RU2456024C2 - Cardioprotection technique - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely cardiology, and may be used for myocardium protection against ischemic and reperfusion injures. That is ensured by the target drug delivery to the ischemic-reperfused myocardium. Aminated silica nanoparticles of the diameter of 10 nm wherein a spacer is grafter are used as a drug carrier. A drug substance is immobilised on a functional group of the spacer by one of the following methods: covalent binding, coordination ion interaction, immobilisation by adsorption.

EFFECT: technique provides the selective accumulation of the drug substance within ischemia-reperfusion after the systemic introduction of the specified complex with the minimum effect of the preparation on intact organs and tissues and good biodegradability of the carrier; surface functionalisation of said nanoparticles also provides a binding ability of the drug substances of different chemical compositions in various proportions.

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Description

The invention relates to medicine, namely to cardiology, and can be used to protect the myocardium from ischemic and reperfusion injury in patients with coronary heart disease.

The effectiveness of myocardial protection against ischemic and reperfusion injury can be significantly increased by providing targeted delivery of drugs with cardioprotective activity to the ischemic myocardium using nanoscale carriers. The targeted delivery of drugs using such carriers solves several urgent problems. It leads to a decrease in toxicity, an increase in the bioavailability, solubility and stability of the drug (Singh R., Lillard J.W. Jr. Nanoparticle-based targeted drug delivery. Exp Mol Pathol 2009; 86 (3): 215-223). A number of drugs with experimentally proven cardioprotective activity have not been widely used in clinical practice due to the presence of serious side effects that can be attenuated or completely eliminated with targeted delivery. Intravenous administration of angiogenic growth factors such as vascular endothelial growth factor and fibroblast growth factor is associated with the development of vasodilation and arterial hypotension (Epstein S.E., Kornowski R., Fuchs S., Dvorak HF Angiogenesis therapy: amidst the hype, the neglected potential for serious side effects. Circulation 2001; 104 (1): 115-119). Systemic administration of the bradykinin myocardial preconditioning trigger is accompanied by the onset of arterial hypotension and bronchospasm (Cyr M., Eastlund T., Blais C. Jr., Rouleau JL, Adam A. Bradykinin metabolism and hypotensive transfusion reactions. Transfusion 2001; 41 (1): 136- 150).

Among the various nanoparticles, drug carriers are currently the most actively studied liposomes, phospholipid and polymer micelles, nanoemulsions, polymer biodegradable nanoparticles, dendrimers and silica nanoparticles.

There are two methods of targeted delivery - passive and active transfer. Active drug delivery to damaged tissues involves labeling the surface of nanoparticles with antibodies or other recognition elements that provide highly selective binding of nanoparticles to antigens expressed on the surface of damaged cells (McNeeley K.M., Karathanasis E., Annapragada AV, Bellamkonda RV Masking and triggered unmasking of targeting ligands on nanocarriers to improve drug delivery to brain tumors. Biomaterials 2009; 30 (23-24): 3986-3995).

There is a method of directed active delivery of vascular endothelial growth factor packed into liposomes into the damaged myocardium by attaching monoclonal antibodies against P-selectin to the surface of liposomes (Scott R.C., Rosano J.M., Ivanov Z., Wang B., Chong PL , Issekutz A.S., Crabbe DL, Kiani M.F. Targeting VEGF-encapsulated immunoliposomes to MI heart improves vascularity and cardiac function. FASEB J 2009; 23 (10): 3361-3367). A method is described for active targeted delivery of ATP to the myocardium using liposomes having monoclonal antibodies against myosin on their surface (Liang W., Levchenko T., Khaw B.A., Torchilin V. ATP-containing immunoliposomes specific for cardiac myosin. Curr Drug Deliv 2004; 1 (1): 1-7).

The disadvantages of these methods are as follows.

1. These methods of cardioprotection were used only on the model of permanent occlusion of the coronary artery, which does not meet modern standards for the treatment of acute coronary syndrome, providing for the speedy restoration of blood flow from a heart attack-dependent artery. The results cannot be compared with current results of treatment of acute coronary syndrome (Brodie B.R., Webb J., Cox DA, Qureshi M., Kalynych A., Turco M., Schultheiss HP, Dulas D., Rutherford B., Antoniucci D ., Stuckey T., Krucoff M., Gibbons R., Lansky A., Na Y., Mehran R., Stone GW, EMERALD Investigators. Impact of time to treatment on myocardial reperfusion and infarct size with primary percutaneous coronary intervention for acute myocardial infarction (from the EMERALD Trial). Am J Cardiol 2007; 99 (12): 1680-1686).

2. The positive effect is only to improve the contractility of the left ventricular myocardium and to reduce the area of the post-infarction scar, and not directly prevent the death of cardiomyocytes in the ischemic zone (Scott RS, Rosano J.M., Ivanov Z., Wang B., Chong PL, Issekutz A.S., Crabbe DL, Kiani M.F. Targeting VEGF-encapsulated immunoliposomes to MI heart improves vascularity and cardiac function. FASEB J 2009; 23 (10): 3361-3367). This is due to the accumulation of drug nanoparticles in the peri-infarction zone, and not in the ischemic zone.

3. The use of monoclonal antibodies as guide ligands rarely allows you to create reproducible results. This is due to the specificity of antibodies, which differs significantly from batch to batch (Fernandes D. Demonstrating comparability of antibody glycosylation during biomanufacturing. Eur Biopharm Rev 2005; 106-110).

4. The use of monoclonal antibodies as a directing ligand significantly increases the cost of production technology for the described dosage forms.

Passive transfer is now well described in relation to tumors and occurs due to the predominant release of drug nanoparticles into the tumor tissue due to a local increase in the permeability of tumor microvessels (Matsumura Y., Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 1986; 46: 6387-6392). Physical factors such as ultrasound and hyperthermia can be used to further increase the permeability of microvessels in the target tissue.

A known method of producing a nanoscale drug delivery system with the addition of surfactants to create medical and veterinary drugs used as target drug delivery systems and to overcome the blood-brain barrier, characterized in that silica nanoparticles are used as a carrier (patent application 2007136310). However, this system does not allow to achieve the desired cardioprotective effect, since the drug substance is adsorbed directly on the carrier, which can lead to undesirable side effects (Epstein S.E., Kornowski R., Fuchs S., Dvorak HF Angiogenesis therapy: amidst the hype, the neglected potential for serious side effects. Circulation 2001; 104 (1): 115-119)

There is a method of targeted drug delivery to the myocardium, which we selected as a prototype, based on the use of adenosine-loaded liposomes with a diameter of 134 nm in a rat myocardial ischemia-reperfusion model (Takahama H., Minamino T., Asanuma H. et al. Prolonged targeting of ischemic / reperfused myocardium by liposomal adenosine augments cardioprotection in rats. J Am Coll Cardiol 2009; 53 (8): 709-717). A suspension of adenosine-loaded liposomes in physiological saline was administered to animals once intravenously in doses (in terms of adenosine) of 50, 150 and 450 μg / kg × min for 10 minutes, starting from the 25th minute of ischemia. Thus, the infusion continued during the last 5 minutes of ischemia and the first 5 minutes of myocardial reperfusion. The use of this method was accompanied by less pronounced side hemodynamic effects and a more pronounced infarction-limiting effect than the use of an equivalent dose of free adenosine.

This method has several disadvantages.

1. Rapid elimination of carrier nanoparticles from the bloodstream by elements of the reticuloendothelial system, which is associated with their diameter exceeding 100 nm (Wang AZ, Gu F., Zhang L. et al. Biofunctionalized targeted nanoparticles for therapeutic applications. Expert Opin Biol Ther. 2008; 8 (8): 1063-1070).

2. The lack of simultaneous targeted delivery of drugs of various chemical structures.

3. The high cost of carrier material.

4. Low cardioprotective effect due to the low release of the drug from the material of the carrier.

5. The inability to control the process of release of the drug from the material of the carrier by varying the method of binding.

The aim of the invention is to increase the efficiency of the method.

This is achieved by the fact that nanoparticles of aminated silica with a diameter of 10 nm are taken as a carrier, to which insert molecules (spacers) are grafted. The drug is immobilized on the spacer. A molecule of the drug is immobilized for each functional group of the spacer. Medicines can have a different chemical structure. The rate of release of the drug from the surface layer of the carrier depends on the method of immobilization. Three options for immobilization of the drug are used: covalent binding, coordination-ion interaction, adsorption immobilization. The complex "drug carrier" is administered intravenously immediately before reperfusion.

The positive effect of the introduction of the proposed method is to increase the cardioprotective effect and reduce side effects of the complex "drug-carrier" on intact organs and tissues. The method allows you to control the rate of release of the drug from the material of the carrier, as well as combine the use of drugs with a different mechanism of action. In clinical practice, the method can be used in patients with unstable angina, as well as in patients with acute coronary syndrome in addition to conducting percutaneous coronary intervention.

The method is as follows.

To achieve the result, the silica surface is modified by amino groups according to the method of chemisorption of 3-aminopropyltriethoxysilane from the gas phase in a flow reactor. The design of the reactor provides evaporation of the reagent at a temperature of 220 ° C, chemisorption, removal of excess reagent and by-products of the reaction. In order to avoid entrainment of silica by the carrier gas stream, the reaction zone of the upper part of the reactor is limited by a porous filter.

The synthesis procedure involves removing physically bound water from the surface of silica at a temperature of 220 ° C for 2 hours, chemisorption of 3-aminopropyltriethoxysilane at a temperature of 220 ° C for 3 hours, removing excess reagents and by-products of the reaction at the same temperature for 2 hours.

The hydrolysis of alkoxy groups that did not react with the silica surface is carried out by treating the sample with water vapor for an hour at a temperature of 200 ° C.

The synthesis of the spacer is as follows. A sample of 100 mg of aminated silica is treated with a solution of symmetric 3- (vosmino) octanoic anhydride in methylene chloride.

Symmetric anhydride is obtained by the carbodiimide method according to the following procedure. Mixed in an equimolar ratio of dicyclohexylcarbodiimide and 3- (octamino) octanoic acid, the solution is left for 10 minutes, then the precipitate of dicyclohexylurea is filtered off. In solution, the symmetric anhydride of 3- (octamino) octanoic acid is obtained.

In the synthesis, a 10-fold excess of 3- (vosamino) octanoic acid anhydride is used in relation to the available amino groups. Next, the grafted amino acid is released for 1 hour using formic acid. Deprotonation is carried out using a 10% solution of triethylamine in methylene chloride. At the final stage, the sample is dried under vacuum at a temperature of 60 ° C.

Then, the drug is immobilized using one of three methods: covalent binding, coordination-ion interaction, or adsorption. Bradykinin was used as a model object, which was covalently immobilized on the surface of aminated silica with a spacer grafted by the carbodiimide method.

0.5 mg of bradykinin is poured into 1 ml of succinic anhydride, filtered off, the resulting solution is placed in a test tube containing 50 mg of aminated silica and left for 2 hours for the reaction to proceed.

A suspension of the drug in sodium hydrochloride is prepared at a concentration of 2 mg / ml. 20 mg of the drug is taken, placed in a glass cup and filled with 10 ml of physiological saline. The resulting mixture is shaken and placed in an ultrasonic dispersant for 2 minutes. The resulting solution of the drug is stored in the refrigerator at a temperature of 4 ° C. The solution is used for intravenous injection.

To confirm the possibility of obtaining a technical result of the claimed method, experiments were conducted on a model of myocardial ischemia-reperfusion with a quantitative assessment of the biodistribution and biocompatibility of silica nanoparticles. All experiments were carried out in accordance with the "Guide for the care and use of laboratory animals" (publication of the National Institute of Health, USA No. 85-23).

Study material. The experiments were carried out on male Wistar rats weighing 220-300 g (Rappolovo kennel), kept under 12/12 hour light conditions and treated with standard food and drinking ad libitum water.

Methods of regional myocardial ischemia-reperfusion. For anesthesia, chloral hydrate was used at a dose of 450 mg / kg intraperitoneally, followed by maintenance intravenous infusion through a catheter placed in the femoral vein. During the experiment, artificial lung ventilation through a tracheostomy (respiratory rate - 50 / min, tidal volume - 3 ml / 100 g body weight) and measurement of systemic hemodynamics parameters (pulse blood pressure (BP) and heart rate (HR)) were carried out using a pressure sensor (Baxter, USA) via a catheter inserted into the aorta through the common carotid artery and recorded on a computer using PhysExp Gold software. Access to the heart was performed by thoracotomy in the fourth intercostal space on the left. The pericardium was stupidly opened, the localization of the common trunk of the left coronary artery (LCA) was determined, under which a thin polypropylene ligature was brought using an atraumatic needle (6-0). The common trunk of the LCA was determined, focusing on the left at the ear of the left atrium, and on the right - on the cone of the pulmonary artery. An occluder was formed to create reversible myocardial ischemia.

Protocol of experiments and measurements. Animals were randomized into three groups:

1. The first group of animals (n = 3) included rats that underwent organ harvesting (heart and liver) without prior surgical procedures.

2. Animals from the second group (n = 5) also had an autopsy of the chest without ligature. At the 25th minute of thoracotomy, intravenous drip infusion of a suspension of silica nanoparticles at a concentration of 2 mg / ml of a 0.9% sodium chloride solution was started, and the infusion duration was 10 minutes. 55 minutes after the end of the introduction of the suspension of nanoparticles, organs were taken out - the heart and liver.

3. The third group of animals (n = 3) included rats that underwent mechanical occlusion of the LCA using the supplied polypropylene ligature. The duration of ischemia was 30 minutes. Then the ligature was removed to restore blood flow. The duration of the reperfusion period is 60 minutes. At the 25th minute of ischemia, intravenous drip administration of a solution of nanoparticles of the same concentration as in group 2 began, and the administration was completed at the 5th minute of reperfusion. After the end of the reperfusion period, the ligature was tightened and a 5% solution of blue Evans was injected intravenously in a volume of 1 ml, then organs — the heart and liver — were taken.

Hemodynamic parameters were recorded in the initial state, after the ligature was placed under the LCA, 30 minutes after the ligature was added, and also 30 and 60 minutes after the start of the reperfusion.

Biodistribution assessment. After organ harvesting, they were washed once with distilled water. In the third group of animals, myocardial samples for subsequent analysis were selected taking into account Evans blue staining (sections of the myocardium that were not stained with dye, i.e., related to the anatomical risk zone, were excised). In the first and second experimental groups, the entire volume of the left ventricle was isolated for further manipulations. In all experiments, similar fractions of the liver were chosen as liver samples. After organ harvesting, the resulting tissues were dried in a thermostat at a temperature of 90 ° C for 24 hours, bringing the samples to constant weight.

The next step was the analysis of the silicon content in the samples using the atomic absorption spectroscopy method, the essence of which is to illuminate the atomized sample with monochrome light, followed by the decomposition of the light transmitted through the sample by a light dispersant and fixing the absorption spectrum. The dried tissue sample was weighed, then its wet mineralization was carried out according to one of two schemes:

1) Decomposition of concentrated nitric acid under pressure in a fluoroplastic beaker (Minotaur microwave system);

2) Decomposition in an open system with a mixture of concentrated nitric acid and hydrogen peroxide (1: 1) in a glassy carbon crucible.

The obtained mineralizate was analyzed on an atomic absorption spectrometer with electrothermal atomization and Zeyman correction of non-selective absorption of MGA-915 (a lamp with a hollow cathode for silicon λ = 251.6 nm; concentrated ammonia solution is used as a chemical modifier). Next, the silicon concentration found in the mineralizate was recalculated to the dry mass of the sample and expressed in μg / g.

Hemodynamic data, as well as the levels of silicon in the tissues were presented in the form of "mean ± standard deviation".

Hemodynamic parameters. The initial mean blood pressure was 113 ± 11 mm Hg. Art. (table 1) in group No. 2 and 120 ± 8 mm RT. Art. in group No. 3. No significant differences in the mean arterial pressure between the two groups of experiments were observed. During the experiments, a gradual decrease in blood pressure was observed in both groups. The introduction of silica nanoparticles did not cause significant changes in blood pressure. The initial heart rate was 359 ± 39 beats / min. (table 2) in group No. 2 and 315 ± 16 bpm. in group No. 3. The values of heart rate did not significantly differ between groups at the corresponding points. Intravenous infusion of a suspension of silica nanoparticles was not accompanied by significant changes in the value of heart rate.

Table 1 Values of mean arterial pressure in experiments with the introduction of silica nanoparticles (mean value ± standard deviation) The introduction of silica nanoparticles (n = 5) Ischemia-reperfusion + introduction of silica nanoparticles (n = 3) The average blood pressure, mm RT. Art. Originally 113 ± 11 120 ± 8 After summing up the ligature / ligation of LCA 91 ± 20 104 ± 11 30 minutes after the ligature 86 ± 3 99 ± 7 60 minutes after ligature 88 ± 15 96 ± 2 90 minutes after ligature 78 ± 21 78 ± 13

table 2 Values of heart rate in experiments with the introduction of silica nanoparticles (mean value ± standard deviation) The introduction of silica nanoparticles (n = 5) Ischemia-reperfusion + introduction of silica nanoparticles (n = 3) Heart Rate Average Originally 359 ± 39 315 ± 16 After summing up the ligature / ligation of LCA 313 ± 30 305 ± 24 30 minutes after the ligature 284 ± 60 298 ± 32 60 minutes after ligature 278 ± 53 282 ± 22 90 minutes after ligature 303 ± 59 298 ± 34

Biodistribution. In FIG. shows the silicon content in the myocardium and liver of animals of various experimental groups according to atomic absorption spectroscopy: 1 - background content; 2 - the introduction of nanoparticles; 3 - the introduction of nanoparticles after ischemia-reperfusion. The background silicon content in the liver was 3.8 ± 2.25 μg / g. In experiments with intravenous infusion of silica nanoparticles, there was a sharp increase in the silicon content in the liver, significantly exceeding its content in the heart, which indicates the active capture of nanoparticles by the elements of the reticulo-endothelial system of the liver, in particular, Kupffer cells. For example, the level of silicon in the liver in the group of falsely operated animals was 229 ± 80 μg / g, and in the group with ischemia-reperfusion of the myocardium, 306 ± 56 μg / g. Moreover, the difference between these groups was unreliable. On the other hand, the introduction of silica nanoparticles was not accompanied by a significant increase in the silicon content in the heart compared to the background value in intact animals (Fig.). On the contrary, there was a tendency toward a decrease in the silicon content in the heart tissue. The introduction of silica nanoparticles on the background of myocardial ischemia-reperfusion led to a significant increase in the silicon content in 2 out of 3 experiments of group No. 3.

Findings:

1. With intravenous administration of silica nanoparticles with an average diameter of 10 nm to animals with myocardial ischemia-reperfusion, their predominant accumulation in the damage zone is observed, which justifies the prospects of using this carrier for passive targeted delivery of drugs to the ischemic myocardium.

2. The effectiveness of the proposed method of cardioprotection significantly exceeds the existing ones, since the intravenous administration of a system of targeted delivery to animals with myocardial ischemia-reperfusion is accompanied by an increase in the content of the carrier material in the ischemia-reperfusion zone by more than 10 times (Fig.), Whereas when using the prototype in the damage zone only a 5-fold excess of the content of the “drug-carrier” complex over control was observed.

3. The proposed carrier for targeted delivery of drugs to the ischemic myocardium, namely, silica nanoparticles with a diameter of 10 nm, has good biocompatibility and biodegradability.

4. The use of various methods of chemical binding of the drug to the surface of silica nanoparticles makes it possible to control the process of drug release from the surface layer of the carrier.

5. The functionalization of the surface of silica nanoparticles provides the possibility of joining drugs of different chemical structures in various ratios.

Claims (1)

A method of cardioprotection by targeted delivery of a drug to a myocardium subjected to ischemia-reperfusion, characterized in that the carrier of the drug substance is aminated silica nanoparticles with a diameter of 10 nm, to which a spacer is grafted, for each functional group of which the drug is immobilized using one of the methods: covalent binding , coordination-ion interaction, adsorption immobilization.

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