RU2454426C1 - Potassium uretic peptide - Google Patents

Abstract

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

Description

The invention relates to the field of physiology, in particular to therapeutically active chemical compounds that affect the urinary activity of the kidneys, and can be used to produce peptides that cause a selective increase in the excretion of potassium ions by the kidney, useful for the treatment of hyperkalemia.

It is known that maintaining a constant concentration of potassium ions in blood plasma and in extracellular fluid is one of the prerequisites for the stability of the cells of various organs and systems, including electro-excitable cells, in particular neurons and heart muscle cells. The extracellular concentration of potassium ions depends on its total amount in the body and the distribution between the intracellular and extracellular spaces of the body [1]. Regulation of potassium excretion by the kidney is the main mechanism for maintaining the balance of potassium ions in the body [2].

Hyperkalemia is a condition in which the concentration of potassium ions in the blood plasma is increased - more than 5.6 mmol / l [3]. Hyperkalemia can develop as a result of an imbalance between the consumption and excretion of potassium ions from the body or the redistribution of these ions between intracellular contents, where a high concentration of potassium ions dominates, and extracellular fluid, in which their concentration is close to the concentration in blood plasma, and sodium ions prevail.

The causes of increased levels of potassium in blood plasma can serve:

1) increased intake of potassium in the body, especially in patients with reduced renal function, when its elimination from the body is delayed;

2) a violation of the elimination of potassium by the kidney in acute and chronic renal failure, which is observed under clinical conditions when taking a number of drugs (potassium-sparing diuretics, cyclosporine, trimethoprim, non-steroidal anti-inflammatory drugs, an angiotensin-converting enzyme inhibitor) in patients with hypoaldosteronism and pseudohyploid hypoaldeal, some forms of congenital adrenal gland pathology;

3) redistribution of potassium in the body - the release of potassium ions from the intracellular contents into the extracellular space, which is observed with acidosis, cell destruction (hemolysis, burns, prolonged compression syndrome, tumor decay, massive blood transfusions), taking a number of drugs (digoxin, β-blockers, osmotic diuretics) [4-5].

Modern approaches to the treatment of hyperkalemia should take into account its degree and causes of development, whether it is observed as an acute violation or is a manifestation of a chronic process. Currently, the main methods of treating hyperkalemia are aimed at:

1) restriction of potassium intake into the body (abolition of oral and parenteral potassium preparations, as well as drugs that enhance hyperkalemia);

2) temporary redistribution of potassium between cells and extracellular fluid - transfer of potassium into cells (glucose infusion with insulin, β-adrenergic agonists and administration of sodium bicarbonate in case of acidosis);

3) non-selective (together with other ions) removal of excess potassium from the body (ion-exchange resins, hemodialysis, peritoneal dialysis, the introduction of loop diuretics).

When using the above methods of treating hyperkalemia, a number of undesirable side effects can be observed. So, for example, in the treatment of loop diuretics [6], correction of hyperkalemia is achieved rather slowly, while patients lose a large amount of sodium ions and, accordingly, a large volume of extracellular fluid.

A number of compounds are described that increase the removal (excretion) of potassium ions by the kidney. For example, pituitary peptide hormone arginine-vasotocin, AVT (Heller, N., 1963), 1-desamino-arginine-vasotocin [7]; 1-desamino-1-monocarbo-arginine-vasotocin, 1d-1-mc-AVT [8]; medicinal diuretic furosemide, 5- (aminosulfonyl) -4-chloro-2 - [(2-furanylmethyl)] aminobenzoic acid (radar drug reference). These compounds cause an increase in the excretion of potassium by the kidney, however, the excretion of sodium and water ions by the kidney is also enhanced, and the increase in the excretion of sodium and water is several times higher than the increase in excretion of potassium.

Derivatives of vasotocin are known [9], which have a high blood pressure coefficient for antidiuretic activity and are specific as vasodilators. The antidiuretic effect (decreased urine output) is due to the removal of the source of the molecule. The combination of amino acids with each other forms the so-called peptide bonds, which also act in the solid phase. Known derivatives of vasotocin cause increased excretion of potassium by the kidney, but this also increases the excretion of sodium ions and water by the kidney, and the increase in sodium and water excretion is several times higher than the increase in potassium excretion.

A known method of maintaining and correcting the water-electrolyte balance in mammals [10], based on the introduction of a synthetic analogue of argenin-vasotocin. The main objective of the method was to develop a method for maintaining and correcting the water-electrolyte balance in mammals, which allows to increase the reabsorption of osmotically free water while maintaining the balance of potassium and sodium and not causing changes in the activity of the cardiovascular system. A known method of maintaining and correcting the water-electrolyte composition enhances the excretion of potassium by the kidney, however, the excretion of sodium and water ions by the kidney is also enhanced, and the increase in sodium and water excretion is several times higher than the increase in potassium excretion.

A known method [11] of maintaining and correcting the water-electrolytic balance in primates, also based on the introduction of a synthetic analogue of argenin-vasotocin. However, the disadvantage of using the known compounds is the increased excretion by the kidney of sodium ions and water, exceeding the excretion of potassium several times.

It is known 1-desamino-8-arginine-vasotocin (1d-AVT), used as a means to increase the excretion of sodium salts by the kidney and enhance reverse selective absorption of water-solvent from the renal tubules into the blood [12], which is closest to the claimed invention and adopted as a prototype. The known compound is used as a means to increase the excretion of sodium salts by the kidney and enhance reverse selective absorption from the renal tubules into the blood of a water-solvent. The tool according to the claimed activity exceeds the known arginine-8-vasotocin and can be used for hypernatremia.

A disadvantage of the known compound is the selective excretion of sodium ions.

As the results of the analysis of scientific and patent literature show, to date, drugs for the selective excretion of potassium ions from the body by the kidneys do not exist.

That is why the development of agents that would facilitate the selective removal of potassium by the kidney is advisable and relevant. These funds can be useful for the treatment of hyperkalemia, a fairly common disease among various age groups of the population.

The technical result of the claimed invention is to obtain a peptide that causes a selective increase in the excretion of potassium ions by the kidney.

The specified technical result is achieved by a new potassium-uretic peptide: 1-desamino-4-arginine-8-arginine-vasotocin (1-d-Arg-AVT):

Mpr-Tyr-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH ₂

The technical result is confirmed by numerous experimental studies on the synthesis of the claimed peptide and the study of its diuretic properties, which were carried out at the laboratory and experimental bases of St. Petersburg State University and the Institute of Evolutionary Physiology and Biochemistry named after I.M.Sechenova RAS.

Example 1

Verification of potassium-uretic action. The experiments were carried out on female Wistar rats with a body weight of 150-220 g in accordance with international standards for working with experimental animals, the form of the experiments was approved by the Ethics Committee of the IEFB RAS. Peptide preparations - arginine-vasotocin (AVT), 1-desamino-arginine-vasotocin (1d-AVT) and 1-desamino-4-arginine-8-arginine-vasotocin (1d-4-Arg-AVT) were dissolved in 0.9 % NaCl solution to a concentration of 0.5 nmol / ml and was administered intramuscularly in a volume of 0.1 ml per 100 g of body weight. The choice of dose is based on the results of our studies of the effect on 1d-AVT kidney activity [12]. Furosemide in the form of a 1% solution was administered intramuscularly in a volume of 0.1 ml per 100 g of body weight. The control was animals from vivarium, which were injected with a 0.9% solution of NaCl in the same volume. Animals were placed in cage-cases with a wire bottom, through the openings of which urine flowed down a funnel into a test tube. Urine excretion was recorded for 4 hours with spontaneous urination, urine volume, Na ⁺ , K ⁺ concentration, and osmolality were measured. Blood was taken at the end of experiments from the carotid artery under ether anesthesia.

The osmolality of blood serum and urine was determined by cryoscopy using a 3300 microosmometer (Advanced Instruments, Inc., USA). The study of the concentration of Na ⁺ , K ⁺ in blood serum and urine was carried out on a Corning-410 flame photometer (UK) in an air-propane flame.

Statistical processing was performed using the Statistica 6.0 program. All data are presented as M ± m. The significance of intergroup differences was evaluated using the ANOVA test and the Newman-Cales test for multiple comparisons (at p <0.05).

Results. Administration to rats of the claimed compound - 1d-4-Arg-AVT - at a dose of 0.05 nmol per 100 g of body weight causes increased excretion of potassium ions, without affecting urination, excretion of sodium ions and osmotically free water (table 1). At the same time, vasotocin (AVT, the hormone of most vertebrates except mammals) and its analogue 1dAVT in the same dose enhance the excretion of both sodium and potassium. Under the action of AVT, 1d-AVT, as well as a loop diuretic furosemide, the kidney excretes several times more sodium ions than potassium. Thus, the influence of currently known substances that enhance the excretion of potassium is non-specific - kidney cells enhance the excretion of various ions and water. In the case of loop diuretics, this is due to the blockade of the cotransporter Na, K, 2Cl in the luminal membrane of the cells of the thick ascending part of the Henle loop.

The effect of the claimed compound 1d-4-Arg-AVT is highly selective - the peptide causes a 3.2-fold increase in potassium excretion compared to the control, while neither diuresis nor sodium excretion significantly changes after its injection (table 1). This compound is the only substance among the studied peptides, after the injection of which the K / Na ratio exceeds 1, i.e. more potassium than sodium is excreted by the kidney (table 1). The known hormone aldosterone, when injected into normal healthy animals, does not affect the initial level of potassium excretion by the kidney, it enhances the excretion of potassium and reduces the excretion of sodium in animals and humans with a decrease in the secretion of this hormone by the adrenal cortex. It should be emphasized that 1d-4-Arg-AVT not only selectively enhances the excretion of potassium by the kidney, but this effect is manifested in very small doses and it manifests itself quickly after its injection.

Table 1 The effect of peptides on the excretion of potassium and sodium ions by rat kidney Experience Conditions V ^2h , ml U _Osm V ^2h , μOcm U _Na V ^{2 h} , μmol U _K V ^2h , μmol C _H2O ^{2 h} , ml K / Na 1d-4-Arg-AVT 0.20 ± 0.02 246 ± 27 35 ± 6 57 ± 9 * -0.63 ± 0.07 1.63 1d-AVT 1.14 ± 0.07 * 1022 ± 30 * 336 ± 10 * 122 ± 8 * -2.29 ± 0.08 * 0.36 AVT 0.53 ± 0.07 * 512 ± 55 * 110 ± 16 * 59 ± 6 * -1.19 ± 0.12 * 0.54 1d-1-mc-AVT 0.75 ± 0.15 * 632 ± 122 * 183 ± 37 * 27 ± 6 * -1.40 ± 0.30 * 0.15 Furosemide 3.35 ± 0.19 * 1069 ± 52 * 385 ± 24 * 83 ± 3 * -0.24 ± 0.05 0.22 The control 0.22 ± 0.03 214 ± 16 26 ± 3 18 ± 3 -0.50 ± 0.05 Note: The indexes of excretion within 2 hours of the experiment 100 g body weight: urine (V ^2h) osmotically active substances (U _Osm V ^24), sodium (U _Na V ^2h), potassium (U _K V ^2h) and of solute free water (C _H2O ^2h ); K / Na is the ratio of potassium excretion to sodium excretion by the kidney. * - significance of differences compared with control (p <0.05).

Example 2

Synthesis of Mpr-Tyr-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH ₂ (1d-4-Arg-AVT)

1d-4-Arg-AVT was synthesized by the solid phase method using the Fmoc / tBu strategy on a 2-chloro-tritylamine resin. To protect the sulfhydryl group of cysteine and β-mercaptopropionic acid, an acetamidomethyl (Acm-) residue was used. The following amino acid derivatives were used: Fmoc-Gly-OH, Fmoc-Arg (HCl) -OH, Fmoc-Pro-OH, Fmoc-Cys (Acm) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Ile-OH , Fmoc-Tyr (tBu) -OH and S-Acm-β-mercaptopropionic acid. The first amino acid (Fmoc-Gly-OH) was attached to the polymer using diisopropylcarbodiimide in the presence of N-hydroxybenzotriazole in dimethylformamide. The removal of Fmoc-protecting groups during the growth of the peptide chain was carried out with a 20% solution of piperidine in dimethylformamide. The condensation of amino acids was carried out with diisopropylcarbodiimide in the presence of N-hydroxybenzotriazole. At each stage of condensation, a 2-fold excess of the added Fmoc amino acid was used; the completeness of the reaction was monitored using ninhydrin (Kaiser test). After complete assembly of the peptide chain, the peptide – polymer complex was treated with a mixture of trifluoroacetic acid – triisopropylsilane – ethanedithiol – water (92.5 / 2.5 / 2.5 / 2.5, vol%). The polymer was filtered off, washed on a filter with trifluoroacetic acid, and dry ether was added to the combined filtrates. The precipitate was filtered off, washed with dry ether, and dried.

The resulting product was dissolved in 80% aqueous acetic acid and treated with a saturated solution of iodine in 80% acetic acid (10-fold molar excess of iodine). The solution was evaporated to ~ ¼ of the original volume, water was added to the residue and evaporated again. The evaporation residue was frozen and freeze dried. The resulting lyophilisate was dissolved and purified by reverse phase HPLC. After chromatographic purification, the target peptide with a purity higher than 98% was obtained. To confirm the structure of the obtained compound, an amino acid analysis was performed (Table 2) and the molecular weight was determined using mass spectrometry (MALDI-TOF). The peak of the molecular ion in the mass spectrum of MALDI-TOF corresponded to mass 1064.4945 (theoretically 1064.26). The yield calculated for Fmoc-Gly-OH was 70-75% in different experiments.

table 2 Amino Acid Results Amino acid Calculated Found Tyrosine one 0.98 Isoleucine one 1.10 Arginine 2 2.00 Aspartic acid one 1.04 Cysteine one 0.95 Proline one 1.02 Glycine one 1.00

As the results of the studies show, the claimed invention achieves the technical result by producing a peptide that causes a selective increase in the excretion of potassium ions by the kidney, useful for the treatment of hyperkalemia.

List of references

1. Rosa RM, Epstein FH Extrarenal potassium metabolism. In: Alpern RJ and Hebert SC eds. The kidney. Physiology and pathophysiology, 4 ^th Edition. Amsterdam, Elsevier, 2008, p. 1277-1300.

2. Malnic G., Muto S., Giebisch G. Regulation of potassium excretion. In: Alpern RJ and Hebert SC eds. The kidney. Physiology and pathophysiology, 4 ^th Edition. Amsterdam, Elsevier, 2008, p. 1301-1348.

3. Natochin Yu.V., Mukhin H.A. Introduction to Nephrology. SPb., “GEOTAR-Media”, 2007, 152 pp.

4. Natochin Yu.V. Bud. The reference book of the doctor. SPb., Publishing house SPbSU, 1997, 208 p.

5. Kutina A.V., Kuznetsova A.A., Natochin Yu.V. Cations in human serum. Usp. fiziol. sciences. 2005.V. 36. Number 3. S.3-32.

6. Lehnhardt A., Kemper M.J. Pathogenesis, diagnosis and management of hyperkalemia. Pediatr. Nephrol. 2010. DOI 10.1007 / s00467-010-1699-3.

7. Heller, H. 1963. Neurohypophyseal hormones. In J. Euler and H. Heller (eds.), Comparative Endocrinology, vol. 1, pp. 26-72. Academic Press, New York.

8. Kanashkina T.A., Shakhmatova E.I., Natochin Yu.V. The effect of 1-desamino-1-monocarbo-arginine-vasotocin on the excretion of sodium and water by rat kidneys. Bull. expert. biol. and honey. 2007, t.143, No. 5, p. 494-496.

9. RF patent No. 2067586 (PCT application: SE 91/00154).

10. RF patent No. 2342100.

11. RF patent No. 2342101.

12. RF patent No. 2342949 (prototype).

Claims (1)

Compound of the formula
Mpr-Tyr-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH ₂ ,
possessing selective potassium-uretic action.