RU2493164C1 - Amide of nonapeptide preventing increase of hyperpermeability of vessel endothelium - Google Patents

Abstract

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

Description

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

Violation of the barrier function of the endothelium is accompanied by an increase in the permeability of the vascular wall and the development of tissue edema, the consequences of which can vary from minor to catastrophic if the lungs, brain and other vital organs are affected. This problem is often observed in a number of pathological conditions, such as acute heart failure, pathology of the heart valves, renal failure, liver cirrhosis, cancer, exposure to toxic substances, etc., and, despite the achievements of modern medicine, it continues to cause high mortality, even in a hospital setting. Diuretics, crystalloids and steroid hormones are used to combat edema. These compounds improve the excretion of fluid from the body, but do not affect the molecular mechanisms of edema development - an increase in the permeability of the endothelial lining of the microvessels arising from the contractile activity of endothelial cells.

One of the key regulators of the contractile activity of cells, including endothelial cells, is myosin light chain kinase (MLC). This enzyme is activated by many edema factors and triggers the reduction of the microvessel endothelium, which leads to a violation of its barrier function and the development of tissue edema. In this regard, an obviously promising approach to reducing the hyperpermeability of blood vessels of the microvasculature in stressful situations is to suppress the contractile reaction of the endothelium by inhibiting endothelial MLCK.

However, most existing MLCK inhibitors are not suitable for human use due to serious side effects. In this regard, the search for MLCK inhibitors characterized by a pharmacologically privileged structure and high specificity is relevant. One of these compounds is a low molecular weight inhibitor based on aminopyridazine [1. Behanna H.A., Watterson D.M., 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., Watterson D.M., Wainwright M.S. MLCK210 gene knockout or kinase inhibition preserves lung function following endotoxin-induced lung injury in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 292 (6), L1327-1334, 2007. 3. Wainwright MS, Rossi J., Schavocky J., Crawford S., Steinhom D., Velentza AV, Zasadzki M., Shirinsky V., Jia Y., Haiech J. , Van Eldik LJ, Watterson DM 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, peptide MLCK inhibitors have been developed in the world for which the effect on the permeability of intestinal epithelium in the experiment was shown in the absence of pronounced toxicity [Clay burgh DR, Barrett T.A., Tang Y., Meddings J. B., Van Eldik LJ, Watterson DM, Clarke LL, Mrsny RJ, Turner JR Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest. 115 (10), 2702-2715, 2005.]. Nonapeptide H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 (L-PIK), created on the basis of the auto-inhibitory site of MLCK [Lukas T.J., Mirzoeva S., Slomczynska U., Watterson D.M. 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.], is able to penetrate through the plasma membrane and affects the in vitro epithelial permeability [Clayburgh DR, Barrett TA, Tang Y., Meddings J. B., Van Eldik LJ, Watterson DM , Clarke LL, Mrsny RJ, Turner JR Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest. 115 (10), 2702-2715, 2005.], but due to its chemical structure, it is extremely susceptible to proteolytic enzymes [Owens SE, Graham WV, Siccardi D., Turmer JR, Mrsny RJ, A Strategy to Identify Stable Membrane-Permeant Peptide Inhibitors of Myosin Light Chain Kinase. Pharm. Res. 22 (5), 703-709, 2005]. This casts doubt on the possibility of its use in practical medicine. Modification of the L-PIK peptide by replacing its L-amino acids with the D-configuration, as expected, leads to increased resistance of the peptide to proteolytic degradation [Clayburgh DR, Barrett TA, Tang Y., Meddings J. B., Van Eldik LJ, Watterson DM, Clarke LL, Mrsny RJ, Turner JR 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-PIK was significantly lower than that of L-PIK, apparently due to the fact that D-PIK is a complete enantiomer of L-PIK and has a different spatial structure [Sekridova AV, Sidorova MV, Azmuko A .A., Molokoedov A.S., Bushuev V.N., Marchenko A.V., Scherbakova O.V., Shirinsky V.P., Bespalova Zh.D. Proteinase resistant peptide inhibitors of myosin light chain kinase. Bioorganic chemistry 36 (4), 498-504, 2010]. Since in the series of peptide analogues there is no clear correlation between structure and biological activity, even small modifications of the peptide can dramatically affect its inhibitory properties.

Closest to the claimed peptide is the peptide PIC1 of the formula H- (W-Me) -Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH ₂ (or [NMe-Arg ¹ ] -PIC). When comparing peptide MLCK inhibitors with various variants of modifying the structure of the initial L-PIK (D-PIK, [NMe-Arg ¹ ] -PIK, [εAsa ¹ ] -PIK, [Cit ¹ ] -PIK, [Cit ¹ , Orn ³ -PIK ) it was shown that in vitro activity comparable to the initial L-PIK only [NMe-Arg1] -PIC (PIC1) possesses a modification of the N-terminal amino acid of the original L-PIC peptide [Sekridova A.V., Sidorova M.V. ., Azmuko A.A., Molokoedov A.S., Bushuev V.N., Marchenko A.V., Scherbakova O.V., Shirinsky V.P., Bespalova Zh.D. Proteinase resistant peptide inhibitors of myosin light chain kinase. Bioorganic chemistry 36 (4), 498-504, 2010]. The lifetime of PIK1 in blood plasma and in vitro was almost 3 times longer than the life of L-PIK, and at a concentration of 100 μM PIK1 reduced the thrombin-induced increase in the permeability of the monolayer of endothelial cells [Marchenko AV, Stepanova E.O., Sekridova A.V. ., Sidorova M.V., Bushuev V.N., Bespalova Zh.D., Shirinsky V.P. New peptide inhibitors of myosin light chain kinase inhibit vascular endothelial hyperpermeability. Biophysics, 55, 1008-1013, 2010]. However, the effect of PIK1 is observed in relatively high concentration ranges (about 100 μM), which from a pharmacological point of view makes it less attractive for in vivo use.

The objective of the invention is to provide effective suppression of hyperpermeability of the vascular endothelium, and the creation of a pharmaceutical composition for injection to combat acute edema of vital organs. The relevance of this task is due to the need to provide practical medicine with effective drugs that suppress the hyperpermeability of the vascular endothelium, in particular to combat acute pulmonary edema in situations critical for the patient.

The technical result consists in obtaining a peptide having enhanced activity in suppressing hyperpermeability of vascular endothelium and increased plasma life time.

The problem is solved by synthesis of methylated (Me) at the amino-terminal group of the nonapeptide amide of the formula H- (W-Me) -Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg ^8- Lys-NH ₂ (PIK2) and its pharmaceutically acceptable non-toxic salts. The problem is also solved by the creation of a pharmaceutical composition for injection containing a therapeutically effective amount of PIC2 and a pharmaceutically acceptable carrier. And also the task is solved by the development of a method of reducing hyperpermeability of the vascular endothelium in the recipient by injection of a therapeutically effective amount of the specified pharmaceutical composition.

The invention is illustrated by 4 (four) illustrations, where, in FIG. 1 and FIG. 2, respectively, fragments of ¹ N-NMR spectra of the prototype (PIC1) and of the claimed peptide (PIK2) in solution and in human blood plasma (minus the blood plasma spectrum) are presented. ) indicating the signals of the corresponding groups of free amino acids. Figure 3 presents the dynamics of phosphorylation of regulatory myosin light chains under the influence of MLCK in vitro in the presence of the claimed peptide PIK2 and prototype peptides L-PIK and PIK1 at a concentration of 100 nM. Figure 4 shows the effect of the claimed peptide (PEAK 2) and prototype (PEAK 1) on the diffusion of albumin conjugated to fluorescein isothiocyanate through a monolayer of endothelial cells of the EA.hy926 line. Presents the relative permeability of the endothelium 4 hours after stimulation with 100 nM thrombin in the presence of 10 μM (white columns), 20 μM (black columns) and 100 μM (shaded columns) of the inventive peptide (right) and prototype (left). The permeability of control endothelial cells after thrombin stimulation was taken as 100% (indicated by a dashed line).

The inventive peptide was obtained by solid-phase peptide synthesis using the 9-fluorenylmethoxycarbonyl (Fmoc) technology described in Example 1 below.

Pharmaceutically acceptable salts of the peptide of the invention include salts formed with inorganic or carboxylic acids. Examples of inorganic acids forming salts include hydrohalic acids such as hydrochloric, hydrobromic acids, phosphoric acid, sulfuric acid. Carboxylic acid salts are formed with acids such as acetic, propionic, malonic, maleic, citric, succinic, malic, benzoic, fumaric and other similar carboxylic acids. Salts with acids are obtained by conventional methods, for example, by neutralizing the PIK2 peptide in the form of a free base with acid. Preferred salts are sulfate and hydrochloride. As indicated above, the present invention includes solvates of the compounds of this invention and their pharmaceutically acceptable salts. The inventive peptide PIK2 or its pharmaceutically acceptable salts can form solvates with water or common organic solvents. Such solvates are included in the scope of the present invention.

The term "pharmaceutically acceptable" means a carrier that is compatible with other ingredients of the pharmacological composition and does not have toxic or adverse effects on patients. Since the claimed peptide is highly soluble in aqueous solutions, for example, isotonic sodium chloride solution 0.25%, sterile water for injection, Ringer's solution for injection, blood substitutes, glucose solution for infusion 5%, other solutions can be used as carriers for PIK2 active preparations for intravenous or intramuscular administration, provided that there are no adverse drug interactions between PIK2 or its pharmaceutically acceptable salts and the second active substance (active substances s).

A therapeutically effective amount of PIK2 as an active principle is defined as the amount necessary to achieve the desired physiological response in the recipient, and may depend on the recipient's body weight, severity of the recipient, individual physiological characteristics of the recipient, conducted by concomitant therapy, etc. The amount of PIK2 in the pharmacological composition will be depend on the form of its release - lyophilisate for solution for injection or solution for injection. Effective single doses of PIK2 for intravenous administration can be from 0.1 to 1.5 mg / kg of the recipient's body weight. With the introduction of the drug intramuscularly, the dose necessary to effectively reduce the hyperpermeability of the vascular endothelium will be higher and can reach up to 5 mg / kg of the recipient's body weight.

Medicines with the claimed peptide or its pharmaceutically acceptable salts as the active substance can be presented in the form of a lyophilisate for the preparation of a solution for injection or in the form of a ready-made solution for injection. A lyophilized form for dissolution is preferred.

Methods for administering a drug with the claimed peptide or its pharmaceutically acceptable salts as an active ingredient include intravascular bolus administration, infusion administration in other administration solutions with the components of which the inventive PIC2 peptide or its pharmaceutically acceptable salts do not form undesirable interactions, intramuscular administration . The preferred route of administration is a bolus intravascular injection, allowing therapeutically effective concentrations of the active substance in the blood of the recipient to be rapidly reached. Indications for use of the claimed peptide or its pharmaceutically acceptable salts should be considered the appearance of clinical symptoms or signs of the development of hyperpermeability of the vascular endothelium, for example, in acute pulmonary edema such symptoms are cyanosis, pallor, profuse sweat, pulse alternation, accent II tone over the pulmonary artery, protodiastolic rhythm gallop. The introduction of PIK2 for symptomatic treatment should be carried out immediately if there are signs of acute edema.

Example 1. Synthesis of the claimed peptide (PIK2)

H- (N-Me) -Arg ¹ -Lys-Lys-Tyr-Lys-Tyr-Arg ⁷ -D-Arg ⁸ -Lys-NH ₂

Derivatives of amino acids (AA) and hexafluorophosphate (benzotriazol-1-yl) hydroxy tris (dimethylamino) phosphonium (BOP, NovaBiochem, Switzerland), N, N'-diisopropylcarbodiimide (DIC), N, N-diisopropylethylamine (DIPEA) were used , hydroxybenzotriazole (HOBt), triisobutylsilane (TIBS) company Fluka, Switzerland, 4-methylpiperidine (4-MePip, Aldrich, USA). For the synthesis, N-methylpyrrolidone, dichloromethane (DCM), trifluoroacetic acid (TFA) from Fluka, Switzerland were used; for chromatography, acetonitrile (Panreac, Spain). Analytical high performance liquid chromatography (HPLC) was carried out on a chromatograph (Gilson, France), a Nucleosil 100 C18 column, 5 μm, (4.6 × 250 mm) (Sigma, USA) was used as eluents, buffer A - 0.1% TFA, buffer B - 80% acetonitrile in buffer A, elution with a concentration gradient of buffer B in buffer A from 0% to 60% in 30 minutes. A flow rate of 1 ml / min, detection at 220 nm. The structure of the obtained peptides was proved by ¹ H-NMR spectra and mass spectrometry data. ¹ H-NMR spectra were recorded on a WM-500 spectrometer (Bruker) 500 MHz (Germany) in deuterated dimethyl sulfoxide (DMSO-d ₆ ) at 300 K, the concentration of peptides was 2-3 mg / ml. Chemical shifts were measured relative to tetramethylsilane. Mass spectra were recorded on a PC-Kompact MALDI instrument (Kratos, England).

For solid-phase synthesis, a copolymer of styrene with 1% divinylbenzene with 4- (2,4-dimethoxyphenyl) -9-fluorenylmethoxycarbonyl-aminomethylphenoxy-anchor group (Rink-amide-polymer) from Nova BoiChem, Switzerland, designed to produce peptide amides containing 0.64 was used mmol / g of amino groups. The nonapeptide amide was synthesized from the C-terminus, stepwise (adding one amino acid each), starting from 0.40 g (0.25 mmol) of the Rink-amide polymer. The first 8 cycles of synthesis were carried out automatically on an Applied Biosystems 431 A peptide synthesizer according to the standard program for a single condensation of Fmoc amino acids. The guanidine groups of the Arg and Arg residues were blocked with 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) (see solid phase synthesis protocol). To attach the N-terminal, N-methyl substituted Arg ¹ residue, the guanidine function was protected with 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), and the highly effective Castro-BOP reagent was used to create the amide bond [Castro B., Dormoy JR , Evin G., Selve C. Peptide coupling reaction with benzotriazol-l-yl-tris (dimethylammo) phosphonium hexafluorophosphate. Tetrahedron Lett. V.14. P.1219-1222, 1975].

Solid Phase Synthesis Protocol No. Operation Reagent Time of processing Cycles 1-8 one Flushing 5x N-methylpyrrolidone (NMP) 3 min 2 The release of α-amino groups 25% 4-MePip / NMP 10 min 3 Flushing 5 × NMP 3 min four Activation 1 mmol Fmoc-AA + 1 mmol HOBT + 1 mmol DIC in NMP 20 minutes 5 Condensation 1 mmol activated derivative Fmoc-AA in NMP 90 min 6 Flushing 5 × NMP 3 min Cycle 9 one Flushing 5 × NMP 3 min 2 The release of α-amino groups 25% 4MePip / NMP 10 min 3 Flushing 5xNMP 3 min four Activation 1 mmol Fmoc- (N-Me) Arg (Mtr) -OH + 1 mmol BOP + 1 mmol HOBT + 2 mmol DIPEA in NMP 5 minutes 5 Condensation 1 mmol activated derivative Fmoc-AA in NMP 90 min 6 Flushing 5xNMP 3 min

The final unblocking and cleavage of the nonapeptide from the polymer was carried out in one step by treating the corresponding nonapeptidyl polymer with 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 of the unlocking mixture, the filtrate was evaporated and the residue dry ether was added. The precipitate was filtered off, washed with DCM (3 × 3 ml), ether (3 × 5 ml), dried in a vacuum desiccator. 0.32 g of a solid-phase synthesis crude product with a basic substance content of 76% was purified using preparative HPLC on a Beckman instrument (USA) using a Diasorb C16 13 RT column (25 × 250 mm), sorbent particle size 10 μm. As eluents used buffer A - 0.01 M solution of ammonium acetate and buffer B - 80% acetonitrile in water. Elution was performed 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. Peptides were detected at a wavelength of 220 nm. Fractions containing the desired product were combined, acetonitrile was evaporated and lyophilized. As a result, 0.16 g (47.1% based on the starting amino acid attached to the polymer) of the nonapeptide amide PIK2 was obtained. Product homogeneity determined by analytical HPLC was 96.8%. Mass spectrum: 1338.6, calculated 1338.7.

The lifetimes of the claimed peptide (PIK2) and the prototype peptide (PIK1) in blood plasma were compared by ¹ H-NMR, as described in example 2. The results of comparison of the claimed peptide (I) and prototype are presented in Figure 1 and Figure 2, respectively .

Example 2. The lifetime of the peptides in blood plasma in vitro

Under aseptic conditions, 100 ml of whole blood was taken from healthy donors in 50-ml tubes with 5 ml of preservative (130 mM citrate in phosphate-buffered saline (PBS), pH 7.4). Blood was held for 15-20 minutes at room temperature and centrifuged at 1500 g for 15 minutes at 18 ° C. The supernatant was transferred to new tubes and centrifuged repeatedly under the same conditions. Blood plasma volume was measured and dry NaBr was added at the rate of 280 mg NaBr per 1 ml of plasma. After NaBr dissolution, plasma (approximately 30 ml) was poured into centrifuge glasses (for Type 45Ti rotor, Beckman, USA) and 20 ml of a solution with a density of 1.216 g / l of the following composition were layered on top: 11.42 g of NaCl and 100 mg of ethylenediaminetetraacetic acid (EDTA) was dissolved in 1 L of distilled water, pH 7.4, then 281.44 g of NaBr was added. Centrifuged at 40,000 rpm for 48 hours. After centrifugation, the upper dark yellow liquid layer containing lipoproteins and a colorless buffer layer were selected, leaving the buffer about 1 cm high to the visible plasma boundary. Plasma obtained from one donor was pooled and dialyzed for 36 h against 4 L of FSB free of Ca ²⁺ and Mg ^{2+ ions} (MP Biomedicals, USA) in dialysis bags with pore sizes of 12-14 kDa (Medicell, UK) . The dialysis procedure was repeated twice for 24 hours against fresh buffer. After dialysis, the plasma was centrifuged at 25,000 rpm in a Type 45Ti rotor (Beckman, USA) for 15 min at 4 ° C. The supernatant was removed and lyophilized. The dry residue was dissolved in D ₂ O (in a volume equal to the volume of the plasma before the introduction of NaBr) and again lyophilized. Then it was dissolved in the same volume and stored in aliquots of 1 ml at -20 ° C until use.

1H NMR spectra were obtained on a WM-500 spectrometer (Bruker, USA) at 37 ° С. The chemical shifts of the signals in the spectra were measured relative to the internal standard, the sodium salt of 2,2-dimethyl-2-silapentane-5-sulfanate. The assignment of signals was performed using the double resonance method. To isolate the signals belonging to the protons of the peptide, difference spectroscopy was used: the free plasma spectrum was subtracted from each spectrum obtained after adding the peptide to the blood plasma. The concentration of the peptide in the sample was 3 mg / ml.

As can be seen from Figure 1, in the case of the prototype peptide PIK1 after 190 min incubation in human plasma in vitro, the signals corresponding to the CαN groups of free arginine and lysinamide are well defined. This indicates the degradation of the C-terminal portion of the peptide, leading to the formation of free amino acids. In this case, the half-life of PIK1 in the blood plasma is about 120 minutes [Sekridova A.V., Sidorova M.V., Azmuko A.A., Molokoedov A.S., Bushuev V.N., Marchenko A.V., Shcherbakova O.V., Shirinsky V.P., Bespalova Zh.D. Proteinase resistant peptide inhibitors of myosin light chain kinase. Bioorganic chemistry 36 (4), 498-504, 2010]. At the same time, in the case of the PIK2 peptide (Figure 2), the initial spectrum does not undergo significant changes even after 420 min of incubation in the blood plasma, indicating a high stability of PIK2 in the blood plasma with a half-life> 420 min. Thus, the claimed peptide is characterized by an increased lifetime in plasma. This allows you to maintain its effective concentration, necessary to reduce hyperpermeability of the vascular endothelium in critical conditions. The increased peptide lifetime also allows the use of lower (compared with homologues) concentrations (5-30 μM in circulating blood) while maintaining the therapeutic effect.

An increase in the lifetime of the peptide when replacing the natural L-configuration of the a-carbon atom of the Arg ⁸ residue with the D-configuration in the structure of the claimed PIK2 peptide was to be expected, since it is known that peptide bonds formed by D-amino acids are more stable than bonds of L-amino acid analogues, since they are not recognized by proteolytic enzymes [Hruby VJ, Qian X. Approaches to the asymmetric synthesis of unusual amino acids. Methods Mol. Biol. 35, 249-286, 1994]. However, along with an increase in the lifetime, modifications can adversely affect the inhibitory activity of the peptide. This is confirmed by our earlier comparative study of the activity of peptide inhibitors of D-PEAK, [NMe-Arg ¹ ] -PIK, [εAsa ¹ ] -PIK, [Cit ¹ ] -PIK and [Cit ¹ , Orn ³ ] -PIK, of which only [NMe-Arg ¹ ] -PIK, (PIK1) had an in vitro activity comparable to the L-PIK peptide [Sekridova A.V., Sidorova M.V., Azmuko A.A., Molokoedov A.S., Bushuev V.P., Marchenko A.V., Shcherbakova O.V., Shirinsky V.P., Bespalova Zh.D. Proteinase resistant peptide inhibitors of myosin light chain kinase. Bioorganic chemistry 36 (4), 498-504, 2010]. Our subsequent studies aimed at solving the problems of this invention showed that the peptides H-DLys-DArg-DArg-DTyr-DLys-DTyr-DLys-DLys-DArg-NH ₂ (retroenantio-PIC), H-Arg (NO ₂ ) -Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH ₂ (with protection of the N-terminal portion of the peptide by introducing a nitro group into the guanidine group of the residue Arg ¹ ), H-Argψ [CH ₂ NH] - Lys-Lys-Tyr-Lys-Tyr-Arg-DArg-Lys-NH ₂ (with protection of the N-terminal portion of the peptide by introducing a pseudopeptide bond in the first position), H- (NαMe) Arg-Lys-Lys-Tyr-Lys -Tyr-Arg-Arg-Lys-Pro-Gly-Pro-OH (with protecting the N-terminal portion of the peptide by introducing a methyl group and protecting the C-terminal portion with by the tripeptide Pro-Gly-Pro), and the peptide H-Argψ [CH ₂ NH] -Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-Pro-Gly-Pro-OH (with N-terminal protection peptides by introducing a pseudopeptide bond in the first position and protecting the C-terminal part with the Pro-Gly-Pro tripeptide) were also significantly inferior to the claimed PIK2 peptide in inhibitory activity in vitro against purified MLCK and in the model for suppressing permeability of the endothelial monolayer in vitro.

The inhibitory activity of the claimed peptide (PIK2) and prototypes (PIK1 and L-PIK), which are the most active of the known peptides of the PIK family, were compared by their effect on the phosphorylation of regulatory myosin light chains in vitro, as described in example 3. The results of the analysis are presented in FIG. .3.

Example 3. Phosphorylation of regulatory myosin light chains in vitro

Myosin regulatory light chains (RLCs) at a final concentration of 20 μM phosphorylated MLCs from turkey stomachs (9 nM) at 30 ° C in a buffer containing 10 mM 3-rM-Morpholino] propanesulfonic acid (MOPS), pH 7.0, 100 mM NaCl, 0.5 mm CaCl ₂ , 1 mm MgCl ₂ , 0.5 mm Mg-ATP, 0.3 μm calmodulin, 1 mm dithiothreitol (DTT) in the presence of 100 nm of the claimed peptide or prototype peptides L-PIK or PIK1. The reaction was initiated by the addition of MLCK. At certain time intervals (0-40 min), aliquots of the reaction mixture (5 μl) were taken and dissolved in the sample buffer. The completeness of the reaction was analyzed by electrophoresis in the presence of urea and glycerol, which allows the separation of proteins based on their charge. Electrophoresis was performed 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% bromphenol blue). A 5.2% concentration gel was used, prepared in 20 mM Tris-HCl buffer pH 6.8 containing 16.5% glycerol and 3.25 M urea; and a 12% separating gel prepared on 20 mM Tris-23 mM glycine buffer pH 8.6 containing 40% glycerol. Electrophoresis was carried out in 20 mM Tris-23 mM glycine buffer, pH 8.6, containing 0.2% β-mercaptoethanol at 330 V for 20 min and 400 V for 180 min. The gels were fixed and stained with Coomassie R-250. The experiments were repeated at least 3 times. The electrophoretic mobility of protein bands corresponding to RLC and phospho-RLC was determined by the mobility of the corresponding standards. The gels were scanned and a quantitative densitometric analysis of the obtained electrophoregrams was performed using the Image J program, version 1.45. The degree of phosphorylation of myosin RLC was calculated as the ratio of phosphorylated RLC to the total number of RLC. The obtained data were processed in Excel and graphs were plotted of the degree of phosphorylation of myosin RLC (mole of phosphate / mole of RLC) versus time in the presence of different concentrations of the peptide. Data were expressed as mean + std. off

It can be seen from the above example that under the given conditions of the reaction in the absence of an inhibitor, complete phosphorylation of myosin RLC is achieved after 15 minutes. During the same time, in the presence of prototype peptides L-PIK and PIK1, the reaction proceeds by 90-95%, while in the presence of the inventive peptide (PIK2), the reaction proceeds only by 50-60%. These data indicate stronger inhibitory properties of the claimed peptide PIK2 in comparison with the peptide prototypes L-PIK and PIK1. It should be noted that under these conditions, the lifetime of the peptides does not affect their inhibitory activity, since the reaction is carried out in the absence of proteolytic enzymes.

The effect of the claimed peptide (PIK2) and prototype (PIK1) on the permeability of the endothelium was evaluated by the diffusion rate of FITC-albumin through the monolayer of endothelial cells EA.hy926, as described in example 4. The results of comparison of the claimed peptide (PIK2) and prototype PIK1 are presented in Figure 4 .

Example 4. Permeability of the endothelial monolayer in vitro

The effect of peptides on endothelial permeability was evaluated by the diffusion rate of albumin labeled with fluorescein isothiocyanate (FITC) through the monolayer of EA.hy926 endothelial cells (Fig. 3). On ThinCerts liner membranes (Greiner Bio One, Austria) with a pore diameter of 0.4 μm and a pore density of 1 × 10 ⁸ / cm ^2, 1 × 10 ⁵ endothelial cells of the EA.hy926 line were planted in 200 μl of Dulbecco’s nutrient medium (DMEM) ) high in glucose in the presence of 20% fetal calf serum. 800 μl of the same cell-free medium was added to the external chamber. Cells were cultured for 4 days. During this time, the cells reached a monolayer. The growth medium was changed to DMEM without embryonic serum and cell deprivation was performed for 30 minutes. After that, the studied peptide was added to the lower and upper chambers at concentrations of 10-100 μM and incubated for 30 minutes. Then, FITC-albumin was added to the upper chamber to a final concentration of 0.5 mg / ml and samples were taken from the lower chamber every 30 min to calculate the basal diffusion rate of FITC-albumin through the endothelial monolayer (relative units of fluorescence intensity / hour). Changes in the permeability of the endothelium were induced by adding 100 nM thrombin to the upper chamber and, at intervals of 30 min, continued sampling of the medium from the lower chamber to analyze the content of FITC-albumin in it. The content of FITC-albumin in the samples was measured using a Victor X3 multifunctional tablet analyzer (PerkinElmer, USA). The results were presented as mean ± standard deviation (Figure 4). Significance of differences was determined by Student t-test.

From the above example it is seen that the claimed peptide (PIK2) reduces the thrombin-induced permeability of the monolayer of endothelial cells more effectively than the prototype (PIK1). So, after the preincubation of cells with 10 μM prototype, the response of endothelial cells to thrombin is reduced by only 10% and is 90% of the response of cells in the absence of inhibitors. At the same time, after preincubation with 10 μM of the claimed peptide (PIK2), the response to thrombin is reduced by 80%, accounting for 20% of the cell response in the absence of inhibitors. At a concentration of 20 μM, the prototype suppresses the response to thrombin by 40%, while the claimed peptide (PIK2) at the same concentration completely cancels the increase in permeability of the monolayer. When the concentration of the prototype equal to 100 μm, a significant (90%) suppression of the reaction to thrombin is achieved. At the same time, after preincubation with 100 μM peptide (PIK2), the barrier function of the monolayer was enhanced and the permeability became 30% lower than the base permeability of the control cells in the absence of thrombin stimulation.

The applicability and effectiveness of drugs, including the claimed peptide PIK2 or its pharmaceutically acceptable salts, to reduce in vivo hyperpermeability of vascular endothelium demonstrate experiments with modeling of acute pulmonary edema caused by bacterial liposaccharide. The final functional link in the development of tissue edema is the enhancement of para- and transcellular transport of substances through the cells of the vascular endothelium. These processes are associated with the activity of endothelial MLCK. Therefore, the use of bacterial liposaccharide allows you to simulate the development of acute pulmonary edema of various etiologies.

Example 5. In vivo model of pulmonary edema.

Male Wistar rats weighing 300-340 g under ketamine anesthesia (100 mg / kg) were inserted with catheters into the carotid artery and jugular vein to record mean arterial pressure (BP), heart rate (HR) and to administer substances. 10 minutes after recording the initial parameters, the rats were injected with S. typhimurium lipopolysaccharide (LPS) at a concentration of 8 mg / kg in physiological saline. After 20 minutes, the claimed peptide (PIK2) was administered to rats at a concentration of 1.5 mg / kg intravenously, bolus in 0.5 ml of physiological saline. 24 hours after the experiment, the rats were killed with an overdose of urethane. The chest was opened, aspirated, and the amount of fluid from the pleural cavities was measured, the lungs were removed and weighed. 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 the wet weight of the lungs plus pleural effusion to the dry weight of the lungs. In the control group of animals, these parameters were determined without the introduction of LPS and PIK2. The results were presented as mean ± error of the mean (tab.). Significance of differences was determined by Student t-test.

The influence of the claimed peptide PIC2 on the severity of pulmonary edema in rats Group Number of animals Animal Weight (g) The ratio of the initial mass and dry weight of the lungs Fluid in the lungs (initial mass - dry matter + effusion, g) the control 6 320 ± 6 4.64 ± 0.06 1,08 ± 0,05 LPS fifteen 323 ± 11 4.92 ± 0.04 1.61 ± 0.07 LPS + PIC2 17 311 ± 10 4.80 ± 0.04 1.35 ± 0.05

As can be seen from the table, the introduction of LPS leads to a significant increase in lung mass due to the intake of water and low molecular weight substances, as well as high molecular weight compounds and blood cells. With the introduction of the claimed peptide (PII), the increase in the amount of fluid in the lungs in response to the effects of LPS is reduced by 2 times. Thus, the claimed peptide (PIK2), administered at a dose of 1.5 mg / kg (concentration in circulating blood of about 30 μM), is able to attenuate acute pulmonary edema caused by exposure to bacterial endotoxin.

Since mechanisms of increasing vascular endothelial permeability are involved in the development of acute pulmonary edema of various etiologies, PIK2, when administered intravenously in physiological saline or other pharmaceutically acceptable carriers, can be an effective means of combating acute critical conditions associated with a pathological increase in vascular endothelial permeability. The most relevant area of application of the claimed pharmaceutical composition is the fight against acute pulmonary edema, which is the cause of high mortality in patients. Acute pulmonary edema can occur during artificial lung ventilation during surgery, with sepsis, intoxication, inflammatory and allergic reactions, injuries (burns, squeezing), ischemia-reperfusion, heart failure and a number of other disorders.

Claims (3)

1. A peptide having enhanced activity against suppressing vascular endothelial hyperpermeability and increased plasma lifetime, having the formula H- (N-Me) -Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys -NH ₂ . 2. A pharmaceutical composition having the ability to suppress vascular endothelial hyperpermeability, comprising a therapeutically effective amount of the peptide according to claim 1 and a pharmaceutically acceptable carrier for injection. 3. A method for suppressing vascular endothelial hyperpermeability in a recipient by injecting a therapeutically effective amount of a pharmaceutical composition according to claim 2.

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