Amide of nonapeptide preventing increase of hyperpermeability of vessel endothelium

FIELD: biotechnologies.

SUBSTANCE: peptide of the formula H-(N-Me)-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH2 is proposed. The proposed peptide may find application as an agent for reduction of pathological hyperpermeability of vessel endothelium in various areas of medicine (in cardiology, toxicology, neurosurgery, oncology, etc.).

EFFECT: prevention of sharp increase of vessel endothelium permeability when injected to a recipient.

3 cl, 4 dwg, 1 tbl, 5 ex

 

The present invention relates to medicines for the treatment of disorders of the extracellular fluid, namely to biologically active peptides that can inhibit the increase in the permeability of the vascular endothelium. Due to this property, this peptide can be used in medicine as a decongestant means, and the means of treatment and prevention of other conditions associated with a pathological increase in the permeability of the vascular endothelium.

Impaired barrier function of the endothelium is accompanied by increased permeability of the vascular wall and the development of tissue edema, the effects of which can range from minor to catastrophic proportions if the lungs, brain and other vital organs. This problem is often observed in several pathological conditions, such as acute heart failure, abnormal heart valves, kidney failure, cirrhosis of the liver, cancer, exposure to toxic substances and other and, despite advances in modern medicine, continues to be a cause of high mortality, even in conditions of inpatient treatment. To combat edema are used diuretics, crystalloids and steroid hormones. These compounds improve the excretion of fluids from the body, but does not impact the molecular mechanisms of edema development - increased permeability of the endothelial lining microvessels, resulting contractile activity of endothelial cells.

One of the key regulators of contractile activity of cells, including endothelial, is a light chain kinase myosin (CLSM). This enzyme is activated by the action of many damaging factors and triggers a reduction in the microvascular endothelium, which leads to disruption of the barrier function and the development of tissue edema. In this regard, obviously, a promising approach to reducing hyperprolinemia vessels terminal vascular beds in stressful situations is the inhibition of the contractile response of the endothelium by inhibiting endothelial CLCM.

However, most of the existing inhibitors CLCM not suitable for use in humans due to serious side effects. In this regard, the actual search inhibitors CLCM characterized pharmacologically elevated structure and high specificity. One of these compounds is a low molecular weight inhibitor, created on the basis of aminopyridazine [1. Behanna N.A., D.M. Watterson, Ranaivo H.R. Development of a novel bioavailable inhibitor of the calmodulin-regulated protein kinase MLCK: a lead compound that attenuates vascular leak. Biochim. Biophys. Acta. 1763 (11), 1266-1274, 2006. 2. Rossi J.L., Velentza A.V., Steinhom D.M., D.M. Watterson, M.S. Wainwright MLCK210 gene knockout or kinase inhibition preserves lung function following endotoxin-induced lung inury in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 292 (6), L1327-1334, 2007. 3. Wainwright M.S., Rossi J., Schavocky J., Crawford, S., Steinhom D., Velentza A.V., Zasadzki M., V. Shirinsky, Y. Jia, J. Haiech, Van Eldik L.J., D.M. Watterson Protein kinase involved in lung injury susceptibility: evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment. Proc. Natl. Acad. Sci. USA. 100 (10), 6233-6238, 20031-3]. In recent years the world designed peptide inhibitors CLSM, which was shown to influence the permeability of the intestinal epithelium in the experiment in the absence of severe toxicity [Clay burgh D.R., Barrett T.A., Tang Y., Meddings J.., Van Eldik L.J., D.M. Watterson, Clarke L. L., R.J. Mrsny, Turner J.R. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest. 115 (10), 2702-2715, 2005.]. The nonapeptide H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 (L-to-PEAK), created on the basis of autoinhibitory plot CLTM [T.J. Lukas, S. Mirzoeva, Slomczynska, U., D.M. Watterson Identification of Novel Classes of Protein Kinase Inhibitors Using Combinatorial Peptide Chemistry Based on Functional Genomics Knowledge. J. Med. Chem. V.42., p.910-919, 1999.], able to penetrate through the plasma membrane and affects epithelial permeability in vitro [Clayburgh D.R., Barrett T.A., Tang Y., Meddings J.., Van Eldik L.J., D.M. Watterson, L.L. Clarke, R.J. Mrsny, Turner J.R. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest. 115 (10), 2702-2715, 2005.], however, the nature of its chemical structure is extremely susceptible to the action of proteolytic enzymes [Owens SE, Graham W.V., Siccardi D., Turmer J.R., R.J. Mrsny, A Strategy to Identify Stable Membrane-Permeant Peptide Inhibitors of Myosin Light Chain Kinase. Pharm. Res. 22 (5), 703-709, 2005]. This casts doubt in the possibility of its application in practical medicine. Modification of the peptide L-PEAK by replacing part of L-amino acids to D-configuration, as expected, increases the resistance of the peptide to proteolytic degradation [Clayburgh D.R., Barrett T.A., Tang Y., Meddings J.., Van Eldik L.J., D.M. Watterson, L.L. Clarke, R.J. Mrsny, Turner J.R. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest. 115 (10), 2702-2715, 2005]. However, the inhibitory activity of D-PEAK is much inferior to that of the L-PEAK, apparently, due to the fact that the D-PEAK is full enantiomer L-PEAK and has a different spatial structure [Seredova A., Sidorova M.V., Asiaco A.A., Molokoedov A.S., Bushuev, VN, Marchenko A.V., O. Shcherbakova, Shirinsky VP, Bespalova RAILWAY Peptide inhibitors of kinases light chains of myosin, resistant to the action of proteases. Bioorganic chemistry 36 (4), 498-504, 2010]. Because in a series of peptide analogs there is no clear relationship between structure and biological activity, even a small modification of the peptide can dramatically affect its inhibitory properties.

Closest to the claimed peptide is a peptide PICK formula H-(W-Me)-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2(or [NMe-Arg1]-PEAK). When comparing peptide inhibitors CLCM with various modifications of the structure of the original L-PEAK (D-PEAK, [NMe-Arg1]-PEAK [ε1]-PEAK [Cit1]-PEAK [Cit1,Orn3PEAK) was shown to that activity in vitro comparable to the original L-PEAK, has only [NMe-Arg1]-PEAK (PIC) modification of the N-terminal amino acids of the original peptide L-PEAK [Seredova A., Sidorova M.V., Asiaco A.A., Molokoedov A.S., Bushuev, VN, Marchenko A.V., O. Shcherbakova, Shirinsky VP, Bespalova RAILWAY Peptide inhibitors of kinases light chains of myosin, resistant to the action of proteases. Bioorganic chemistry 36 (4), 498-504, 2010]. Time life PIC in plasma t vitro almost 3 times the lifetime of the L-PEAK, and at a concentration of 100 μm PIC reduced the thrombin-induced increase in the permeability of the monolayer of endothelial cells [Marchenko A.V., Stepanov EO, Sekigawa A., Sidorova M.V., Bushuev, VN, Bespalova RAILWAY, Shirinsky VP New peptide inhibitors of kinases light chains of myosin suppress hyperproliferate vascular endothelium. Biophysics, 55, 1008-1013, 2010]. However, the effect PIK observed in relatively high concentration ranges (about 100 microns)that pharmacological point of view makes it less attractive for use in vivo.

The objective of the invention is the provision of effective suppression of hyperprolinemia vascular endothelium, and the creation of a pharmaceutical composition for injection for control of acute edema of the vital organs. The urgency of this problem is caused by the need to provide practical medicine is effektivnye drugs, suppressing hyperproliferate vascular endothelium, in particular for the control of acute pulmonary edema in a critical patient situations.

The technical result is to obtain a peptide having enhanced activity against suppression hyperprolinemia vascular endothelium and increased life time in the blood plasma.

The problem is solved by the synthesis of methylated (Me) on aminobenzoyl group nonapeptide amide of the formula H-(W-Me)-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg8-Lys-NH2(PIC) and its pharmaceutically acceptable non-toxic salts. The problem is solved also by the creation of a pharmaceutical composition for injection containing a therapeutically effective amount PIC and a pharmaceutically acceptable carrier. And the problem is solved by providing a method of reducing hyperprolinemia vascular endothelium in the body through injections of a therapeutically effective quantity of a specified pharmaceutical compositions.

The invention is explained in 4 (four) illustrations, in which figure 1 and Figure 2, respectively, presents fragments of1N-NMR spectra of the prototype (PIC) and the claimed peptide (PIC) in solution and in plasma of human blood (minus the spectrum of blood plasma) showing signals of the respective groups of free amino acids. Figure 3, not only the dynamics on the phosphorylation of the regulatory light chains of myosin under the action of CLSM in vitro in the presence of the claimed peptide PIC and peptides prototype L-PEAK and PICK at a concentration of 100 nm. Figure 4 presents the influence of the proposed peptide (PEAK 2) and prototype (PEAK 1) on the diffusion of albumin conjugated with fluoresceinisothiocyanate through a monolayer of endothelial cell line EA.hy926. Presents the relative permeability of the endothelium after 4 hours after stimulation with 100 nm thrombin in the presence of 10 μm (white bars), 20 μm (black bars) and 100 μm (hatched columns) of the inventive peptide (right) and prototype (left). Permeability control endothelial cells after stimulation by thrombin is accepted for 100% (dotted line).

The inventive peptide was obtained by a solid phase method of peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-technology described below in example 1.

Pharmaceutically acceptable salts of the peptide of the invention include salts formed with inorganic or carboxylic acids. Examples of inorganic acids, forming salts include halogen acids such as hydrochloric, Hydrobromic acid, phosphoric acid, sulfuric acid. Salts of carboxylic acids are formed with such acids as acetic, propionic, malonic, maleic, citric, succinic, malic, benzoic, fumaric, and other carboxylic acids. Salts with acids receive conventional methods, for example, by using a neutralizing peptide PIC in formatvorlage base with acid. Preferred salts are sulfate and hydrochloride. As indicated above, the present invention includes a solvate of the compounds of this invention and their pharmaceutically acceptable salts. The inventive peptide PIC or its pharmaceutically acceptable salt may form a solvate with water or common organic solvents. Such a solvate is included in the scope of the present invention.

The term "pharmaceutically acceptable" means a carrier that is compatible with other ingredients of a pharmaceutical composition and not having toxic or adverse effects on patients. Because the inventive peptide is highly soluble in aqueous solutions, as carriers for PICK can be used, for example, isotonic sodium chloride solution 0.25%, sterile water for injection, ringer's solution for injection, blood substitutes, glucose solution for infusion 5%, solutions of other active drugs for intravenous or intramuscular injection in the absence of adverse drug-drug interactions between PIC or its pharmaceutically acceptable salts and second active substances (active substances).

Therapeutically effective amount PIC as active principle is defined as the amount needed to achieve the desired physiological about the reply from the recipient, and may depend on the body weight of the recipient, the severity of the condition of the recipient, the individual physiological characteristics of the recipient conducted by concomitant therapy and other Number PICK in the pharmaceutical composition will depend on the shape of its release - the lyophilisate for solution for injection or solution for injection. Effective single dose PIC for intravenous administration can be from 0.1 to 1.5 mg/kg of body weight of the recipient. With the introduction of the drug intramuscularly at the dose required for effective reduction of hyperprolinemia vascular endothelium, will be higher and can be up to 5 mg/kg of body weight of the recipient.

Drugs with the claimed peptide or its pharmaceutically, priemlimymi salts as the active substance can be represented in the form of a lyophilisate for solution for injection or in the form of a ready solution for injection. Preferred is liofilizovannye form for dissolution.

Methods of administration of the medicinal product with the claimed peptide or its pharmaceutically acceptable salts as an active ingredient include intravascular bolus, the introduction of infusion part of other solutions for introduction, components of which the claimed peptide PIC or its pharmaceutically acceptable salt is not the way the t unwanted interactions, introduction intramuscularly. The preferred method of administration is by bolus intravascular injection, allowing you to quickly achieve therapeutically effective concentrations of the active substance in the blood of the recipient. Indications for use of the inventive peptide or pharmaceutically acceptable salts should consider the appearance of clinical symptoms or signs of development hyperprolinemia vascular endothelium, for example, in acute pulmonary edema such symptoms are cyanosis, pallor, profuse sweat, the alternation of the pulse, accent II tone of the pulmonary artery, protodiastolic gallop rhythm. Introduction PIC for symptomatic treatment should be done immediately if signs of acute edema.

Example 1. The synthesis of the inventive peptide (PIK)

H-(N-Me)-Arg1-Lys-Lys-Tyr-Lys-Tyr-Arg7-D-Arg8-Lys-NH2

In the paper, we used a derivative of the amino acid (AA) and hexaflurophosphate (benzotriazol-1-yl)oxy-Tris(dimethylamino)phosphonium (BOP, NovaBiochem, Switzerland), N,N'-diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIPEA), hydroxybenzotriazole (HOBt), triisobutylene (TIBS) the company Fluka, Switzerland, 4-methylpiperidine (4-MePip, Aldrich, USA). For the synthesis of applied N is an organic, dichloromethane (DCM), triperoxonane acid (TFA) of the company Fluka, Switzerland, for chromatography - acetonitrile(Panreac, Spain). Analytical high performance liquid chromatography (HPLC) was performed on the chromatograph (Gilson, France)was used column Nucleosil 100 C18, 5 μm, (4.6×250 mm) (Sigma, USA) as eluents used buffer A - 0.1% TFA, buffer B was 80% acetonitrile in buffer A, elution with a concentration gradient of buffer B in buffer And from 0% to 60% in 30 min flow Rate 1 ml/min, detection at 220 nm. The structure of these peptides proved spectra1H-NMR data and mass spectrometry.1H-NMR spectra were taken on the spectrometer WM-500 (Bruker) 500 MHz (Germany) in deuterated dimethylsulfoxide (DMSO-d6) at 300 K, the concentration of the peptides was 2-3 mg/ml Chemical shifts were measured relative to tetramethylsilane. Mass spectra were recorded on the instrument PC-Kompact MALDI (Kratos, UK).

For solid-phase synthesis used is a copolymer of styrene with 1% divinylbenzene, 4-(2,4-acid)-9-fluorenylmethoxycarbonyl-aminomethylphenol - anchor group (Rink-amide-polymer) company Nova BoiChem, Switzerland, designed to obtain amides of peptides containing 0.64 mmol/g of amino groups. The synthesis of the nonapeptide amide conducted with end stage (adding one amino acid), on the basis of 0.40 g (0.25 mmol) Rink-amide-polymer. The first 8 cycles of synthesis was performed in automatic mode on the peptide synthesizer, Applied Biosystems 431 And the standard program for onocr the things of condensation of Fmoc-amino acids. The guanidine group of residues Arg and Arg blocked using 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) (see Protocol for solid-phase synthesis). To attach the N-terminal, N-methylsiloxane residue Arg1the guanidine function defended using 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), and to create amide bond used a highly effective reagent Castro - THIEF [Castro C., J. R. Dormoy, Evin G., C. Selve Peptide coupling reaction with benzotriazol-l-yl-tris(dimethylammo)phosphonium hexafluorophosphate. Tetrahedron Lett. V.14. P.1219-1222, 1975].

Protocol for solid-phase synthesis
No.OperationReagentProcessing time
Cycles 1-8
1Flushing5x N-organic (NMP)3 min
2The release of α-amino groups25% 4-MePip/NMP10 min
3Flushing5×NMP3 min
4Activation1 m the ol Fmoc-AA+1 mmol of NOUT+1 mmol of DIC in NMP 20 min
5Condensation1 mmol of activated derivative Fmoc-AA in NMP90 min
6Flushing5×NMP3 min
Cycle 9
1Flushing5×NMP3 min
2The release of α-amino groups25% 4MePip/NMP10 min
3Flushing5xNMP3 min
4Activation1 mmol of Fmoc-(N-Me)Arg(Mtr)-OH+1 mmol THIEF+1 mmol of NEWT+2 mmol DIPEA in NMP5 min
5Condensation1 mmol of activated derivative Fmoc-AA in NMP90 min
6Flushing5xNMP3 min

Final released is the formation and cleavage of the nonapeptide from the polymer were carried out in one stage by treatment of the corresponding nonparticipation a mixture of 10 ml of TFA, 0.25 ml of deionized water and 0.25 ml of TIBS for 16 hours Then the polymer was filtered off, washed with 2×2 ml deblokiruyuschee mixture, the filtrate was evaporated and to the residue was added dry ether. The precipitate was filtered off, washed with DCM (3×3 ml), ether (3×5 ml), was dried in a vacuum desiccator. 0.32 g of crude product of solid-phase synthesis with a basic substance content of 76% was purified using preparative HPLC on a Beckman instrument (USA), using a column of diasorb C16 13 FROM (25×250 mm), the size of the sorbent particles 10 microns. As eluents used the buffer And 0.01 M solution of ammonium acetate and buffer B was 80% acetonitrile in water. The elution was performed with a gradient of 0.5% per minute buffer B in buffer And from 100% buffer And at a rate of 10 ml/min, the Peptides were detected at a wavelength of 220 nm. The fractions containing the desired product were combined, the acetonitrile was evaporated and liofilizovane. In the end I was 0.16 g (47.1% (based on the starting amino acid attached to the polymer) nonapeptide amide PIC. The homogeneity of the product was determined using analytical HPLC, is 96.8%. Mass spectrum: 1338.6 calculated 1338.7.

The life time of the claimed peptide (PIC) and peptide-prototype (PIC) in plasma were compared by the method of1H-NMR as described in example 2. The results of the comparison of the claimed peptide (I) and the prototype presented in figure 1 and figure 2, respectively.

Example 2. The lifetime of the peptides in the square is the ZMA blood in vitro

Under aseptic conditions from healthy donors were taken in 100 ml of whole blood in a 50 ml test tubes with 5 ml of preservative (130 mm citrate phosphate-buffered saline (FSB), pH 7.4). The blood kept for 15-20 min at room temperature and centrifuged at 1500 g for 15 min at 18°C. the Supernatant was transferred into a new tube and centrifuged again under the same conditions. Measured the amount of blood plasma was added dry NaBr based 280 mg of NaBr per 1 ml of plasma. After the dissolution of NaBr plasma (approximately 30 ml) was poured into the centrifuge glasses (for rotor Type 45Ti, Beckman, USA) and were layered on top of 20 ml of a solution with a density of 1,216 g/l of the following composition: 11,42 g NaCl and 100 mg ethylenediaminetetraacetic acid (EDTA) was dissolved in 1 l distilled water, pH 7.4, was then added 281,44 g NaBr. Centrifuged at 40,000 rpm for 48 hours After centrifugation was selected the top dark yellow liquid layer containing lipoproteins, and colorless layer of the buffer, leaving the buffer to a height of approximately 1 cm below the visible border of the plasma. Plasma obtained from a single donor, were combined and were dialyzed for 36 h against 4 l of the FSB, free from ions of CA2+and Mg2+(MP Biomedicals, USA) in dialysis bags with a pore size of 12-14 kDa (Medicell, UK). The dialysis procedure was repeated twice for 24 h against fresh buffer. After dialysis plasma to powergeneration at 25000 rpm rotor Type 45Ti (Beckman, USA) for 15 min at 4°C. the Supernatant was collected and was liofilizovane. The residue was dissolved in D2O (in the same volume of plasma to make NaBr) and again liofilizovane. Then was dissolved in the same volume, and kept the aliquot 1 ml at -20°C.

Spectra 1H-NMR were obtained on a spectrometer WM-500 (Bruker, USA) at 37°C. the Chemical shifts of the signals in the spectra were measured relative to internal standard sodium salt of 2,2-dimethyl-2-silapentane-5-sulfonate. The assignment of the signals was performed using the double resonance. For selection of signals belonging to the protons of the peptide was applied differential spectroscopy: from each spectrum obtained after addition of the peptide in blood plasma of a read range of free plasma. The concentration of peptide in the sample was 3 mg/ml

As can be seen from Figure 1, in the case of peptide-prototype PIC through 190 min incubation in plasma of human blood in vitro, well defined signals corresponding αN-groups of free arginine and lisened. This indicates a degradation of the C-terminal part of the peptide, leading to the formation of free amino acids. While the half-life of PIC in plasma is about 120 min [Seredova A., Sidorova M.V., Asiaco A.A., Molokoedov A.S., Bushuev, VN, Marchenko A.V., O. Shcherbakova, Shirinsky VP, Bespalova RAILWAY Peptide inhibitors is Inez light chains of myosin, resistant to proteases. Bioorganic chemistry 36 (4), 498-504, 2010]. At the same time, in the case of peptide PIC (2) the original spectrum unchanged even after 420 min of incubation in plasma, indicating high stability PIC in plasma with a half-life >420 min Thus, the inventive peptide is characterized by increased life time in the blood plasma. This allows you to maintain an effective concentration required to reduce hyperprolinemia vascular endothelium in critical States. The increased lifetime of the peptide also allows the use of lower, when compared with homologues concentrations (5-30 μm in circulating blood) while maintaining therapeutic effect.

The increase in the lifetime of the peptide when replacing the natural L-configuration and the carbon atom of residue Arg8on the D-configuration in the structure of the claimed peptide PIC was to be expected, since it is known that the peptide bond formed by D-amino acids are more stable than the ties of L-amino acid analogs as are not recognized by proteolytic enzymes [Hruby VJ, X. Qian Approaches to the asymmetric synthesis of unusual amino acids. Methods Mol. Biol. 35, 249-286, 1994]. However, along with increasing the life time of modification may adversely affect the inhibitory activity of the peptide. Proof of this is is our previous comparative study of the activity of the peptide inhibitors of the D-PEAK, [NMe-Arg1]-PEAK [ε1]-PEAK [Cit1]PEAK and [Cit1,Orn3]PEAK, of which only [NMe-Arg1]PEAK, (PIC) had activity in vitro that is comparable to the peptide L-PEAK [Seredova Century A., Sidorova M.V., Asiaco A.A., Molokoedov A.S., Bushuev VP, Marchenko, A.V., O. Shcherbakova, Shirinsky VP, Bespalova RAILWAY Peptide inhibitors of kinases light chains of myosin, resistant to the action of proteases. Bioorganic chemistry 36 (4), 498-504, 2010]. Our subsequent studies aimed at addressing the objectives of this invention showed that the peptides H-DLys-DArg-DArg-DTyr-DLys-DTyr-DLys-DLys-DArg-NH2(retrognathia-to-PEAK), H-Arg(NO2)-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2(protected N-terminal part of the peptide with the introduction of the nitro-group in the guanidine group of Arg residue1), H-Argψ[CH2NH]-Lys-Lys-Tyr-Lys-Tyr-Arg-DArg-Lys-NH2(protected N-terminal part of the peptide by administering pseudopeptides connection in the first position), H-(NαMe)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-Pro-Gly-Pro-OH with N-terminal part of the peptide by introducing a methyl group and protection of the C-terminal part with Tripeptide Pro-Gly-Pro), and peptide H-Argψ[CH2NH]-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-Pro-Gly-Pro-OH with N-terminal part of the peptide by administering pseudopeptides connection in the first position and the protection of the C-terminal part with Tripeptide Pro-Gly-Pro) also was significantly lower than the claimed peptide PIC on inhibitory aktivnosti vitro against purified CLCM and model of the suppression of permeability of endothelial monolayer in vitro.

The inhibitory activity of the inventive peptide (PIC) and prototypes (PICK and L-to-PEAK), which is the most active of the known peptides of the family of the PEAK, were compared for their effect on the phosphorylation of the regulatory light chains of myosin in vitro as described in example 3. The results are shown in Figure 3.

Example 3. Phosphorylation of the regulatory light chains of myosin in vitro

Regulatory light chain (RLC) myosin at a final concentration of 20 μm was fosforilirovanii CLCM from the bowels of turkeys (9 nm) at 30°C in buffer containing 10 mm 3-RM-Morpholino]propanesulfonic acid (MOPS), pH 7.0, 100 mm NaCl, 0.5 mm CaCl2, 1 mm MgCl2, 0.5 mm Mg-ATP, 0.3 μm calmodulin, 1 mm dithiothreitol (DTT) in the presence of 100 nm of the claimed peptide or peptides prototype L-PEAK or PIC. The reaction was initiated by adding CLCM. After a certain period of time (0-40 min), aliquots of the reaction mixture (5 μl) was collected and dissolved the sample buffer. The complete passage of the reaction were analyzed by electrophoresis in the presence of urea and glycerin that is used to separate proteins on the basis of the magnitude of their charge. The electrophoresis was carried out according to [Persechini A, Kamm KE, Stull JT. Different phosphorylated forms of myosin in contracting tracheal smooth muscle. JBC, 261, 6293-6299, 1986] with modifications. Protein preparations were dissolved in sample buffer (20 mm Tris-HCl, pH 6.8, 9 M urea, 10 mm DTT, 0.05% of bromophenol is ini). Used 5,2% concentrating gel prepared in 20 mm Tris-HCl buffer pH 6.8, containing 16.5% of glycerol and 3.25 M urea; and 12% separating gel, prepared in 20 mm Tris-23 mm glycine buffer pH of 8.6, containing 40% glycerol. The electrophoresis was carried out in 20 mm Tris-23 mm glycine buffer, pH of 8.6, containing 0.2% β-mercaptoethanol at 330 20 min and 400 V for 180 min, the Gels were fixed and stained, Kumasi R-250. The experiments were repeated at least 3 times. Electrophoretic mobility of the protein bands corresponding to RLC and phospho-RLC, were set according to the mobility of the respective standards. Gels were scanned and quantitative densitometric analysis of the obtained electrophoregram was performed using the program Image J, version 1.45. The degree of phosphorylation RLC myosin was calculated as the ratio of phosphorylated RLC to the total number RLC. The obtained data were processed in Excel and graphics built according to the degree of phosphorylation RLC myosin (mol phosphate/mol RLC) from time to time in the presence of different concentrations of peptide. Data were expressed as mean + STD. off.

From the above example shows that under these conditions the reaction in the absence of inhibitor full phosphorylation RLC myosin is achieved after 15 minutes At the same time in the presence of peptides prototype L-PEAK and PICK the reaction proceeds at 90-95%,while in the presence of the claimed peptide (PIC) the reaction proceeds only at 50-60%. These data suggest a stronger inhibitory properties of the claimed peptide PIC compared with peptides-prototypes L-PEAK and PIC. It should be noted that under these conditions, the lifetime of the peptides have no effect on the inhibitory activity, since the reaction is carried out in the absence of proteolytic enzymes.

The influence of the proposed peptide (PIC) and prototype (PIC) on the permeability of the endothelium was assessed by the rate of diffusion of FITZ-albumin through a monolayer of endothelial EA.hy926 cells as described in example 4. The results of the comparison of the claimed peptide (PIC) and prototype PIC presented in figure 4.

Example 4. The permeability of the endothelial monolayer in vitro

The effect of peptides on the permeability of the endothelium was assessed by the rate of diffusion of labeled fluoresceinisothiocyanate (FITZ) albumin through a monolayer of endothelial EA.hy926 cells (figure 3). On membrane inserts ThinCerts (Greiner Bio One, Germany) with a pore diameter of 0.4 µm and density of the pores is 1×108/cm2planted 1×105endothelial cell line EA.hy926 to 200 ál of nutrient medium Needle in the modification of Dulbecco (DMEM) with a high content of glucose in the presence of 20% fetal calf serum. In the outer chamber made of 800 ál of the same medium without cells. Cells were cultured for 4 days. During this time the cells reached monolayer. The growth medium was changed to DM is M without fetal serum and were deprived cells within 30 minutes. Then in the lower and upper chambers were added to the analyzed peptide at concentrations of 10-100 μm and incubated for 30 minutes. Then in the upper chamber were added FITZ-albumin to a final concentration of 0.5 mg/ml every 30 min samples were taken from the bottom of the camera to calculate the basal rate of diffusion of FITZ-albumin through an endothelial monolayer (relative units of fluorescence intensity per hour). The change in the permeability of the endothelium induced by addition of 100 nm thrombin in the upper chamber and at intervals of 30 min continued sampling of the medium from the lower chamber to analyze the content FITZ-albumin. Content FITZ-albumin in the samples was measured using multi-function tablet analyzer Victor X3 (PerkinElmer, USA). The results were presented as mean±standard deviation (Figure 4). Reliability of differences was determined by t-criterion of student.

From the above example shows that the inventive peptide (PIC) reduces thrombin-induced permeability of the monolayer of endothelial cells is more efficient than the prototype (PIC). So, after inactivated cells with 10 μm of the prototype response of endothelial cells by thrombin is reduced by only 10% and 90% of the response of cells in the absence of inhibitors. At the same time after inactivated with 10 μm of the claimed peptide (PIC) response to thrombin is reduced by 80%, accounting for 20% of the CTE is and cells in the absence of inhibitors. At a concentration of 20 μm prototype suppresses the response to thrombin by 40%, whereas the inventive peptide (PIC) in the same concentration completely abolishes the increased permeability of the monolayer. When the concentration of the prototype, equal to 100 μm, is achieved substantial (90%) inhibition of the response to thrombin. At the same time after inactivated with 100 μm peptide (PIC) increased barrier function of the monolayer and the permeability was 30% below the baseline permeability control cells in the absence of stimulation by thrombin.

The applicability and effectiveness of drugs, including the claimed peptide PIC or its pharmaceutically acceptable salt, to reduce hyperprolinemia vascular endothelium in vivo experiments demonstrate with experimental acute pulmonary edema caused by a bacterial liposaccharides. The final functional element in the development of tissue edema is strengthening the pair - and transcellular transport of substances through the cells of the vascular endothelium. These processes involve the activity of endothelial CLCM. Therefore, the use of bacterial liposaccharide allows you to simulate the development of acute pulmonary edema of various etiologies.

Example 5. Model of pulmonary edema in vivo.

The male Wistar rats weighing 300-340 g under ketamine anesthesia (100 mg/kg) was inserted catheters in the carotid artery and jugular vein of your registration file average blood pressure (BP), heart rate (HR) and for the introduction of substances. After 10 min of registration original settings rats were injected lipopolysaccharide (LPS) of S. typhimurium at a concentration of 8 mg/kg in saline. After 20 min the rats were administered the inventive peptide (PIC) at a concentration of 1.5 mg/kg intravenous bolus of 0.5 ml of saline. At 24 h after the experiment the rats were scored by an overdose of urethane. The chest was opened, was aspirated and measured amount of fluid from the pleural cavities were removed and weighed lungs. The lungs were dried for 3 days at a temperature of 54°C to constant weight. The total water content in the lungs was determined as the ratio of wet weight of the lung plus the pleural effusion to the dry weight of the lung. In the control group of animals was determined these parameters without the introduction of FSC and PIC. The results were presented as mean ± error of the mean (see table). Reliability of differences was determined by t-criterion of student.

The influence of the proposed peptide PIC on the severity of pulmonary edema in rats
GroupQty animalsThe weight of animals (g)The ratio of the initial mass and dry in the society of lightThe liquid formed in their (initial weight - dry + effusion, g)
control6320±6with 4.64±0,061,08±0,05
FSC15323±11to 4.92±0,041,61±0,07
LPS+PIC17311±104,80±0,041,35±0,05

As can be seen from the table, the introduction of LPS leads to a significant increase in the quantity of light caused by the ingress of water and solutes, as well as high-molecular compounds and formed elements of blood. With the introduction of the claimed peptide (PIUS) increase the amount of fluid in the lungs in response to exposure to LPS reduced in 2 times. Thus, the claimed peptide (PIC), introduced in the dose of 1.5 mg/kg (concentration in circulating blood about 30 μm), able to reduce acute pulmonary edema caused by exposure to bacterial endotoxin.

Because the mechanisms for increasing the permeability of the vascular endothelium is involved in the development of acute pulmonary edema of various etiologies, PICK when administered intravenously in saline or other pharmaceutically acceptable carrier which may be an effective way of dealing with acute critical conditions associated with a pathological increase in the permeability of the vascular endothelium. The most important field of application of the inventive pharmaceutical composition seems to struggle with acute pulmonary edema, which is the reason for the high mortality rate of patients. Acute pulmonary edema may occur during the carrying out of an artificial lung ventilation during surgery, sepsis, poisoning, inflammatory and allergic reactions, injuries (burns, squeezing), ischemia-reperfusion injury, heart failure and other disorders.

1. Peptide with enhanced activity against suppression hyperprolinemia vascular endothelium and increased life time in the blood plasma, having the formula H-(N-Me)-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH2.

2. Pharmaceutical composition having the ability to suppress hyperproliferate vascular endothelial containing a therapeutically effective amount of a peptide according to claim 1 and a pharmaceutically acceptable carrier for injection.

3. The method of suppressing hyperprolinemia endothelium of blood vessels in the body through injections of a therapeutically effective amount of the pharmaceutical composition according to claim 2.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to molecular biology, pharmacology and medicine. Disclosed is an oligopeptide KNNMINNDLG isolated from oligopeptide PB1 (531-540), which is active against both human influenza H1N1 (EC50=28.6 mcg/ml) and bird influenza H5N2 (EC50=35.3 mcg/ml).

EFFECT: invention can be used for vaccination against influenza virus subtypes H1 and H5.

2 cl, 1 tbl, 2 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular medicine and a target-directed drug delivery. There are presented new peptide sequences sized 10 or less amino acids containing at least a continuous amino acid sequence D(LPR) which selectively influence on the VEGFR-1 and NRP-1 expressing cells.

EFFECT: target-directed molecules according to the inventions are applicable for treating and diagnosing the neovascular and angiogenic VEGF-associated disorders, such as cancer, obesity, diabetes, asthma, arthritis, cirrhosis and eye diseases.

16 cl, 4 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: disclosed are peptides which are isolated from the FOXM1 protein and are capable of activating cytotoxic (killer) human T cells by forming an antigen-presenting complex with a HLA-A2 molecule. Disclosed are compositions which contain the disclosed peptides, use of a peptide to produce an agent for inducing cancer immunity, treating and preventing cancer, and for producing antibodies which selectivity bind the disclosed peptides. The invention describes an exosome and an isolated antigen-presenting cell, which present a complex of the disclosed peptide with a HLA-A2 molecule, for inducing cytotoxic T cells, methods of inducing a antigen-presenting cell and a cytotoxic T cell, as well as a method of damaging FOXM1 and HLA-A2 expressing cells.

EFFECT: invention can further be used in treating tumours that are characterised by high FOXM1 expression.

14 cl, 2 ex, 1 dwg, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biochemistry and immunology, and may be used in pharmacology for preparing medicines with anticancer activity. What is described is a peptide representing a fragment (351-359) of polypeptide 1 associated with the cell division cycle (CDCA1), and possessing ability to induce cytotoxic (killer) T cells, as well as analogues thereof, including a replacement of an amino acid the second next to an N-terminal by methionine and/or an C-terminal amino acid by valine or leucine and preserving its inducing action on killer T cells.

EFFECT: what is presented is using the peptides according the invention both directly, and as an ingredient of immunogenic compositions (vaccines), for preventing and/or treating cancer diseases associated with HLA-A2 and CDCA1 expression.

21 cl, 1 tbl, 16 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: there are presented versions of the peptide (A) or (B) with the amino acid sequence of SEQ ID NO: 1 or 2 respectively presented in this description. The peptide has activity to induce a cytotoxic T-cell when an antigen-presenting cell carrying HLA-A2 (A*0201) presents it. There are described the versions of the peptide antibodies prepared by immunisation by the proper peptide. There are presented: an agent, methods for inducing: a cytotoxic (killer) T-cell, an antigen-presenting cell, CDH3 expressing cancer immunity; as well as a method of treating CDH3 expressing cancer on the basis of the peptide. What is presented is an isolated cytotoxic T-cell induced by said method on the basis of the peptide. There are described: the antigen-presenting cell and exosome presenting the complex containing the peptide and HLA-A2 (A*0201).

EFFECT: higher effectiveness of the use of the invention in treating CDH3 expressing cancer.

15 cl, 5 dwg, 2 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: peptide is presented, which is produced from protein WT1, which is able to bind with a molecule HLA-A*1101 and to induce CTL, having a sequence SEQ ID NO:6 or SEQ ID NO:7, presented in the description. Besides, a peptide dimer is described, which is used for the same purposes and comprises two peptide monomers selected from a group of peptides that includes SEQ ID NO:7, SEQ ID NO:3, SEQ ID NO:8 and SEQ ID NO:9, available in the description. A nucleic acid is presented, which codes the specified peptide and the expression vector, which contains the specified nucleic acid. A pharmaceutical composition is described for treatment or prevention of cancer of an individual, positive by HLA-A*1101, which contains the specified peptide, nucleic acid or vector. A WT1-specific CTL is described, which is induced by the specified peptide or dimer, and an antigen-presenting cell that presents the peptide. There is data on the method and set for induction of the WT1-specific CTL and for induction of the antigen-presenting cell. The method of in vitro diagnostics of cancer in an individual positive by HLA-A*1101 is presented, which includes incubation of the specified CTL or the antigen-presenting cell with a sample received from an individual positive by HLA-A*1101, and detection of the quantity of the specified CTL or antigen-presenting cell.

EFFECT: invention makes it possible to expand assortment of peptides that bind with HLA-A*1101 and are produced from the antigen WT1.

14 cl, 1 tbl, 14 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing a peptide, which is characterised by that it involves conversion of the -SH group of the peptide which contains an amino acid residue having an -SH group to an -OH group, said method comprising the following steps from (a) to (c): (a) reaction of the -SH group in the peptide with a methylating agent to convert the -SH group to an -SMe group; (b) reaction of the -SMe group formed at step (a) with a cyanating agent to obtain an intermediate reaction product in form of an ester; (c) converting the intermediate reaction product obtained at step (b) to a peptide which contains an amino acid residue having an -OH group in more basic conditions than conditions at step (b).

EFFECT: improved method.

20 cl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. What is presented is a peptide having an ability to induce cytotoxic T-cells wherein the peptide consists of amino acid sequences SEQ ID NO:3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74, what is also presented is a peptide having an ability to induce cytotoxic T-cells wherein the peptide consists of amino acid sequences SEQ ID NO:3-5, 7-9, 12, 17, 67 or 74 wherein 1 or 2 amino acids are substituted or added. What is presented is a peptide having an ability to induce cytotoxic T-cells wherein the peptide consists of amino acid sequences SEQ ID NO:15-19, 22, 24, 27-30 and 37 wherein 1 or 2 amino acids are substituted or added. What is presented is a pharmaceutical agent used for regulation of T-reg cells containing 1 or more types of said peptides. What is disclosed is a method for inducing antigen-presenting cells. What is presented is a recovered cytotoxic cell and an antigen-presenting cell. What is disclosed is a method for regulation of T-reg cells; what is also disclosed is the used of said peptides for producing a vaccine. There are presented polynucleotides coding said peptides.

EFFECT: invention enables producing the peptides used in the pharmaceutical agent for regulation of T-reg cells.

21 cl, 10 dwg, 4 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biologically active peptide complexes with immunomodulating and antiviral activity. The disclosed peptide complexes have a three-dimensional structure wherein X1 is absent or contains at least 1 amino acid; R1 and R2 are peptide chains which contain amino acid residues, His or Cys, capable of reacting with transition metal ions, wherein R1 contains up to 5 amino acid residues or is absent; R2 contains up to 3 amino acid residues or is absent.

EFFECT: peptide complexes rich in histidine and, primarily alloferon peptides with Zn ions, enable to produce preparations with a directed mechanism of action and enable their design in accordance with the understanding of the structure of the medicinal target.

3 cl, 7 dwg, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to peptides having antibacterial and endotoxin-neutralising activity, having general formula (Xaa1)M-(Xaa2)o-Xaa3-(Xaa4)p-(Xaa5)Q-(Xaa6)M-(Xaa7)R-(Xaa8)S.

EFFECT: obtaining peptides with antibacterial and endotoxin-neutralising activity.

43 cl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and deals with creation of medication of vegetable origin based on biologically active substances of Linaria vulgaris, which has diuretic and anticoagulation activity. Method of obtaining medication, which has diuretic and anticoagulation activity, consists in the following: extraction of milled grass of Linaria vulgaris with ethyl alcohol is carried out with successive drawing with 3 parts of extractant by method of remaceration with heating and connecting vacuum at the stage of discharge, under specified conditions.

EFFECT: method makes it possible to obtain medication, which has high diuretic and anticoagulation activity.

3 tbl, 1 ex

Diuretic agent // 2487714

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is presented is an agent of diuretic action of a group of aquaretics, phenyl-α-O-D- glucopyranoside. The agent may be used both to treat greater and lesser circulation congestions caused by cardiac failure, and to intensify water release thereby helping to reduce oedemas. The agent has high and low saluretic activity unlike the existing diuretics and expands the range of products of synthetic origin possessing diuretic activity.

EFFECT: agent is applicable for treating the number of cardiovascular diseases accompanied by excessive hydration and developing oedema syndrome.

1 tbl

Diuretic agent // 2484826

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is presented is a diuretic of the aquaretic group that is 4-nitro-phenyl-O-D-glucopyranoside. The agent may be used both to treat greater and lesser circulation congestions caused by cardiac failure, and to intensify water release thereby helping to reduce oedemas. The diuretic agent has high and low saluretic activity unlike the existing diuretics and expands the range of products of synthetic origin possessing diuretic activity.

EFFECT: agent is applicable for treating a number of cardiovascular diseases accompanied by excessive hydration and developing oedema syndrome.

1 tbl

FIELD: medicine.

SUBSTANCE: what is described is a new pharmaceutically solid preparation of a vasopressin antagonist which contains: (a) 7-chlor-5-hydroxy-1-[2-methyl-4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro- 1H-benzoazepine and/or its salt; (b) hydroxypropylcellulose containing a hydroxypropoxyl group in the amount of 50% or more; and (c) at least one of the ingredients specified in a group consisting of carmellose, sodium carboxymethyl starch, crospovidone and hydroxypropylcellulose with a lower degree of substitution with an average diameter of particles 30 to 70 mcm and 90% of the cumulative diameter of 100 to 200 mcm.

EFFECT: pharmaceutical solid preparation shows excellent desintegration properties and solubility leading to adequate gastrointestinal absorbability of the active ingredients.

17 cl, 4 tbl, 21 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new benzimidazole derivatives of general formula (I) or to its pharmacologically acceptable salts wherein R1 represents a C6-aryl group which can be substituted by 1-3 groups optionally specified in a group of substitutes (a), or a heterocyclic group which represents pyridyl, dihydrobenzofuranyl, 1,3-benzodioxolyl, tetrahydropyranyl, tetrahydrofuranyl which can be substituted by 1-3 groups optionally specified in a group of substitutes (a), R2 represents a C1-C6 alkyl group, R3 represents a C6-aryl group which can be substituted by 1-2 groups optionally specified in a group of substitutes (a), Q represents a group represented by formula =CH-, or a nitrogen atom and a group of substitutes (a) represents a group consisting of a halogen atom, a C1-C6 alkyl group, a C1-C6 halogenated alkyl group, a carboxyl group, a C2-C7 alkylcarbonyl group, a C2-C7 alkoxycarbonyl group, a C1-C6 alkoxy group, a C1-C6 halogenated alkoxy group, an amino group, a 4-morpholinyl group and a di-C1-C6 alkyl)amino group. Also, the invention refers to a pharmaceutical composition based on a compound of formula (I), to a PPARγ activator/modulator based on the compound of formula (I), to using the compound of formula (I), to a method of reducing blood glucose, to a method of activating PPARγ, a method of treating and/or preventing said pathological conditions.

EFFECT: there are produced new benzimidazole derivatives showing PPARγ modulatory activity.

41 cl, 2 dwg, 6 tbl, 76 ex

Diuretic // 2456002

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to experimental medicine, namely to pharmacology. The object in view is achieved by the fact that 0.5% humic acids additionally containing magnesium sulphate (II) are applied as a diuretic in the following proportions: 0.5% humic acids - 40 ml; magnesium sulphate - 0.1 g.

EFFECT: higher clinical effectiveness and eliminated side effects.

1 cl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to therapeutically active chemical compounds which influence uriniparous activity of kidney and specifically a peptide which causes selective increase in release of potassium ions by kidneys.

EFFECT: obtaining a compound for treating hyperkalaemia.

1 cl, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to biologically active peptides able to inhibit myosin light chain kinase and thereby regulate the variability of vascular endothelial permeability. What is offered is a cyclic nonapeptide of formula cyclo[-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-].

EFFECT: peptide can find application as a decongestant in various fields of medicine.

2 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to surgery and can be used for treatment of distal postmastectomy lymphedema. For this purpose at the background of standard treatment 6 procedures of retrograde lymphotropic introduction of drug mixture are carried out. Lymphotropic introduction is performed in the following way: in area of middle third of forearm applied is cuff, with creating in it pressure of 60 mm Hg, after which in two points on internal surface in area of wrist introduction of mixture of medications is performed. Composition of introduced mixture alternates every second day: 1, 3, 5 days - actovegin 80 mg, ketonal 1 ml, marcaine 0.5% 4 ml; 2, 4, 6 days - chymotrypsin 100 mg, marcaine 0.5% 4 ml.

EFFECT: method ensures efficient treatment of postmastectomy lymphedema due to enhancing of lymphatic drainage of distal parts of extremity.

3 tbl, 2 ex, 1 dwg

Diuretic // 2408367

FIELD: medicine, pharmaceutics.

SUBSTANCE: there is offered the administration of the preparation Histochrome (echinochrome, 2,3,5,6,8-pentahydroxy-7-ethyl-1,4-nahthochine, formerly known as an antioxidant) as a diuretic.

EFFECT: histochrome exhibits an evident diuretic action equal thiazide-type diuretics in intensity, does not cause associated hypokaliemia, has an absolute advantage over thiazide-type diuretics.

2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine and deals with application of mimotopes for treatment of β-amyloidosis, including Alzheimer's disease, where said mimotopes, are polypeptides, which contain from 4 to 20 amino-acid residues and are able to induce in vivo formation of antibodies to complete Ap 1-40/42 and short-cut from N-end forms AβpE3-40/42, Aβ3-40/42, Aβ11-40/42, AβpEl 1-40/42 and Aβ14-40/42 without impairment of physiological function of FHT.

EFFECT: group of inventions ensures reduction of risk of autoimmune diseases when introduced to subject.

29 cl, 3 ex, 4 tbl, 7 dwg

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