) after ischemia reperfusion process, and FIG. 9B is a histogram of Western blot detection of activation level of apoptosis pathway. FIG. 9C is a TUNEL staining pattern of cardiomyocytes wherein C1-C4, C5-C8, C9-C12, C13-C16, C17-C20 are cardiomyocytes in HTK asystole with 15mg/L, 30mg/L, 45mg/L and 60mg/L coenzyme Q 10 TUNEL staining patterns, DAPI (cell nucleus), TUNEL (apoptotic cells) and Actinin (cell skeleton) of HTK asystole after ischemia reperfusion process, with a scale of 50 mu m; fig. 9D is a histogram of TUNEL staining for detection of cardiomyocyte apoptosis rate.
FIGS. 9A-B show that the level of activation of the myocardial apoptosis signaling pathway is at coenzyme Q compared to HTK asystole without any additional components 10 The concentration began to drop at 15 mg/L and was significantly lower at 30mg/L. While the level of apoptosis did not change significantly as the coenzyme concentration continued to increase. FIG. 9C-D shows that the rate of myocardial apoptosis is at coenzyme Q compared to HTK asystole without any additional components 10 The concentration of the coenzyme Q begins to fall at 15 mg/L 10 The concentration was significantly decreased at 30mg/L. While when coenzyme Q 10 The rate of apoptosis of cardiomyocytes did not significantly decrease as the concentration continued to increase.
From the results of fig. 8 and 9, it can be seen that: coenzyme Q in HTK asystole 10 Can achieve good myocardial protection effect when the content of the coenzyme Q in the asystole is 30mg/L 10 Is preferably 30mg/L.
The advantages of the invention are further demonstrated by the examples described above:
1. said coenzyme Q 10 Q prepared by dissolving agent 10 The solution can be well dissolved in HTK stopping liquid, and aggregation and precipitation do not occur.
2. Said coenzyme Q 10 Thiourea can be stabilized in HTK liquid without oxidative deterioration.
3. Containing coenzyme Q 10 Is used for treating myocardial cells after ischemia reperfusion Has protective effect.
Through the use of a dissolving agent and a reducing agent, the coenzyme Q is solved 10 Problems of solubility and stability in cardioplegic solutions. Verification of coenzyme Q-containing according to the invention by cell experiments 10 The cardioplegic liquid composition is safe and nontoxic, and can obviously enhance the protective effect of the cardioplegic liquid on myocardial cells under the condition of not affecting the cardioplegic effect of the cardioplegic liquid.
Coenzyme Q to be prepared 10 After the solution is added into heart protecting liquid, the solution is added into heart protecting liquid through coenzyme Q 10 And/or the self-assembly of the analogue and the surface active substance forms micelle-like structure particles, so that the drug loading process is always carried out in a micelle-like structure with nanometer scale, and therefore, coenzyme Q in the micelle-like structure can be greatly increased by only a small amount of the surface active substance 10 Is a load of (a) in the vehicle. Is beneficial to reducing the dosage of auxiliary materials in the solution so as to improve the compatibility of the solution with heart protecting liquid when the solution is used for protecting cardiac muscle. Meanwhile, coenzyme Q is carried out by adopting the preparation method 10 During loading, the micelle-like structure dispersed in water also has a limiting effect on particle growth, so that coenzyme Q 10 It has not been possible to aggregate large particles, i.e., to effectively load the particles into micelle-like structures in the form of molecules or crystallites. The microcrystalline state medicine is favorable for greatly improving the medicine carrying quantity, further improving the medicine carrying stability and avoiding coenzyme Q 10 Precipitation in cardioprotective fluids. Thus, the coenzyme Q 10 The particle size of the coenzyme Q was not detected in the solution 10 Dispersed in a molecular state in a solution, but coenzyme Q is added to 10 After the solution is added to the cardioprotective solution, uniform nanoparticles can be formed.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.
Claims (9)
1. Coenzyme Q-containing 10 The cardioplegic solution composition of (2) is characterized by comprising coenzyme Q 10 The solution and the cardioplegia solution,
said coenzyme Q 10 The solution, the dissolvent of which comprises 33 to 60 parts by weight of polyoxyethylene nonionic surfactant which is acceptable in pharmacy as solubilizer and 40 to 67 parts of water-soluble polyhydroxy compound which is acceptable in pharmacy as cosolvent, coenzyme Q 10 The concentration is 14.8-58.8 mug/mg, the obtained solution is clear solution with HLB value of 9-29, and the solution passes through coenzyme Q 10 Self-assembling with a surface active substance to form micelle-like structure particles, so that the loading process of the drug is always carried out in a micelle-like structure with nano scale; the concentration of the dissolving agent in the cardioplegia is not more than 10mg/mL, and the coenzyme Q 10 The concentration of (2) is 15-60 mg/L;
the polyoxyethylene nonionic surfactant is used as a solubilizer and comprises one or more of polyoxyethylene hydrogenated castor oil, polyoxyethylenated 12-hydroxystearic acid, caprylic/capric acid polyethylene glycol glyceride, poloxamer, lauroyl polyoxyethylene-6 glyceride, polyoxyethylene castor oil, polyoxyethylene-8 glyceryl behenate, lauroyl polyoxyethylene-32 glyceride, stearoyl polyoxyethylene glyceride, oleoyl polyoxyethylene glyceride and linoleoyl polyoxyethylene-6 glyceride;
the water-soluble polyhydroxy compound is taken as a cosolvent and comprises one or more of glycerol, polyethylene glycol, polyglycerol, a copolymer of polyethylene glycol and polypropylene glycol;
the solubilizer and cosolvent in the dissolvent are mixed with coenzyme Q at 40-50 DEG C 10 Uniformly mixing to obtain coenzyme Q 10 Solution, coenzyme Q obtained 10 The solution exists in semisolid form at low temperature, after heating in water bath, it is recovered to clear solution, and added into heart protecting solution, and self-assembled with surfactant to form coenzyme Q 10 Micelle-like self-assembled structure of (a) to make coenzyme Q 10 Stably dispersed in cardioprotective solution.
2. The coenzyme Q-containing composition according to claim 1 10 The cardioplegic solution composition is characterized in that the weight average molecular weight of the polyethylene glycol is 200-1000.
3. The coenzyme Q-containing composition according to claim 1 10 Wherein the concentration of the solubilizer in the cardioplegia solution is 1.1. 1.1 mg/mL-10 mg/mL.
4. The coenzyme Q-containing composition according to claim 1 10 The cardioplegic solution composition of (2) is characterized in that the coenzyme Q 10 The concentration of (C) is 15-30 mg/L.
5. The coenzyme Q-containing composition according to claim 1 10 The cardioplegic solution composition of (2) is characterized by further comprising thiourea with a concentration of 0.06 mg/mL-0.54 mg/mL.
6. The coenzyme Q-containing composition according to claim 1 10 The cardioplegic solution composition is characterized in that the cardioplegic solution is intracellular fluid type cardioplegic solution.
7. The coenzyme Q-containing composition according to claim 6 10 The cardioplegic solution composition of (2) is characterized in that the cardioplegic solution is HTK cardioplegic solution.
8. Use of a composition according to any one of claims 1 to 7 for ex vivo donor heart preservation in heart transplantation.
9. Use of a composition according to any one of claims 1 to 7 for the preparation of a cardioplegic drug for perfusion in establishing extracorporeal circulation.
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