Crystalloid cardioplegic solutions, containing dodecapeptides (versions)

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine and deals with a crystalloid cardioplegic solution, which contains salt solution, including sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium hydrogen carbonate, water for injections and a structural analogue of natural apelin X-Arg(NGY)-Pro-Arg-Leu-Ser-His-Lys-Cly-Pro-Nle-Pro-Phe-Z, where X=CH3, Y=H, Z=OH. The group of inventions also deals with the crystalloid cardioplegic solution, containing salt solution, including sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium hydrogen carbonate, water for injections and structural analogue of natural apelin X-Arg(NGY)-Pro-Arg-Leu-Ser-His-Lys-Cly-Pro-Nle-Pro-Phe-Z, where X=H, Y=NO2, Z=NH2.

EFFECT: group of inventions provides the recovery of the coronary flow, cardiac contractile and pump function in case of the reperfusion and the reduction of injury to membranes of cardiomyocytes.

2 cl, 2 dwg, 8 tbl, 4 ex

 

The invention relates to medicine, namely to the development of solutions intended for use during operations on the "open" heart on cardiopulmonary bypass.

One of the main methods of myocardial protection in cardiac surgery practice is the use of pharmacological cold crystalloid cardioplegia.

Known cardioplegic solutions the Consol, Custodiol, Celsior, containing potassium chloride, calcium gluconate, magnesium sulfate, sodium bicarbonate, sodium chloride, water for injections with the addition of various components.

(Ostrovsky Y. P. surgery of the heart. M: Honey. Lit., 2007. - 576 p.)

The closest to the claimed cardioplegic solution is saline hospital St. Thomas (CRC GTS) produced by the company Abbott Laboratories (USA) under the name Plegisol, which comprises: sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium bicarbonate. In Russia and abroad this drug is most often used for cardioplegia.

Gharagozloo F., Bulkley V. N., Hutchins G. M., T. J. Bixler, H. V. Schaff, J. T. Flaherty, T. J. Gardner Potassium-induced cardioplegia during normothermic cardiac arrest. Morphologic study of the effect of varying concentrations of potassium on myocardial anoxic injury. J Thorac Cardiovasc Surg. 1979; 77(4):602-607.

Common drawback listed cardioplegic solutions can be attributed to incomplete prevention of metabolic and page�Chernyh changes developing in the myocardium during prolonged ischemia and subsequent restoration of coronary blood flow during surgery. As you know, the main damaging factors during ischemia are abnormalities in myocardial energy supply of cells and generation of reactive oxygen species associated with changes in intracellular ion homeostasis. Their effects on ischemic myocardium is able to block oxidative phosphorylation, to damage the structure of mitochondrial and plasma membranes, to cause contraction of myofibrils and cause intracellular swelling and death of cardiomyocytes. The consequence of the above drawback used cardioplegic drugs is the development of intraoperative heart attacks, reduced recovery of cardiac pump function when resuming circulation, which is complicated by the occurrence of reperfusion arrhythmias and heart failure.

The object of the invention is to develop effective original crystalloid cardioplegic solutions containing dodecapeptide, can reduce the risk of cardiovascular interventions, improving energy metabolism and stabilizing the sarcolemma of cardiomyocytes, as well as optimization of the ionic composition and osmolarity rustboro�.

The technical result of the invention is to restore coronary flow, contractile and pump function of the heart during reperfusion and reducing the damage of the membranes of cardiomyocytes.

This is achieved in that in the claimed crystalloid cardioplegic solution according to the first embodiment, containing a salt solution, comprising, mmol/l: sodium chloride 119-121, potassium chloride 15-17, magnesium chloride 15-17, calcium chloride 1,1-1,3, water for injection, according to the invention, further comprises a structural analogue of the natural apelin X-Arg(NGY)-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-Z, where X=CH3, Y=N, Z=HE's in the amount of 0.13 to 0.15 mol per 1 liter of the salt solution.

According to the second embodiment in the inventive cardioplegic solution containing a salt solution, comprising, mmol/l: sodium chloride 119-121, potassium chloride 15-17, magnesium chloride 15-17, calcium chloride 1,1-1,3, water for injection, according to the invention, further comprises a structural analogue of the natural apelin X-Arg(NGY)-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-Z, where X=N, Y=NO2, Z=NH2in the amount of 0.13 to 0.15 mol per 1 liter of the salt solution.

In recent years, attracted the attention of researchers endogenous adipokine apelin - 77-membered polypeptide and its APJ receptor, involved in the regulation of the tonus of the coronary vessels and myocardial contractility. It is shown that exogenous C-terminal fragments of apeli�and (apelin-36, -13 and -12) are able to limit the size of myocardial infarction and improve the recovery of cardiac function in experimental animals after a period of total or regional ischemia. In cardiomyocytes of rats in the simulation of stress and oxygenation under conditions of energy deficiency of these peptides blocked the opening of mitochondrial pores and apoptosis. The decline of the death of cardiomyocytes during reperfusion under the action of C-terminal fragments of apelin is associated with activation of cascades, including the PI3-Akt kinase and MAP kinase, targets which are endothelial NO-synthase (eNOS), ribosomal p70S6-kinase and apoptotic protein BAX/BAD.

It is known that the peptide apelin-12 (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A12), completely identical to the C-terminal fragment of apelin in different animal species and humans is minimal active part of the molecule the source of apelin-77; it has a high affinity for the APJ receptor and cardioprotective properties. However, the high activity of enzymes (amino - and carboxypeptidases) in the blood hinders the use of apelin-12 in the clinic. In addition, the composition of the A12 comes with a methionine, which is extremely easily oxidized by atmospheric oxygen to the corresponding sulfoxide, which reduces its stability during storage. We have synthesized structural analogues A12, combining high cardioprotective actively�you with increased proteolytic stability and storage stability of the - the peptides (I) and (II).

The molecules of the peptide oxidation-prone methionine is replaced by norleucine - natural non-protein amino acid, is absolutely resistant to oxidation by oxygen. The N-terminal portion of the peptides contains either the Nα-alkyl amino - Nα-methylarginine (peptide I), or NG-the nitro group (peptide II), which increases resistance to the action of aminopeptidase. The C-terminal part of the peptide (II) is additionally protected from the action of carboxypeptidases amide function.

The synthesis of peptides.

Used abbreviations:

Used abbreviations recommended by the Commission IUPAC-IUB (Eur. J. Biochem. (1984). V. 183. P. 9-37), as well as: BOC-tert-butyloxycarbonyl, Buttert-butyl, DCM - dichloro methane, DIC - N,N'-diisopropylcarbodiimide, Fmoc - 9-fluorenylmethoxycarbonyl, HOBt is 1-hydroxy-benzotriazole, 4-MePip - 4-demerol, NMP - N-methylpyrrolidone, Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl, TIBS - triisopropylsilane, TFA is trifluoroacetic acid, Trt is trityl.

We used the derivatives of L-amino acids (Fluka and Bachem, Switzerland), DIC, DIPEA, HOBt, TIBS, (Fluka, Switzerland). For the synthesis used the N-methylpyrrolidone, dichloro methane, 4-demerol, methanol and TFA (Fluka, Switzerland). Analytical HPLC was performed on a chromatograph (Gilson, France), used column Kromasil 100 C18 5 µm, 4.6×250 mm (Sweden), mobile phase: buffer A - 0.1% TFA, buffer B - 80% acetonitrile in buffer a, elution with a concentration gradient of buffer B in buffer And 10% to 70% over 30 min; flow rate 1 ml/min, detection at 220 nm. The structure of the obtained peptides proved spectra of1H-NMR data and mass spectrometry. Mass-spectra were registered on instrument Ultrafex TOF/TOF (Bruker Daltonics, Germany) with time-of-flight base by the method of MALDI (matrix-assisted mass spectrometry laser desorption/ionization).

Solid-phase synthesis of peptide (I) was carried out on an automatic synthesizer Applied Biosystems 431 A (Germany) in accordance with a standard Protocol for single condensation of Fmoc-amino acids. For blocking of functional groups of the side chains of amino acids used the following protective groups: Pmc - for the guanidine group of arginine, Butfor actigraphy serine, Boc - ε - amino group of lysine, Trt - for imidazole of histidine. As the carrier used a Wang resin is a copolymer of styrene with 1% divinylbenzene 4-hydroxymethoxypethidine anchor group. The synthesis was carried out With the end on the basis of 0.4 g (0.25 mmol) of Fmoc-Phe-polymer company Bachem (Switzerland) containing phenylalanine - 0.67 mmol/g. the Following is a standard Protocol for the TFS. The dipeptide Fmoc-Nle-Pro-OH was obtained by classical methods of peptide chemistry in solution,purified by chromatography on silica gel and characterized by TLC data and 1H-NMR spectroscopy.

Table 1.
The Protocol of solid-phase synthesis of peptides (I) and (II).
No.OperationReagentProcessing time
Cycle 1
1Flushing5×NMP3 min
2The release of α-amino groups25% 4-MePip/NMP10 min
3Flushing5×NMP3 min
4Activation1 mmol of Fmoc-Nle-Pro-OH+1 mmol TBTU+1 mmol, NOUT+2 mmol DIPEA in NMP,3-5 min
5Condensation1 mmol of activated derivative Fmoc-dipeptide in NMP90 min
6 Flushing5×NMP3 min
7Test with ninhydrin
Cycle 2-10

1Flushing5×NMP3 min
2The release of α-amino groups25% 4-IU Pip/NMP10 min
3Flushing5×NMP3 min
4Activation1 mmol of Fmoc-AA-OH+1 mmol HOBT+1 mmol DIC in NMP/DMF20 min
5Condensation1 mmol of activated derivative Fmoc-AA in NMP90 min
6Flushing5×NMP3 min

Final release and cleavage of the peptide (I) of the polymer was carried out in one stage by about�processing corresponding peptidylarginine 10 ml of a mixture of 90% TFA, 2.5% deionized water, 5% thioanisole and 2.5% TIBS for 2-3 h. Then the polymer was filtered, washed with 2×2 ml unlocking of the mixture, the filtrate was evaporated and to the residue was added dry ethyl acetate or ether. The precipitate was filtered, washed with DCM (3×3 ml), ether (3×5 ml), dried in a vacuum desiccator. "Raw" solid-phase synthesis product was purified using preparative HPLC, using a column diasorb C16 130T (25×250 mm), the size of the sorbent particles 10 microns. As allentow used buffer A - 0.1% aqueous TFA and buffer B - 80% acetonitrile in water. The elution was conducted with a gradient of 0.5% per minute of buffer B in buffer A from 100% buffer A at a rate of 10 ml/min, the Peptides were detected at a wavelength of 220 nm. Fractions containing the desired product were pooled, the acetonitrile was evaporated and lyophilized. The homogeneity of the product was determined by analytical HPLC, and the structure was confirmed by the data of mass-spectrometry and1H-NMR spectroscopy. The yield of peptide, HPLC data and mass spectrometry are shown in table 3.

For solid-phase peptide synthesis (II) used a copolymer of styrene with 1% divinylbenzene 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxymethyl-anchor group (Rink-amide-resin) company Nova BioChem, Switzerland, designed for producing amides of peptides containing 0.60 mmol/g of amino groups. Synthesis of amide (II) p�bodily with C-end in accordance with the above solid-phase synthesis Protocol (see table.2) on the basis of 0.40 g (0.24 mmol) of Rink-amide-polymer. The synthesis was carried out automatically on a peptide synthesizer Applied Biosystems A under the standard program for a single condensation of Fmoc-amino acids.

The cleavage of the protection was performed using 10 ml of a mixture of 85% TFA, 5% DMB, 2.5% TIBS, 5% deionized water and 2.5% of thioanisole for 3 h; the purification and identification of peptide (II) was performed as described for compounds (I). The data presented in table 2.

Table 2.
Characteristics of peptides.
No.Formula peptideMcalculation.Outputand, %HPLC*MALDI-MS, m/z
Rt,min%
(I)H-(NαMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH1418.77717.06981418.9
(II)H-Arg(NGNO2)-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-NH2 1448.750b16.92981448.8; 1403.8(-NO2)
and Outputs of the peptides are given in the calculation of the starting amino acid attached to the polymer.
* Column Kromasil 100 C18, 4.6×250 mm, 5 µm; buffer A - 0.1% TFA, buffer B - 80% acetonitrile, gradient from B to A 10 to 70% over 30 min; flow rate 1 ml/min; 220 nm.

Preparation of cardioplegic solution consists of two stages: 1) preparing a salt solution of the hospital of St. Thomas); 2) preparation of the CRC-1 and CRC-2 with a given concentration of peptides.

1) Preparation of CRC GTS. In 0,95 liters of water for injection, dissolve successively with stirring NaCl - 5,85 g, KCl - 1.19 g, MgCl2·6H2O - 3.25 g, CaCl2- 0.13 g, NaHCO3- 0,84 g. the pH of the solution was adjusted to 7.8 2n NaOH. The volume of the solution was adjusted to 1.0 l with water and check pH. The final concentration of ingredients are in mol/l: NaCl - 120; COP - 16,0 mm; MgCl2·6H2O - 16,0 mm; CaCl21,2; NaHCO3- 16,0.

2) Preparation of the CRC-1 and CRC-2. In 1.0 l CRC GTS-2 at room temperature and stirring was dissolved 198,6 mg of peptide I or of 202.8 mg of peptide II. Cardiopatici the solution is adjusted to pH 7.8 with 5 n NaOH at 25°C and filtered through a filter with a pore size of 5 µm (Millipore, Bedford, USA). Horse�nye the concentration of peptide I and II in the CRC-1 and CRC-2 are 140,0 μmol/L. The concentration of other ingredients are the same as the CRC of the GTS.

Developed fluids are crystalloid cardioplegic solutions extracellular type designed to protect the human heart from global ischemia and reperfusion injury during cardiac operations. Composition and physico-chemical characteristics of solutions of cardioplegic solution (option 1) CRC-1 and cardioplegic solution (option 2) CRC-2 in comparison with cardioplegic solution hospital St. Thomas - CRC GTS, selected as a prototype, is shown in Table.3.

Table 3.
The compositions and physico-chemical characteristics of the proposed cardioplegic agents (CRC-1 and CRC-2), and drug comparisons CRC GTS.
CRC-1CRC-2CRC GTS (prototype)
Sodium chloride, mmol/l120,0120,0120,0
Potassium chloride, mmol/l16,016,016,0
Magnesium chlorine�, mmol/l16,016,016,0
Calcium chloride, mmol/l1,21,21,2
Sodium bicarbonate, mmol/l10,010,010,0
Peptide I, µmol/l140,0--
Peptide II, mmol/l-140,0-
Water for injection, l1,01,01,0
pH (25°C)7,87,87,8
Osmolarity, mOsm/l285-300285-300285-300

Proposed tools for cardioplegia have a number of advantages compared with the CRC of the GTS (the prototype). First, they provide a better recovery of coronary flow and contractile and pump function of the heart during reperfu�AI. Secondly, contribute to a more effective restoration of aerobic metabolism in reperfusion the myocardium. Thirdly, in a greater degree reduces damage to the membranes of cardiomyocytes during early reperfusion. Advantages of the inventive cardioplegic solutions demonstrated in the examples below.

The compositions of the CRC-1 and CRC-2 original and not described in the available literature. Bibliography of patents on modified analogs of C-terminal fragment of apelin-12 does not contain information on the subject of the invention is the development of cardioplegic funds through the use of peptides such chemical structure. Moreover, before carrying out experiments on perfusion heart of laboratory animals rationality of peptides I and II as a useful addition to salt cardioplegic solution was not obvious. This is due to the originality of the chemical structure of compounds and the lack of data on the influence of natural C-terminal fragments of natural apelin-36, -13 and -12 on the metabolism and function in hearts subjected to cardioplegic stop. Experimental evidence of biological activity of peptides I and II in terms gipertonicheskoj salt cardioplegia open the possibility of the creation of the original water-soluble compositions for intraoperative myocardial protection OS�ove this class of compounds.

Example 1. The restoration of cardiac function and coronary blood vessels.

To study the cardioprotective properties of the CRC-1 and CRC-2 used a model of global ischemia and reperfusion of the isolated perfused rat heart. Such models are used to evaluate the effectiveness of different cardioplegic solutions. They provide an adequate assessment of the impact of drugs on the function and metabolism of the heart and damage of cell membranes by reperfusion. All the 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).

Perfusion of the isolated rat heart. Experiments performed on the heart of male rats Wistar (290-340 g). In an anesthetized by urethane (intraperitoneally 1.25 mg per g of body weight), animals were removed his heart and perfesional retrograde for 15-20 min with a solution of Krebs-henseleit solution (RCH) with 11 mm glucose, saturated with Carbogen (95% O2±5% CO2) pH 7,4±0,1 at 37°C, at a constant perfusion pressure of 60 mm Hg.PT. Thereafter, the hearts were perfesional antegrade on Nile at constant filling pressure of the left atrium 15 mm Hg.PT. and average perfusion pressure in the aorta 60 mm Hg.PT. The pressure in the aorta and left ventricle were recorded using strain gauges R 50, monitor SP1405 Registrar SP 2010 (Gould Statham, USA). Index of the intensity of contractile function (SF) of the left ventricle was the product of frequency of reductions of heart in developed pressure (difference between systolic and minimum diastolic pressure). The pumping function of the left ventricle was assessed by the size of the minute (MO - the amount of coronary flow and aortic volume) and impact (the ratio of the minute volume of the heart rate) volumes.

The Protocol of the experiment. After perfusion for Nile for 20 min (initial state) hearts subjected to cardioplegic stop. The control used CRC GTS in experimental groups - CRC-1 and CRC-2. Cardioplegic solutions were injected retrograde perfusion for 5 min with a constant speed of 4 ml/min; temperature of the solutions in all groups was 25°C. the Stopped hearts were subjected to normothermic global ischemia for 40 min, and then reperfusion standard RCH - first 5 min of retrograde at a rate of 4 ml/min, 25-min antegrade on Nile. By the end of the experiments the hearts were frozen in liquid nitrogen for determination of macroergonomic phosphate and lactate in reperfusion the myocardium. In a separate series of hearts were frozen after 20-min perfusion at Nili to determine the initial concentrations of metabolites. Statistical processing of the obtained�'s results was performed using the program Systat SigmaPlot 11.

In table.4 mapped to the restoration of coronary flow, the intensity of SF and minute and the stroke volume by 30 minutes of reperfusion compared with these indices in the initial state. In experiments drug comparison CRC GTS (prototype) these indicators were recovered, respectively, to 74±2%, 44±1%, 26±1% and 33±1% of the original value. The inclusion in the CRC GTS peptides I or II significantly increased the recovery of function of the heart and blood vessels. So, when using CRC-1 and CRC-2 coronary flow recovered to an average of 90%, the recovery of the SF intensity increased on average 1.7 times a minute and the stroke volume by 2.8 and 2.5 times, respectively.

Table 4.
The influence of the CRC-1 and CRC-2 for reconstruction of basic indicators of function of heart and vessels at the end of reperfusion.
Coronary flow, ml/minThe intensity of the SF, mm Hg.PT./minMinute volume (ml/min)Stroke volume, ál/min
Initial state
18±232493±669 46±1148±1

30 min reperfusion
Control (prototype)47+2and
13±1and13158±301and11±1and
CRC-117±1b25038±1641AB33±3AB114±6AB
CRC-218±1b26470±1208AB35±2AB120±4AB
Data are presented as M±M for a series of 12 experiments. Differs significantly (P<0.05) from: a - initial state, b - control (CRC GTS).

Figure 1 shows the evolution of diastolic pressure in the left ventricle (Pdust.LV) in the control and in the experiments with the proposed cardioplegic solutions. Pdust.LV �is one of the most important indicators of contractile and pump function of the heart, characterizing the relaxation of the myocardium and filling the cavity of the left ventricle in diastole. The original value of Pdust.LV amounted to 4 mm Hg.PT. In control at the beginning of reperfusion Pdust.LV was high, stepping down from 13 to 10 mm Hg.PT. When using CRC-1 and CRC-2 Rdust.LV was significantly decreased and amounted to an average of 3.5 mmHg.PT at the beginning of reperfusion and 0 mm Hg.St the end of the experiment, respectively.

Thus, the addition of peptides I and II in the CRC GTS significantly improves recovery of the functions of the ischemic rat heart during reperfusion. Differences in effectiveness between the CRC-1 and CRC-2 were not statistically significant.

Example 2. The metabolic status of the heart during reperfusion.

Treatment of heart tissue. My heart was chilled frozen in liquid nitrogen forceps of Wollenberger in the initial state or at the end of reperfusion. The tissue is homogenized in cold 6% HClO4(10 ml per g tissue) in a homogenizer Ultra-Turrax T-25 (IKA-Labortechnik, Germany). Proteins were precipitated by centrifugation at 3000 g and 4°C for 10 minutes. The supernatants neutralized with 5 M K2CO3up to a pH of 7.4. The precipitate KClO4was separated by centrifugation in the same conditions. Protein-free extracts were stored at -20°C until the determination of metabolites. Dry weight of the homogenized tissue was determined after drying about�ascov during the day at 110°C. ATP and phosphocreatine (FCR) in tissue extracts was determined spectrophotometrically using glucose-6-phosphatedehydrogenase, geksokinazou and ck; the content of ADP and AMP using myokinase, pyruvate, and lactate dehydrogenase, creatine quantity (Cu) - with the help of creatine kinase, phosphoenolpyruvate and lactate dehydrogenase, lactate using lactate dehydrogenase. The content of metabolites was expressed in µmol/g dry weight tissue.

At the end of reperfusion in the hearts of the studied groups were identified levels of actinolite (ATP, ADP, AMP) and FKR and Cu. Based on these data were calculated as indicators of the energy state of postischemic hearts: the energy charge of cardiomyocytes EZ=(ATP+0.5 ADP)/Σ; the degree of phosphorylation and ATP FKR - ATP/ADP, and FKR/Cu. To assess the intensity of anaerobic glycolysis/glycogenolysis in reperfusion heart was determined lactate content. Comparing the received data with the metabolic state of the heart in the initial state (before the simulation of ischemia and reperfusion) is shown in table.5.

Table 5.
The impact of the use of the CRC-1 and CRC-2 for the restoration of metabolism in perfused rat heart during reperfusion.
MetaboliteInitial state30 min reperfusion
Control (prototype)CRC-1CRC-2
EZE0,97+0,010,76±0.02and0,79±0.02and0,83±0.01AB
ATP/ADP13,00+0,982,48±0,02and2,38±0,03and2,95±0,04ABC
RKFthe 24.75±1.6317,88±0.46and19,04±0,9323,14±1,12BV
FKR/Cu0,55+0,040,37±0,02and0,42±0,020,52±0,03BV
Lactateof 1.53±0.936,03±2.15andof 1.64±0.28ba 1.77±0.83b
Data are presented as M±M for series �W 12 experiments. Differs significantly (P<0.05) from: a - initial state, b - control (CRC GTS-2), - CRC-1.

The use of CRC-1 and CRC-2 increased the recovery of energy metabolism in reperfusion heart. It was confirmed a higher value of VC postischemic cardiomyocytes, increased content of FCR and higher relations of ATP/ADP and FKR/Cu compared to these parameters in the control. Most effective recovery of energy metabolism occurred under the action of peptide II with the introduction of the CRC-2. Lactate content when using CRC-1 and CRC-2 to the end of reperfusion was reduced on average by 3.5 times compared with controls and did not differ significantly from the original values. This showed the best recovery of aerobic utilization of glucose - the main energy substrate perfoirmance heart under the action of peptides I and II. The obtained data confirm the improvement of the energy state reperfusion heart under the action of the CRC-1 and CRC-2.

Example 3. Damage to the membranes of cardiomyocytes.

Damage to the membranes of cardiomyocytes was assessed by the increase in the activity of lactate dehydrogenase (LDH) in flowing from the heart perfusate, which was collected in chilled tubes in ice for 5 min before ischemia (baseline) and during the first 5 min �perfusia. LDH activity was determined using as substrate pyruvate, spectrophotometer Yanako UO-2000 (Japan) at λ=340 nm.

In the initial state, the LDH release was similar in all groups (Tab.6). In the control (when using CRC GTS-2) LDH release during early reperfusion was almost doubled compared with the initial state. When you stop the heart before ischemia cardioplegic solutions CRC-1 and CRC-2 this figure was significantly less (on average 40%, P<0.05) and did not differ significantly from the release of LDH into the perfusate in the initial state.

Table 6.
The effect of the inclusion of peptides in the composition of the cardioplegic solution of the hospital of St. Thomas No. 2 on LDH activity (IU/g dry. weight) in the myocardial outflow of the isolated perfused rat heart during early reperfusion.
Initial stateReperfusion
Control(prototype)5,66±1,15is 10.51±1,53and
CRC-14,32±0,776,03±1,22b
CRC-2 of 5.55±0.566,54±0,78b
Given M±M for a series of 10 experiments. Initial state - LDH activity in the perfusate collected within 5 min prior to ischemia. Reperfusion - LDH activity in the perfusate collected within 5 min after ischemia. Differs significantly (P<0.05) from: a - initial state, b - control (CRC GTS-2).

Thus, the use of a marker of damage to cell membranes LDH clearly indicates greater stability of the sarcolemma postischemic cardiomyocytes in the case of solutions of the CRC-1 and CRC-2.

Example 4. The variation of concentrations of peptides in the CRC.

Were tested by modified mortar compositions CRC-1 and CRC-2 containing lower and higher concentrations of peptides I and II. The final solution pH and osmolality remained unchanged (tab.7). The efficacy of these drugs was evaluated on the model of isolated perfused rat heart to restore its function and coronary flow during reperfusion. The Protocol of the experiments was as above. As an example, figure 2 shows the effect of the concentration of peptides in the cardioplegic solution on the recovery of the minute volume of the heart during reperfusion.

Table 7.
The compositions and physico-chemical characteristics of modified cardioplegic solution CRC-1.
CRC-1-70CRC-1-280
Sodium chloride. mmol/l120,0120,0
Potassium chloride, mmol/l16,016,0
Magnesium chloride, mmol/l16,016,0
Calcium chloride, mmol/l1,21,2
Sodium bicarbonate, mmol/l10,010,0
Peptide I, µmol/l70,0280,0
Water for injection, l1,01,0
pH (25°C)7,87,8
Osmolarity, mOsm/l285-300285-300
Note. Ionic composition and physico-chemical characteristics of modified cardioplegic solution CRC-2, containing 70 or 280 μmol of peptide II/l were the same.

From the data presented in the figure 2 it follows that for peptides I and II maximum recovery of minute volume was achieved by using a concentration of 140 μm. When a two-fold reduction or increase in the concentration of peptides in the cardioplegic solution recovery efficiency decreased. When using CRC with concentrations of peptide I or II 70 and 280 μm was also observed a decrease in the recovery of coronary flow and intensity of the SF compared with the values of these indices for the concentration of peptide 140 µm (PL.4, 8). The results indicate that the optimal to protect the heart from ischemia and reperfusion injury is the concentration of peptides I or II in salt cardioplegic solution, equal to 140 microns.

Table 8.
The effect of varying concentrations of peptides I and II in the cardioplegic solution on recovery of coronary flow and intensity of the contractile function of the heart at the end of reperfusion.
Coronary flow, ml/minThe intensity of the SF, mm Hg.PT./min
Initial state
18±232311±782
30 min reperfusion
Control (prototype)
13±1and13158±301and
CRC-1-7013±1and15479±1019and
CRC-1-24015±119545±1835AB
CRC-2-7015±119969±1585AB
CRC-2-24018±2b27784±536AB
Data are presented as M±M for a series of 6 experiments. Differs significantly (P<0.05) from: a - initial state, b - control (CRC GTS).

Thus, the use of cardioplegic means CRC-1 and CRC-2 provides the best recovery of function of the heart and vessels during reperfusion after a period�and total ischemia. This is combined with a more efficient restoration of the metabolic state of the heart and less damage to the membranes of postischemic cardiomyocytes compared with traditionally used cardioplegic solution hospital St. Thomas.

1. Crystalloid cardioplegic solution containing saline solution comprising sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium hydrocarbonate and water for injection, characterized in that it further comprises a structural analogue of the natural apelin X-Arg(NGY)-Pro-Arg-Leu-Ser-His-Lys-Cly-Pro-Nle-Pro-Phe-Z, where X=CH3, Y=N, Z=HE, with the following component ratio, mmol/l:
sodium chloride 119-121,
potassium chloride 15-17,
magnesium chloride 15-17,
calcium chloride 1,1-1,3,
sodium bicarbonate 10,
a structural analogue of the natural apelin 0,13-0,15,
water for injection.

2. Crystalloid cardioplegic solution containing saline solution comprising sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium bicarbonate, and water for injection, characterized in that it further comprises a structural analogue of the natural apelin X-Arg(NGY)-Pro-Arg-Leu-Ser-His-Lys-Cly-Pro-Nle-Pro-Phe-Z, where X=N, Y=NO2, Z=NH2, with the following ratio of components, mmol/l:
sodium chloride 119-121,
potassium chloride 15-17,
magnesium chloride 15-17,
calcium chloride 1,1-1,3,
sodium hydrocar�ONAT 10,
a structural analogue of the natural apelin 0,13-0,15,
water for injections.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to cardiac surgery, and represents cardioplegic solution, which contains sodium chloride - 3.41-3.62, potassium chloride - 1.092-1.156 g, magnesium chloride - 3.190-3.485 g, calcium gluconate - 0.0105-0.0130 g, mannite - 4.365-4.520 g, L-carnosine - 20.1504-24.1650 g, N-acetylcarnosine - 8.056-11.032 g, L-histidine - 0.705-0.820 g, water for injections to 1000 ml.

EFFECT: invention ensures prevention of reduction of amplitude, speed of front and speed of pulse-wave reduction, as well as increase of diastolic pressure in left heart ventricle during reperfusion with preservation of buffer capacity and osmolarity of cardioplegic solution with physiological pH parameters.

4 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of organic chemistry, namely to novel derivatives of pyrazole pyridine of formula , as well as to its tautomers, geometrical isomers, enantiomers, diastereomers, racemates and pharmaceutically acceptable salts, where G1 represents H; G2 represents -CHR1R2; R1 and R2 independently on each other are selected from H; C1C6-alkoxy-C1C6-alkyl; C1-C6-alkyl; optionally substituted phenyl; optionally substituted phenyl-C1-C6-alkyl; optionally substituted morpholine-C1-C6-alkyl; or -CHR1R2 together form a ring, selected from an optionally substituted C3-C8-cycloalkyl and substituted piperidine; G3 is selected from an optionally substituted C1C6-alkoxy -C1-C6-alkyl; C1-C6-alkyl; substituted phenyl; substituted phenyl-C1C6-alkyl; G4 is selected from a substituted acyl-C1C6-alkyl, where acyl represents a group -CO-R and R stands for H or morpholine; optionally substituted C1-C6-alkyl; optionally substituted phenyl or indene; substituted phenyl-C1-C6-alkyl; optionally substituted pyridine- or furanyl-C1C6-alkyl; morpholine- or piperidine-C1-C6-alkyl; G5 represents H; where the term "substituted" stands for the groups, substituted with 1 to 5 substituents, selected from the group, which includes a "C1-C6-alkyl," "morpholine", "C1-C6-alkylphenyl", "di-C1-C6-alkylamino", "acylamino", which stands for the group NRCOR", where R represents H and R" represents a C1-C6-alkyl, "phenyl", "fluorine-substituted phenyl", "C1-C6-alkoxy", "C1-C6-alkoxycarbonyl", "halogen". The invention also relates to a pharmaceutical composition based on the formula (I) compound and particular compounds.

EFFECT: obtained are the novel derivatives of pyrasole pyridine, useful for the treatment and/or prevention of disorders or states, associated with NADPH-oxidase.

12 cl, 3 tbl, 21 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a citrate of a compound described by formula (II) below, and a pharmaceutical composition containing said citrate.

EFFECT: experimental results of the present inventions prove that said citrate can inhibit activity of phosphodiesterase type 5 and can be used for treating erectile dysfunction, for inhibiting thrombocyte aggregation and treating thrombosis, for reducing pulmonary hypertension and treating cardiovascular diseases, asthma and diabetic gastroparesis.

2 cl

FIELD: medicine.

SUBSTANCE: group of inventions relates to field of therapy and/or prevention of diseases in mammals, in particular humans. Group of inventions includes medication for treatment and/or prevention of cardiovascular disease, and/or inflammatory disease, and/or liver disease, and/or neurological disease, and/or steatosis by increasing content of polyunsaturated fatty acids in mammal's blood, representing dairy product of ruminants with reduced cholesterol content, where cholesterol content constitutes from 10 mg/100 g of fat to 150 mg/100 g of fat, as well as application of dairy product of ruminants with reduced cholesterol content, in which cholesterol content constitutes from 10 mg/100 g of fat to 150 mg/100 g of fat, for treatment and/or prevention of cardiovascular disease, and/or inflammatory disease, and/or liver disease, and/or neurological disease, and/or steatosis by increasing content of polyunsaturated fatty acids in mammal's blood.

EFFECT: obtaining medication for treatment and/or prevention of cardiovascular disease, and/or inflammatory disease, and/or liver disease, and/or neurological disease, and/or steatosis.

18 cl, 5 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula I, wherein R1 and R2 are identical or different and specified in an alkyl or alkenyl hydrocarbon chain; the R3 group values split by lipase are specified in the patient claim. R4 and R5 are independently hydrogen or C1-C7alkyl; R6 represents hydrogen or C1-C7alkyl; and R7 and R8 are independently hydrogen or C1-C7alkyl. The invention also refers to using compounds of formulas ,

which are introduced into the mammalian biological system and increase the cell concentrations of specific sn-2 substituted ethanolamine-plasmalogens.

EFFECT: compounds are applicable in treating or preventing the age-related disorders associated with high membrane cholesterol, high amyloids and low plasmalogens, such as neurodegeneration, cognitive disorder, dementia, cancer, osteoporosis, bipolar disorder and vascular diseases.

11 cl, 18 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to cardiology, and represents a method of the drug treatment of patients in late rehabilitation period after aortocoronary bypass surgery, consisting in the combined administration to the patient of medications thrombo ASS 100 mg, atorvastatin 20 mg and additionally amlodipine in a daily dose from 5 to 10 mg and losartan in a daily dose from 25 to 100 mg.

EFFECT: invention ensures the long-term preservation of results of surgical myocardium revascularisation.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to fluorinated aminotriazole derivatives of formula

,

wherein A represents a group specified in furanyl, oxazolyl and thiazolyl, wherein two attachment points of the above group are found in 1,3-position; R1 represents phenyl, which is unsubstituted, mono- or disubstituted, wherein the substitutes are independently specified in a group consisting of halogen, methyl, methoxy group, trifluoromethyl, trifluormethoxy group and dimethylamino group; and R2 represents hydrogen, methyl, ethyl or cyclopropyl. Besides, the invention refers to a pharmaceutical composition containing the compound of formula (I), and to using the compound of formula (I) for preparing a therapeutic agent.

EFFECT: compounds of formula (I) possessing the agonist activity in relation to ALX receptor.

26 cl, 2 tbl, 36 ex

FIELD: medicine.

SUBSTANCE: method involves taking general baths and prescribing mud applications on lower extremities with underlying standard drug therapy. The patient takes prepared silicate-carbonate baths with the concentration of sodium salt of metasilicic acid of 100-150 mg/l and the content of carbon dioxide of 1.2 g/l. The bath water temperature is 36°-37°C. The length of the procedure is 10-15 minutes. Taking the baths is followed by having a 30-40-minute rest. Sulphide silt mud sock- or boot-like applications are performed. The mud temperature is 32°-36°C; the length of procedure is 8-20 minutes. The procedures are performed 2 days running, with a pause on the 3rd day. The therapeutic course makes 6-10 procedures.

EFFECT: method provides the further development of the symptom-free involvement of target organs: heart, kidneys, vessels by taking the antihypertensive, antianginal and antiarrhythmic effects, promotes the energetic and adaptation possibilities of the body.

5 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and concerns a method for producing progenitor cells from mammalian myocardial samples. A presented method involves milling the samples, implanting them on culture dishes coated so that to provide adhesion; culturing the samples in the hypoxic environment so that to provide the sample adhesion to form a cell culture; recovering cells from the cell culture by enzymatic treatment; culturing the recovered cells until forming cell aggregates, and processing the cell aggregates with an enzyme solution to produce the progenitor cells of myocardium.

EFFECT: presented invention can be used in medicine and veterinary science for studying the myocardium regeneration mechanisms and stem cell biology, as well as for treating cardiac diseases.

10 cl, 2 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: herbal tea has the hypolipidemic, antiaggregant, vasoprotective and adaptogenic action. The herbal tea contains common liquorice roots, blood-red hawthorn fruit, wild camomile blossom, blood-red hawthorn blossom, elevated part of meadow crane, elevated part of clump speedwell, clove tree flower buds, sweet flag rhizome, turmeric rhizome, anomalous peony root, common valerian rhizomes, elevated part of common sweet clover, common willow leaves, field caraway fruit, elevated part of tripartite bur-marigold, elevated part of common St. John's wort, elevated part of garden sage, meadowsweet blossom, elevated part of field mint, elevated part of cudweed in certain proportions. The above herbal tea extends the range of products for preventing primary ischemic stroke in the patients suffering cerebrovascular disease. It promotes mobilising all links of self-protection: immune protective, antioxidant and detoxification, neuroendocrine system.

EFFECT: more effective prevention.

1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed is a pharmaceutical, ear, sterile, preservative-free composition in the form of a transparent aqueous solution, containing 0.01-0.025 % of fluocinilone acetonide optionally in a combination with 0.1-0.8% of ciprofloxacin or its pharmaceutically acceptable salt, a non-ionic surface-active substance, a tonicity-regulating agent and a viscosity-increasing agent.

EFFECT: composition is useful for the prevention and/or treatment of ear inflammation, optionally accompanied with a bacterial infection, and for the introduction of a single dose from a package.

15 cl, 8 ex

FIELD: medicine.

SUBSTANCE: invention represents a balanced infusion solution containing sodium, potassium and magnesium chlorides, a solvent and sodium L-arginine succinate of formula: Na+[NH=C(NH2)NH(CH2)3CH(NH2)COOH]+[OOC(CH2)2COO]2-. The ingredients in the solution are found in certain proportions, wt %.

EFFECT: invention provides enhanced detoxification activity, low toxicity and wide range of clinical applications.

11 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to production of medications for treating dermatosis. Medication according to invention, made in form of cream, contains mometasone furoate, preservative, hydrophilic no-aqueous solvent, emulsifying agent of 1st kind, emulsifying agent of 2nd kind, emollient, disodium edetate (trilon B), pH-regulating agent, and purified water in quantities, given in invention formula.

EFFECT: invention can be applied for treating inflammatory diseases and itching in case of dermatosis, yielding to glycocorticosteroid therapy.

9 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to cardiac surgery, and represents cardioplegic solution, which contains sodium chloride - 3.41-3.62, potassium chloride - 1.092-1.156 g, magnesium chloride - 3.190-3.485 g, calcium gluconate - 0.0105-0.0130 g, mannite - 4.365-4.520 g, L-carnosine - 20.1504-24.1650 g, N-acetylcarnosine - 8.056-11.032 g, L-histidine - 0.705-0.820 g, water for injections to 1000 ml.

EFFECT: invention ensures prevention of reduction of amplitude, speed of front and speed of pulse-wave reduction, as well as increase of diastolic pressure in left heart ventricle during reperfusion with preservation of buffer capacity and osmolarity of cardioplegic solution with physiological pH parameters.

4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: composition has an action of the central nervous system; it is presented in the form of a solution, contains glycine, glycerol, a preserving agent and water. The invention also concerns a therapeutic agent and a biologically active addition based on the above composition.

EFFECT: group of inventions provides high bioavailability as the composition is presented in the liquid and ease of dosing.

12 cl, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics, namely represents pharmaceutical formulations containing 9-cis-retinyl esters in a lipid excipient. The pharmaceutical formulations containing 9-cis-retinyl esters are described to be applicable in a retinoid replacement therapy for treating retinal degenerations in individuals.

EFFECT: using the formulations for the retinoid replacement therapy for treating retinal degenerations in individuals.

73 cl, 14 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine. What is described is a method for preparing a high-purity drug preparation for treating degenerative-dystrophic diseases of peripheral synovial joints and spinal column. The method provides introducing L-proline, an amino acid in an amount of 10-70 g/l into aqueous chondroitin sulphate solution containing no more than 11.5% of an active ingredient, and filtering the solution at temperature 20-50°C through ZetaCarbon (Cuno) R54SLP, R51SLP, R53SLP or AKS (Pall) - AKS 1, 2, 6, 7 filters.

EFFECT: method provides preparing the high-grade 99% pure liquid or lyophilised chondroprotective preparation stabilised with L-proline with the residual content of organic purities: protein - no more than 0,2%, lipids (per 1mg of chondroitin sulphate) - no more than 0,05 mcg, bacterial endotoxins (per 1mg of chondroitin sulphate) - no more than 0,05 EU.

6 cl, 3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to a method for Helicobacter pylori eradication of a gastroduodenal zone by a silver nitrate monotherapy consisting in administering an electrolyte solution of silver ions in the concentration of 300 mcg/l in a daily volume of 960 ml, for the first three days - in an amount of 120 ml every 3 h, 160 ml - every 4 hours and for the following three days - every 6 hours in an amount of 240 ml.

EFFECT: achieving the stable eradication of the vegetative and coccal forms of Helicobacter pylori, reducing the length of treatment by the early recovery of the involved gastric and duodenal mucosa.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: object of the invention is a method and a pharmaceutical composition in the form of a water-alcohol solution of ethanol 30-60° and water 40-70%, wherein at least one hypoglycaemic active substance is stably and completely dissolved, to be used by administering through the oral mucosa as a therapeutic agent in accurate treatment of postprandial hyperglycemia accompanying type II diabetes mellitus in a human or animal; wherein the water-alcohol solution in the composition has a volume of less than 2 ml, wherein an amount of 250mg or less of the above active substance is stably and completely dissolved, while the hypoglycaemic active substance is specified in lipophilic or amphiphilic active substances, such as gliclazide, glinides, incretins and glyphins. The invention also refers to a method for preparing this dosage form.

EFFECT: preparing the therapeutic agent for treating postprandial hyperglycemia accompanying type II diabetes mellitus.

9 cl, 20 ex

FIELD: chemistry, pharmaceutics.

SUBSTANCE: invention relates to method of treating proliferative disease in subject, including introduction to subject of (a) therapeutically effective quantity of AC220 or its salt in dose from approximately 27 to 1000 mg/day, and of (b) second agent, selected from azacitidine, cytarabinum, etoposide, daunorubicin, cladribine, where azacitidine is introduced in dose 50-100 mg/m2/day, cytarabinum is introduced in dose from 5 mg/m2/day to 3 mg/m2/day, etoposide is introduced in dose 10-150 mg/m2/day and daunorubicin is introduced is dose 10-60 mg/m2/day.

EFFECT: invention makes it possible to extend arsenal of medications for treating proliferative diseases, including cancer.

39 cl, 10 dwg, 26 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to the field of pharmaceutics and medicine and deals with the application of cysteamine in the treatment of a microbial infection, caused by a microbial biofilm, as well as to a product, containing at least two anti-biofilm agents, where at least one anti-biofilm agent represents an antimicrobial peptide, and the second anti-biofilm agent represents cysteamine. Also claimed are the application of the said product in the treatment of a microbial infection and a method of preventing the formation of the microbial biofilm in a medium.

EFFECT: group of inventions provides higher antibacterial activity in comparison with the activity of any compound separately.

13 cl, 33 dwg, 2 tbl

Up!