ES2220185B1 - NEW INHIBITOR PEPTIDE OF THE NA + / H + EXCHANGER (PINHE), AND ITS APPLICATIONS. - Google Patents

Abstract

New Peptide inhibitor of the Na + / h + exchanger (PINHE), and its applications. The present invention describes a peptide (PINHE) consisting of 28 amino acids obtained by fractionation and purification of the culture supernatant of Haloferax gibbonsii alicante SPH7. PINHE acts as a natural physiological inhibitor of the Na + / H + exchanger in eukaryotes. It is active in wide ranges of saline concentrations, temperature and pH, trypsin resistant and pronase sensitive. From its reversible action on the Na + / H + exchanger derives: (1) its use for the treatment or prophylaxis of pathologies such as cardiovascular, renal, cerebral, metabolic diseases, pathological proliferative processes, hyperproduction of hydrochloric acid etc., (2) in other diseases or pathologies that occur with ischemia and post-ischemic reperfusion injury due to the hyperactivity of NHE such as ischemia due to primary or secondary vascular alterations in various tissues, post-infarction reperfusion, organ or tissue transplantation, etc. Also as a contraceptive (in male mammals), antibiotic or immunoregulatory agent, and in plants as a saline stress modulator.

Description

New peptide exchanger inhibitor Na + / H + (PINHE), and its applications.

Technical sector

The present invention is framed in several sectors, the most important being the Pharmacist where Applications as protective treatment of various processes pathological diseases of great importance such as ischemic pathology and by reperfusion (regardless of the production mechanism), arrhythmias, hypertension, etc. It could also be applied as contraceptive (in male mammals), as an antibiotic and as an agent immunoregulatory In the Chemical sector, as a regulatory reagent of Na + / H + exchanger activity. Y potentially in agriculture, as a stress modulator saline.

State of the art

The sodium-proton exchanger (Na + / H +) (sodium-proton exchanger or NHE), is a mechanism that is present in the membranes of all cell types, both prokaryotic and eukaryotic, and catalyzes a exchange of intracellular H + electroneutro with extracellular Na + (Putney et al . 2002, Annu. Rev. Pharmacol. Toxicol . 42 : 527-552; Counillon and Pouyssegur, 2000, J. Biol. Chem . 275 : 1-4, Orlowsky and Grinstein, 1997, J. Biol. Chem . 272 : 22373-76; Grinstein et al , 1989, Biochim. Biophys. Acta , 988 : 73-97). It has been known for years that it participates at the cellular level in vital processes that depend on transmembrane phonic exchange such as homeostasis, intracellular pH, cell volume, adhesion, shape determination, migration and proliferation (Motais et al . 1991. Biochim Biophys Acta , 1075 (2): 169-180; Maidurna et al , 1993, Br J Cancer , 67 (2): 297-303; Harguindey and Craoge 1992. Med Hypotheses , 39 (3): 229-237; Putney et al . 2002 , Annu. Rev. Pharmacol. Toxicol . 42 : 527-552).

The NHE exchangers family includes six isoforms (NHE1-NHE6). The NHE1 isoform, which is located in the cytoplasmic membrane, is ubiquitously expressed and plays a central role in the regulatory processes of intracellular pH, cell volume and homeostasis (Orlowski J, Grinstein S. 1997. J. Biol. Chem . 272 : 22373- 76; Counillon L, Pouysségur J. 2000. J. Biol. Chem . 275 : 1-4). In contrast, the NHE2, 3, 4 and 5 exchangers show a limited tissue distribution and a more specialized function. Thus NHE2, 3 and 4 are predominantly expressed in the kidney and in the intestinal tract. In these places, the cellular location of NHE2 is apical, and it is mainly involved in the reabsorption of Na + ions (Tse CM, Levine SA et al . 1993. J. Biol. Chem . 268 : 11917-24). NHE2 can act in collaboration with NHE4 which has a basolateral location (Orlowski J, Kandasamy RA, Shull GE. 1992. J. Biol. Chem . 267 : 9331-39; Pizzonia JH, Biemesderfer D, Abu-Alfa AK et al . 1998 Am J. Physiol . 275 : F510-17; Bookstein C, Xie Y et al . 1997. Am J. Physiol . 273 : C1496-505; Bai L, Collins JF, Muller YL, Xu H, Kiela PR, Ghishan FK , 1999. Am J. Physiol 277 : R1112-19) to promote osmoregulation of the internal renal medullary cells. The NHE3 exchanger has been identified in the apical membrane of the proximal renal tubule epithelium (Biemesderfer D, Pizzonia J, et al . 1993. Am J. Physiol . 265: F736-42) and in the microvilli of the mature intestinal epithelium (Hoogerwerf WA , Tsa SC et al . 1996. Am J. Physiol . 270 : G29-41; Bookstein C, DePaoli AM, et al . 1994. J. Clin. Investig . 93 : 106-13). In the kidney, NHE3 is important in the reabsorption of Na <+> and HCO <3> -, contributing significantly to the maintenance of blood osmolarity and acid-base balance (Moe 0W. 1999. J. Am Soc. Nephrol . 10 : 2412-25). The NHE5 exchanger is predominantly expressed in the brain (Klanke CA, Su YR et al . 1995. Genomics 25 : 615-22; Baird NR, Orlowski J et al . 1999. J. Biol: Chem . 274 : 4377-82) and It is thought to regulate intracellular pH in neurons (Raley-Susman KM, Cragoe EJ, Saplosky RM, Kopito RR. 1991. J. Biol. Chem . 266 : 2739-45). NHE6 is the newest member of the NHE family. This molecule has the most divergent sequence showing only 20% identity with the other isoforms. NHE6 is located exclusively in the mitochondria, giving the highest expression in tissues and organs that have a high metabolism, such as the heart, brain and skeletal muscle (Numata M, Petrecca K, Lake N, Orloski J. 1998. J. Biol. Chem . 273 : 6951-59).

The NHE family of genes has been evolutionarily conserved. Genes encoding NHEs have been identified in all kinds of living organisms. In prokaryotic organisms from archaea such as Halobacterium salinarum (Ng WV, Kennedy SP, et al . 2000. Proc, Nat. Acad. Sci. USA . 97 : 12176-181) to bacteria such as Streptococcus faecalis (Harold FM, Papineau D. 1972 J. Membr. Biol . 8 : 45-62) and Escherichia coli ( nhaA and nhaB genes. Padan E, Maisler N, Taglicht D, Karpel R, Schuldiner S. 1989. J. Biol. Chem . 264 : 20297-302 ), where these exchangers are electrogenic and show very little homology with the NHE of higher organisms. NHE genes have also been identified in plants, specifically an NHE5 homologue in Arabidopsis thaliana (Apse MP, Aharon GS, Snedden WA, Blumwald E. 1999. Science . 285 : 1256-58) and in Schizosaccharomyces pombe yeasts ( sod2 gene , Jia ZP, McCullogh N, Martel R, Hemingsen S, Young PG, 1992. EMBO J. 11 : 1631-40) and Saccharomyces cerevisae (genes nhx1 , nha2 , Nass R, Cunningham KW, Rao R. 1997. J. Biol Chem . 272 : 26145-52; Numata M, Petrecca K, Lake N, Orloski J. 1998. J. Biol. Chem . 273 : 6951-59). The product of the nhxl gene, the Nhx1p protein, is predominantly located in a prevacuolar compartment and is important in the endocytic trafficking process (Bowers K, Levi BP, Patel FI, Stevens TH. 2000. Mol. Biol. Cell . 11 : 4277 -94). NHE family genes have also been identified in Caenorhabditis elegans (Marra MA, Prasad SS, Baillie DL. 1993. Mol Gen. Genet . 236 : 289-98) and in Drosophila melanogaster (Adams MD, Cleniker SE et al . 2000. Science 287 : 2185-95), although specific isoforms have not been identified or their functions characterized. Finally, NHE genes have been identified in a number of higher organisms such as various vertebrates including humans (Klanke CA, Su YR et al . 1995. Genomics 25 : 615-22; and 24), rat (Orlowski J, Kandasamy RA , Shull GE. 1992. J. Biol. Chem . 267 : 9331-39; Collins JF, Honda T, et al . 1993. Proc. Nat. Acad. Sci. USA 90 : 3938-42), mouse (Praetorius J. Andreasen D, Jensen BL, Ainsworth MA, Friis UG, Johansen T. 2000. Am. J. Physiol. Gastrointestinal. Liver Physiol . 278 : G197-206), pig (Reilly RF, Hildebrandt F, et al . 1991. Am J Physiol . 261 : F1088-94), Xenopus laevis (Busch S, Rosskopf D, Lang HJ, Weichert A, Siffert W. 1998. Pflugers Arch-Eur : J Physiol . 436 : 828-33), and trout (Borgese F, Sardet C, Cappadoro M, Pouyséegur J., Motays R. 1992. Proc Nat. Acad. Sci. USA 89 : 6765-69).

Recently, it has been determined that NHE also functions as a membrane anchor for the cytoskeleton through actin and that this function is independent of ion translocation (Putney et al . Annu. Rev. Pharmacol. Toxicol . 42 : 527- 552, 2002). This character of the NHE together with the effects on the regulation of intracellular pH, homeostasis and cell volume, indicate that the NHE regulates a large number of cellular activities that include adhesion, shape determination, migration and proliferation. The normal activity of NHE is therefore of vital importance for the cell (Pouyssegur et al . 1984. Proc. Natl. Acad. Sci. USA , 81 : 4833-37; Kapus et al , 1994 J. Biol. Chem . 269 : 23544-52; Delvaux et al 1990, Am. J. Physiol . 259 : G842-49. Horvat et al . 1992, Eur. J. Cancer 29th : 132-37; Ritter et al. , 1998. Br. J. Pharmacol. 124 : 627-38).

However, there are situations in which there is an abnormal process of hyperactivity of the NHE that lead to a pathological process. From the tissue point of view, NHE is involved in various physiological and pathological processes of vital importance such as ischemia and reperfusion, diuresis, arterial hypertension, the division of tumor cells, gastric acidity, epithelial transport, activation of macrophages, etc. (Rodríguez-Soriano 2000, Pediatr. Nephrol ., 14 : 1121-36; Sastrasinh et al 1995, Am. J. Phisiol . 268 : C1227-34; Szabó et al , 2000 J. Biol. Chem . 275 : 6302-7 ; Cournillon et al , 1993, Mol. Pharmacol . 44 : 1041-45; Scholz et al , 1995, Cardiovasc. Res . 29 : 260-68; Scholz et al . 1999, J. Thromb. Thrombolysis , 8 : 61-70 Rolfe et al . 1992. Am J Respir. Cell. Mol. Biol . 6 (6): 576-582) have indicated that the NHE participates in the release of cytokines by macrophages that take place in the activation processes immune Likewise, NHE activity is increased in essential hypertension in humans and in animal models of hypertension (Canessa et al , 1991, Hypertension . 17 (3): 340-348; Rosskopf et al . 1993. Hypertension , 21 ( 5): 607-617). In pathologies associated with cholesterolemia, an increase in activity of the NHE exchanger or an increase in the number of them in the cells is described (Scholz W .; Albus U .; Linz W .; Schölkens BA 1991. Effect of Na * / H * exchance inhibition in cardiac ischaemia Eur Herat J. 12 (Suppl.), 124 (Abstr.)). The frequent diabetes-hypertension association would have as a common mechanism the increase of the activity of said exchanger. Inhibitor of the exchanger may improve insulin resistance (Dudeya et al . 1992 Kidney Int . 42 (5): 1184-1190; Dudeja PK et al . 1991 J. Clin. Invest . 87 (5): 1755-1762; Dusing et al . 1993 Clin. Invest . 71 (2): 119-125). Finally, the NHE would participate in reperfusion processes after a period of ischemia. During the reperfusion of the myocardium, a gradient of H + is produced due to the increase in extracellular pH versus intracellular pH due to washing. As a consequence, an accumulation of intracellular Na + occurs, which forces the Na + / Ca ++ exchanger to operate in the reverse direction, and an increase in cytosolic Ca ++ occurs to toxic levels that lead to cell death. The use of a good Na + / H + exchanger blocker can improve said frame.

The treatment of all these processes pathological mentioned above could be improved in case of having a good NHE inhibitor.

At present there are no physiological inhibitors of natural origin specific to the NHE exchanger. NHEs are inhibited by synthetic compounds such as amiloride (Cragoe EJ Jr, Woltersdorf OW Jr, Bicking JB, Kwong SF, Jones JH. 1967 J. Med. Chem. Jan ; 10 (1): 66-75) and their analogues such as EIPA (Vigne P, Frelin C, Cragoe EJ Jr, Lazdunski M. 1983 Biochem Biophys Res Commun . 14 ; 116 (1): 86-90) and benzoyl guanidine derivatives such as cariporide (HOE 642) (Scholz, W., Albus, U., Counillon, L., Gögelein, H., Lang, HJ, Linz, W., Weichert, A. and Schölkens 1995 BA Cardiovas Res .; 29 : 260-268; Hoechst AG, Cardiovascular Research H 821, 65926 Frankfurt / Main, Germany: Scholz, W., Albus, U., Gögelein, H., Lang, HJ, Linz, W., Weichert, A. and Schölkens, BA; Center de Biochimie, University of Nice-Sophia Antipolis, Parc Valrose, Nice, France: L Counillon), HOE 694 (Counillon, L., Scholz, W., Lang, HJ, Pouyssegur, 1993. J. Mol Pharmacol . Nov; 44 (5): 1041-5; Scholz, W., Albus, U., Lang, HJ, Linz, W., Martorana, PA, Englert, HC and Schölkens, BA. 1993. Br. J. Pharmacol . 109 ( 2): 562-568; Hoechst AG, Cardiovasc Research H 821, 65926 Frankfurt / Main, Germany: Scholz, W., Albus, U., Gögelein, H., Lang, HJ, Linz, W., Weichert, A. and Schölkens, BA), EMD 85131 (Baumgarth, M., Beier, N. and Gericke, R. 1997. J. Med. Chem . 40 : 2017-2034) and others such as the zoniporide (Guzman-Perez, A., Wester, RT, Allen, MC , Brown, JA, Buchholz, AR, Cook, ER, Day, WW, Hamanaka, ES, Kennedy, SP, Knight, DR, Kowalczyk, PJ, Marala, RB, Mularski, CJ, Novomisle, WA, Ruggeri, RB, Tracey , WR and Hill, RJ 2001. Bioorg Med Chem Lett . Mar 26; 11 (6): 803-7).

Some of these substances such as amiloride, inhibit the NHE exchanger but do not do it specifically, inhibiting other exchangers at the same time, such as Na + / Ca ++ and the nonspecific Na + channels. Despite of these limitations amiloride is being used in Therapeutic as diuretic. However, the number of its side effects. On the other hand, some amiloride derivatives that have shown a more specific effect on the exchanger NHE, with some exceptions, have the serious drawback of being hard to reverse. Greater specificity have shown the cariporide and zoniporide, the latter being in addition to greater power.

The sensitivity to all these drugs varies greatly among the NHE isoforms (Yu et al , 1993. J. Biol. Chem ., 268 : 25536-41; Cournillon et al , 1993, Mol. Pharmacol. 44 : 1041-45; Cournillon et al , 1993, Proc. Natl. Acad. Sci. USA , 90 : 4508-12; Scholz et al , 1995, Cardiovasc. Res . 29 : 260-68; Scholz et al , 1999, J. Thromb. Thrombolysis , 8 : 61-70; Baumgarth et al , 1997, J. Medicinal Chem . 40 : 2017-34). The apparent affinities of the plasma membrane isoforms for an inhibitor can vary in a range of four orders of magnitude, generally following the order: NHE1> NHE 2 >> NHE 3> NHE 4 (Putney et al , 2002, Annu. Rev Pharmacol Toxicol 42 : 527-552). In contrast, mitochondrial NHE is relatively insensitive to amiloride, but is very effectively inhibited by its benzamil analogue (Sastrasinh et al , 1995, Am. J. Phisiol . 268 : C1227-34; Brierley et al , Biochemistry, 28 : 4347- 54; Kapus et al , 1988, Biochim Biophys Acta, 944: 383-90).

Other pharmacological agents such as cimetidine , clonidine , and harmaline also exhibit different affinities for NHE isoforms. However, these compounds, which are not chemically related to amiloride or cariporide, have an imidazole or guanidine part that can explain some structural similarity (Orlowski and Grinstein. 1997. J. Biol. Chem . 272 : 22373-22376).

Some of the NHE inhibitors have been shown to be effective in decreasing the size of the necrosis zone in experimental myocardial infarction (Gumina et al . 1998 J. Pharm. Experim. Therapeutics , 286 : 175-183; Miura et al . 1997. J. Am. Coll. Cardiol . 29 : 693-701; Munch-Ellingsen et al . 1998. Mol. Cell Biochem . 186 : 13-18; Otani et al . 2000. Clin. Exp. Pharmacol. Physiol . 27 : 387-393).

Some of them are subject to patents international (e.g. WO0183470 "Sodium-hydrogen exchanger type 1 inhibitor ", published on November 8, 2001, headlines: Chen Weichao George, Cox Eric David, PFIZER PROD INC, Guzmán Perez Angel; US6348476 "Pharmaceutical combination preparation of an inhibitor of the sodium / hydrogen exchanger and a medicament for the treatment of cardiovascular diseases ").

Against these substances of synthetic origin, to date, halocin H7 (formerly H6), is the only known substance of biological origin that shows a specific inhibitory effect of NHE. Halocin H7 is a protein substance that has been the subject of two previous patents ( inventors: 1 Meseguer Soria, M Torreblanca Calvo, B Soria Escoms, F Colom Valiente and 1 Alba Carballo. No. of patents. ES 2 094 695 and ES 2 094 696 published both on January 16, 1997, headline: University of Alicante ). Said halocin has a molecular weight of 30,500 ± 500 D and is obtained from the culture of strain CECT 4547 ( protected in the patent: ES 2 094 695 ) and is extracted using protein purification procedures such as column chromatography with concanavalin A and high pressure chromatography with ion exchange column. The complete purification process is long, expensive and complex.

The most outstanding advantages of halogen H7 versus the other NHE inhibitors are (as described in the patent ES 2 094 696):

-

To be a product of a peptide nature has reversibility of action therapeutic and the ease to metabolize itself of the protein substances

-

Is a natural substance of antibiotic type, its microbicidal power is based on its inhibitor action of the exchanger sodium-proton

-

Is a specific inhibitor, active at very low concentrations what decreases side effects

-

May operate in a wide range of salt concentrations, temperature and pH, which makes it the drug of choice in certain processes

-

Is resistant to the action of trypsin, which allows its use in the intestinal tract.

-

To the being a pronase sensitive peptide its effect can be terminated  when desired

The importance of having inhibitors of sodium-proton exchanger (NHE), resides in the consequences that derive from the activity of this mechanism in various pathological processes Except for halogen H7, I don't know knows any natural and / or peptide nature products capable of inhibit NHE. Although the H7 halocin already had a series of advantages over synthetic type inhibitors, the great disadvantage that we could highlight in front of them is that the process of obtaining and purification is too long, complex and expensive, and also that since it is a 30.5 kD protein, it could induce immune response.

The PINHE, object of this patent, in addition to possess all the advantages mentioned above for the Halocin H7 with respect to the rest of known NHE inhibitors, It presents new advantages that allow to ensure that we are before a substance of choice and with greater potential from the point of sight applied.

The main advantages offered by the PINHE Regarding halogen H7 are:

-

Is a small peptide, with a molecular weight 11 times less than that of Halocin H7, which could imply a lower risk of antigenic capacity

-

Higher ease of handling

       \ newpage
    

-

Though it is obtained from the culture of the same microorganism, it has a purification process much faster, easier and economic.

With regard to the activity of the PINHE, at Like halogen H7, it specifically inhibits NHE.

Description of the invention brief description

The present invention describes a peptide (PINHE), derived from halogen H7, of small molecular weight and defined by its amino acid or other analog sequence that acts as a natural physiological inhibitor of the Na + / H + exchanger ( NHE) in eukaryotes. PINHE is obtained by fractionation (which includes a precipitation with cold acetone) and purification of the culture supernatant of Haloferax gibbonsii alicante SPH7, which produces it naturally or, on the basis of its amino acid sequence, can be synthesized or be modified and expressed by molecular or genetic engineering techniques. PINHE is stable and active in wide ranges of saline concentrations, temperature and pH, trypsin resistant and pronase sensitive and can also be lyophilized and reconstituted without loss of activity. The use of said NHE inhibitor peptide, or its pharmaceutically acceptable salts, in the preparation of a pharmaceutical composition constitutes another additional object of this invention. From its reversible action as an inhibitor of the Na + / H + exchanger of eukaryotes of both the peptide and its salts, its uses are derived: (1) for the treatment or prophylaxis of pathologies in mammals, preferably human, caused due to hyperactivity or unwanted NHE activity, such as cardiovascular diseases (eg hypertension, myocardial infarction, arrhythmias, angina pectoris, etc.), brain diseases (eg cerebral infarction, cerebral edema, embolisms, etc.), kidney diseases (eg renal failure), metabolic diseases (eg diabetes mellitus, hyperlipidemias, etc.), diseases with hyperproduction of gastric mucosa hydrochloric acid, diseases with alterations in epithelial transport, diseases with alterations of cell volume, pathological proliferative processes (eg cancer, tumors, fibrosis, etc.), and alterations associated with excessive activity of immune system cells (eg inflammation, hypersensitivity) and t Ambien its use in (2) other diseases or pathologies that present with ischemia and post-ischemic reperfusion injury due to the hyperactivity of the NHE, among others: ischemia due to primary or secondary vascular alterations in various tissues and attached organs, due to post-reperfusion infarction, by transplantation of organs or tissues, by cardiac and vascular surgery, by ischemia in physiological situations such as physical exercise, pregnancy, childbirth, menstruation, etc., by trauma, or any pathology that involves tissue hypoperfusion (hemorrhagic, thermal shock, neurogenic, etc.) and in any surgical process in general. Other uses, derived from its activity, are as contraceptive (in male mammals), antibiotic or immunoregulatory agent and in another field as a modulator of saline stress in plants.

Detailed description

The invention provides a small peptide Inhibitor size of Na + / H + exchanger, hereinafter NHE or PINHE inhibitor peptide, which comprises a sequence of amino acids selected from:

to)

the amino acid sequence identified as SEQ ID NO 1 (PINHE) or a fragment thereof; Y

b)

a amino acid sequence analogous to the sequence defined in to)

As used in the present invention, the term "NHE" refers to the different forms of NHE present in different prokaryotic or eukaryotic organisms.

In the sense used in this invention, the term "analogous" is intended to include any sequence of amino acids (aás.) that can be isolated or constructed based on the aás sequence. shown in SEQ ID NO 1, for example, by the introduction of aás substitutions, including the insertion of one or more years., the addition of one or more years. in any of the ends of the molecule or the deletion of one or more years. in any end or inside the sequence.

In general, a sequence of years. analogous is substantially homologous to the aás sequence. identified as SEQ ID NO 1. In the sense used in this description, the "substantially homologous" expression means that sequences of aás. in question they have a degree of identity, at the level of a., of at least 40%, preferably of at least 85%, or more preferably of at least 95%.

The PINHE peptide of the present invention is synthesized by an extreme halophilic microorganism belonging to the Archaea Domain also known as haloarchaea. Specifically, the PINHE is produced by the "Alicante SPH7" strain of the Haloferax gibbonsii haloarquea, which was deposited in the Spanish Type Culture Collection (CECT), University of Valencia, Research Building, Burjasot Campus, 46100 Burjasot, Valencia , Spain, corresponding to the deposit number CECT 4547.

Almost all haloarqueas produce peptide substances with antagonistic activity compared to other members of the group. These substances are generically referred to as halokines (Rodriguez-Valera F, Judge G, Kushner DJ, 1982, Can. J. Microbiol . 28 : 151-154) and so far only a small number of them have been studied (O'Connor EM, Shand RF. J Ind Microbiol Biotechnol . 2002 28 : 23-31). Only the complete sequence of halogen H4 (Cheung J, Danna KJ, O'Connor EM, Price LB, Shand RF. 1997 J. Bacteriol 179 : 548-51) and halogen S8 (Price LB, Shand RF. 2000. J. Bacteriol . 182 : 4951-8). With regard to the mechanism of action, only that of halogen H7 (formerly H6, Meseguer I, Torreblanca M, Konishi T. 1995. J. Biol. Chem . 270 : 6450-5) is derived from which the PINHE object of this invention.

Given the broad antagonistic activity of haloarqueas and the fragility of these microorganisms before a inhibition of your NHE exchanger, this constitutes a target perfect for a mechanism of competition or antagonism in the fight for population survival. It is therefore more than likely that, among the wide range of halocins, peptides can be found PINHE counterparts that may or may not be, naturally.

In a particular embodiment, the aás molecule. of the invention is a molecule of aás. of the "Alicante SPH7" strain of Haloferax gibbonsii of SEQ ID NO 1 (PINHE peptide). PINHE is a peptide derived from halogen H7 that has all its activity, so an important aspect of the invention is the relationship between halogen H7 and PINHE, and how it is obtained from halocin. At present there are no more studies on halogen H7 or PINHE than those performed by the inventors and at the moment the peptide sequence of halogen H7 is unknown, of which only the amino terminal end is known. We also know that cells excrete an NHE inhibitor substance in the form of H7 halocin into the medium and that PINHE, as such, is not detected in the medium. It is known that PINHE is somehow part of the H7 halocin, however, it is unknown if Halocin H7 is a monomer with a unique peptide sequence that includes the PINHE sequence or if it is actually a dimer formed by two peptides , one small and hydrophobic responsible for the inhibitory activity of NHE, and another larger and hydrophilic that would act as a means of transport for the PINHE. This second option would be the most logical considering that the treatment to separate the PINHE, with cold acetone, is not too aggressive to break peptide bonds. Whatever the case, the way to obtain halocin or PINHE has a series of steps in common (production, obtaining the solution without cells, clarification and concentration) until obtaining a protein concentrate containing H7 halocin and then in Depending on the purification procedure used, halocin or PINHE is obtained. The key step in obtaining one or the other, as already mentioned, is precipitation with acetone. The separation of the PINHE from the rest of the halocin molecule requires that the precipitation with acetone be done cold and for a more or less prolonged time, preferably several hours. Without the treatment of acetone, or another that allows the separation of PINHE, halogen 1-17 can be purified using the procedures already described (Torreblanca M, Meseguer 1, Rodriguez-Valera F. 1989, J. Gen. Microbiol 135 : 2655-2661).

PINHE is a peptide derived from halogen H7 that owns all its activity, so it follows that the PINHE is corresponds to the peptide fraction where the activity resides of halogen H7.

The PINHE peptide of the invention is derived from the "Alicante SPH7" strain of Haloferax gibbonsii and can be found in similar forms in other species of microorganisms, where they may be naturally occurring or otherwise, they could also be as a result of a transformation process gene by which the transformed organism expresses said NHE inhibitor peptides. The amino acid molecule of the present invention can be isolated, among others, by conventional techniques, from protein extracts of microorganisms containing it, or by the use of mono- or polyclonal antibodies, prepared thanks to the information in the sequence of aás . provided in this invention.

The NHE inhibitor peptide of the invention it includes the fragments of the same that present said activity inhibitor of the Na + / H + exchanger and which can be easily identified from existing knowledge in the technical sector.

Aás molecule. of the present invention can be expressed, in general, by genetic engineering techniques that allow recombinant expression of the inhibitory peptide of the present invention through the use of an expression vector, which contains a nucleotide sequence encoding the sequence SEQ ID NO 1, in a wide range of host cells. In general, said expression vector comprises at least one DNA sequence encoding the inhibitory peptide of the present invention and at least one promoter that directs the transcription of said nucleotide sequence of interest, to which it is operably linked, and other sequences necessary or appropriate for transcription and its appropriate regulation in time and place, for example, start and end signals, cutoff sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case among plasmids, artificial yeast chromosomes (YACs), artificial bacterial chromosomes (BACs), artificial chromosomes based on bacteriophage P1 (PACs), cosmids or viruses, which may also contain a bacterial or yeast origin of replication so that it can be amplified in bacteria or yeasts, as well as a usable marker to select transfected cells other than the gene or genes of interest. This vector can be obtained by conventional methods known to those skilled in the art (Sambroook, J., Russell DW 2001 Molecular Cloning: A laboratory manual . Third edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor NY.).

"Artificial synthesis" means the use of physicochemical synthesis techniques that do not involve the use of microorganisms or other beings alive to obtain a compound that contains total or partially the sequence of the inhibitor peptide of the present invention and having inhibitory activity on NHE.

The term "mixed procedures" such as used in the present invention means utilization joint biological manipulation and synthesis procedures artificial for obtaining a compound that contains total or partially the sequence of the NHE inhibitor peptide of the present invention and having inhibitory activity on the NHE

Within the scope of this invention, find the pharmaceutically acceptable salts of the peptide NHE inhibitor provided by this invention. The term "pharmaceutically acceptable salts" includes salts usually used to form metal salts or salts of acid addition. The nature of salt is not critical, as long as when pharmaceutically acceptable. These salts can Obtained by conventional methods well known to technicians in the field reacting the acid or base appropriate with the NHE inhibitor peptide.

The NHE inhibitor peptide of the present invention, as well as its pharmaceutically acceptable salts, can be part of various types of compositions for application in the body of a mammal, preferably the human being. In a particular embodiment, said composition is a composition Pharmaceutical The pharmaceutical composition provided by this invention comprises a therapeutically effective amount of a NHE inhibitor peptide together with at least one excipient pharmaceutically acceptable.

The use of said NHE inhibitor peptide, as well as its pharmaceutically acceptable salts, in the preparation of said pharmaceutical composition constitutes another additional object of this invention.

The pharmaceutical compositions containing the NHE inhibitor peptide can occur in any form of administration, for example, solid or liquid, and can be administered by any appropriate route, for example, by route oral, parenteral, rectal or topical, for which they will include pharmaceutically acceptable excipients necessary for the formulation of the desired administration form.

A further object of the present invention is the use of said pharmaceutical compositions in the preparation of a medication for the treatment or prophylaxis of processes pathological of mammals, preferably human, caused by a hyperactivity or unwanted activity of the NHE, such as, among others, cardiovascular diseases (e.g. hypertension, myocardial infarction, arrhythmias, angina pectoris, etc.), brain diseases (e.g. cerebral infarction, cerebral edema, embolisms, etc.), kidney diseases (e.g. renal failure, kidney disease diabetic, etc.), metabolic diseases (diabetes mellitus, hyperlipidemias, etc.), diseases with acid hypersecretion hydrochloric in the intestinal mucosa, diseases with alterations in epithelial transport, diseases with alterations of the cell volume, pathological proliferative processes (e.g. cancer, tumors, fibrosis, etc.), and alterations associated with an activity excessive immune system cells (e.g. inflammation, hypersensitivity).

A particular object of the present invention is the use of said pharmaceutical compositions in the preparation of medications for the treatment or prophylaxis of processes pathological of mammals, preferably human, that occur with ischemia and post-ischemic reperfusion injury due to the hyperactivity of the NHE. Among these processes pathological are included as notable ischemic lesions produced by vascular alterations (both primary and secondary) in tissues belonging to the systems cardiocirculatory (heart and blood and lymphatic vessels), respiratory, digestive, genito-urinary, nervous (central and peripheral), musculoskeletal, hematic, endocrine and skin, and in general all the organs attached to the systems previously mentioned, ischemic reperfusion processes post-infarction, ischemic processes after transplantation of organs and / or tissues and ischemic processes in situations physiological such as physical exercise, pregnancy, childbirth, menstruation, etc. On the other hand, these pharmaceutical compositions can be administered as prophylaxis of ischemia or hypoxia that takes place during cardiac and vascular surgery (during and after these surgeries) and also as a result of trauma including crush syndrome and destruction of members, as well as in any pathology that involves tissue hypoperfusion (hemorrhagic, cardiac, thermal shock, neurogenic, anaphylactic, etc.).

Another particular and additional object of the present invention is the use of the pharmaceutical composition of the present invention in the contraception. The estrogenic alpha receptor is essential for adequate fertile function in male individuals since its activity is necessary for the maintenance of cytoarchitecture in the efferent ducts and for the concentration of sperm in the head of the epididymis. Zhou et al . ( PNAS 2001; 98 (24): 14132-14137) have shown that the estrogenic alpha receptor is responsible for the expression of an isoform of the sodium-proton exchanger (Na + / H +) in the efferent tubules of the reproductive system in male mice, and therefore, influences sodium reabsorption and passive water transport, these effects, necessary for sperm concentration. Likewise, these authors suggest that the interference in the function of this sodium-proton exchange system (Na + / H +), would be able to produce infertility in males, without altering the synthesis of testoterone and therefore the physiological effects of this, such as the maintenance of libido. Thus, the application to contraception would be of exceptional importance, among other situations, because of the reversibility of the effects of said compound.

Another particular object of the present invention is the use of the pharmaceutical composition of the present invention for its antibiotic activity in the preparation of an antibiotic for the treatment of infectious diseases, among others, infectious diseases caused by microorganisms (eg Helicobacter pylori ).

Another particular object of the present invention is the use of effective amounts of the compound of the present invention as a modulator of the expression of the Na + / H + exchanger in plants having an increased overexpression (constitutive or not) of the Na + / H + exchanger or the like. In the efforts made to have salt-tolerant plants, it could serve to modulate the expression of the Na + / H + vacuolar exchanger of plants such as that patented by Eduardo Blumwald (Overexpression of a Vacuolar Na ^ {+} / H + Antiport in Arabidopsis . Apse MP, Aharon GS, Snedden WA, Blumwald E. Science. 1999 Aug 20; 285 (5431): 1256-8.), Or any other, performing the same function, was inhibited by the peptide object of the present invention. In this sense, the application or not of this Na + / H + exchanger inhibitor could modulate the expression of said gene allowing a greater or lesser enzymatic activity of the protein encoded by it. This would mean that in conditions where there was no excess of ClNa, the plant that overexpressed the transporter could have less undesirable effects (or lack thereof). Such undesirable effects could be produced, for example, by excessive acidification of the cytoplasm. Although the Na + / H + exchanger uses Na +, it cannot be ruled out that a high presence of this enzyme can use other cations, extruding protons into the cytoplasm. Commercial use could reside in the addition of this inhibitor in controlled amounts to plants that have an increased expression (constitutive or not) of the above-mentioned vacuolar Na + / H + exchanger or similar enzymes.

Descriptions of the figures

Figure 1A.- Electrophoresis in SDS polyacrylamide (12%) of a sample containing PINHE after purification with acetone. Street 1 and 2: Standard proteins. Street 3: Sample containing the PINHE. Staining with silver nitrate.

Figure 1B.- Electrophoresis in SDS-polyacrylamide (12%) of a sample containing Semi-purified H7 first by Hydroxyapatite and second place by Sephadex G50. Lane 1: standard proteins. Street 2: Sample containing H7. Staining with silver nitrate.

Figure 1C.- Purification of H7 (formerly H6) (figure published in Torreblanca M, Meseguer I, Rodriguez-Valera F. 1989, J Gen. Microbiol . 135: 2655-2661) Staining with Comassie blue.

Figure 2.- Halos of inhibition showing the PINHE inhibitory activity on the double indicator strain cap.

Figure 3A.- Optical photomicrograph of untreated Halobacterium salinarum cells (X 2000).

Figure 3B.- Optical photomicrograph of Halobacterium salinarum cells treated with PINHE (X 2000).

Figure 4.- Relationship between cytometric measurement of the cytoplasmic acidity of 293HEK cells (Ratio) and their pH intracellular The ratio is the measure of BCECF fluorescence in the cytoplasm expressed as a ratio between fluorescence orange (FL3) and green (LF1) detected by the cytometer. PH intracellular is the pH of the medium where cells are suspended permeabilized with nigericin. To do this the cells (1 x 10 5 cells per ml) were incubated at 37 ° C for 15 minutes with medium DMEM with glucose and without bicarbonate which in turn contained 10% FBS, 25 mM HEPES and BCECF 2 µg / ml and whose pH was adjusted with HC1. PH intracellular was balanced with the external by presence in the medium of 2 µg / m1 of nigericin. After incubation the Fluorescence was measured in the cytometer.

Figure 5.- Recovery of intracellular pH of 293HEK cells after acidification with sodium propionate. The trial was performed in the presence (right) and absence (left) 1000 U / ml of the NHE inhibitor peptide as described in the Figure 4 but without the presence of nigericin. On the abscissa axis the ratio is represented and in time the time elapsed in the test (the maximum time outlined in the figure, (1023, is equivalent to 300 seconds)

Examples of embodiment of the invention Example 1 Procedure for obtaining and purifying the PINHE. Comparison  with the obtaining and purification of halogen H7

PINHE, like halogen H7, is obtained at from strain CECT 4547 and following the same conditions of culture described in the patent ES 2 094 695. Next the cells are removed, the supernatant is concentrated and dialyzed with distilled water by filtration techniques and tangential ultrafiltration also following the protocol there indicated. At this point, he tripped over the main inconvenience that showed the halogen H7 which was the process of purification, which consisted of several stages in which they were required Slow and expensive protein purification techniques.

A) Obtaining the PINHE

A culture of the producing strain SPH7 is prepared (CECT 4547) in a medium containing 2.66 M ClNa, 0.13 M Cl 2 Mg x 6H 2 O, 0.16 M SO 4 Mg x 7H 2 O, 6.64 mM Cl 2 Ca, 53.3 mM ClK, 1.6 mM CO 3 HNa, 4.48 mM BrNa, supplemented with 0.5% yeast extract and pH 7.2. Incubate at 37 ° C with aeration until reaching an optical density between 0.9 and 1 at 600 nm. The cells are then separated by using a tangential filtration system with pore size filters of 0.45 µm. Subsequently, large macromolecules are removed by ultrafilters of 100,000 Daltons. Then the Filtering is concentrated using 1000 Daltons ultrafilters. He concentrate is dialyzed against distilled water and proceed as follow:

Precipitation with three or more volumes of acetone cold for 18-24 hours.

The PINHE remains in the supernatant and the precipitate containing impurities by centrifugation.

The supernatant is taken and the acetone is removed (e.g. by a rotary evaporator).

The degree of purity of the PINHE is checked.

If a degree of purity has not been reached satisfactory (approximately 90%) the repeat is repeated process.

The determination of the degree of purity of the samples containing PINHE was performed by analysis in polyacrylamide gels in SDS (Figure 1A) following the protocol described by Laemmli (Laemmli UK, 1973. J. Mol. Biol . 80 : 575-599) . The PINHE thus obtained is capable of being treated to obtain its peptide sequence indicated in SEQ ID NO 1.

B) Obtaining H7 Halocin

From the dialysate concentrate, we can obtain halogen H7 following another procedure that does not include the precipitation with acetone. Thus a sample of the concentrate is applied in a hydroxyapatite column in buffer containing 10 mM potassium phosphate, 2.5 M NaCl, pH 7.2 and eluted using a linear gradient of potassium phosphate buffer 10-400 mM, 2.5 M NaCl, pH 7.2. 10 ml fractions were collected and from each one test was performed to determine its activity inhibitory

Fractions containing the highest inhibitory activity were concentrated to a final volume of 3 ml. They were subsequently dialyzed against distilled water and filtered using a Sephadex G-50 column previously equilibrated in 0.05 M TRIS / HC1 buffer, pH 6.7. The 1 ml fractions were eluted in the same buffer. Those containing the maximum activity were collected and concentrated to a volume of 1.5 ml. At this point the degree of purity of the sample was determined by means of a polyacrylamide gel electrophoresis in SDS (Figure 1B) following the protocol described by Laemmli (Laemmli UK, 1973. J. Mol. Biol. 80 : 575-599 ).

If a higher degree of purity is desired, the process must be continued using additional methods of protein purification. Using high pressure chromatography, as described for example in Torreblanca et al. (Torreblanca et al 1989, J. Gen. Microbiol . 135 : 2655-2661), a good degree of purity can be achieved, although the process performance It is low (Figure 1C shows the result of this purification procedure).

Physical-chemical characteristics of PINHE

It has a molecular weight of 2733.8 Daltons calculated by mass spectrometry that corresponds to the weight calculated as the sum of the weights of the amino acids that make up.

From its sequence, it follows that the PINHE It is a peptide with hydrophobic character. This is in accordance with the difficulty he has shown to dissolve in water after having been lyophilized.

To study other properties physicochemical, samples of PINHE were taken, determined its activity and underwent various treatments after which was determined again the remaining activity. How As a result, the following was determined:

PINHE is resistant to denaturation thermal, being able to withstand 100ºC for several minutes and is 10 min treatment needed. at 121 ° C to destroy totally its activity by heat.

The PINHE keeps all its activity in a wide range range of saline solutions ranging from distilled water to saturated salt solutions. It also maintains its activity in a great diversity of aqueous solutions, not having been found so far lost of activity for these reasons in any case.

The PINHE supports a wide pH range of solvent, being active at least, at pH values between 5 and 9.

Proteases such as trypsin do not affect you, without However, it is sensitive to other enzymes such as pronase.

It is susceptible to lyophilization without loss of activity although for subsequent rehydration requires previously dissolved in a solvent such as DMSO.

It is also a very stable substance, able to conserve all its activity in aqueous solutions sterile for long periods of time. It also supports well the low temperatures, even the freezing, although it has observed a partial decrease in its activity after successive freezes and defrosts.

Example 2 PINHE activity determination

PINHE activity is determined from it so that of halogen H7, using the system called de double layers containing the indicator strain.

A solid culture medium is prepared such that it contains the same components as the liquid medium indicated in Example 1 by adding 2% agar-agar. It is distributed in Petri dishes at a rate of 10 ml per plate and allowed to solidify. Tubes with 5 ml of the same medium are prepared and kept at 50 ° C to avoid solidification. To these tubes a microbial suspension containing approximately 10 6 cells of the indicator strain of Halobacterium salinarum is added and quickly poured onto the plates 15 prepared above and also allowed to solidify, thus generating the double layer. Serial dilutions of the PINHE sample to be tested are prepared. Of these dilutions, 10 µl are deposited on the double layer and left at room temperature until completely absorbed. The plates are then incubated at 37 ° C for four days. The maximum dilution of the PINHE showing a detectable inhibition halo is considered to contain 1 arbitrary unit, that is, 100 arbitrary units (AU) per milliliter (Figure 2).

Example 3 Antibiotic effect on Halobacterium salinarum

It has been proven that the PINHE as well as the Halocin H7, has antibiotic activity against microorganisms from the haloarchaeas group. These 25 microorganisms that inhabit in hypersaline environments (approx. 4 M of Na + in the middle external versus 2 M of intracellular Na +), have an NHE Unidirectional very efficient to remove this ion from the medium intracellular taking advantage of the proton motive force. Inhibition NHE is lethal to these microorganisms, since in a medium where the concentration of Na + is so high, if it doesn't work NHE, this ion accumulates intracellularly causing a osmotic imbalance Water enters the cell interior, the cell It swells and finally smooths and dies. One way to detect this Lethal effect is by observation under the microscope of the cells before and after treatment.

For this purpose, a culture of the indicator strain of Halobacterium salinarum is prepared, centrifuged and resuspended in a solution containing the same concentration of salts as in example 1. Proteins are quantified and adjusted to reach the concentration of 1 mg of proteins per milliliter. . Photos of the cells are taken using a phase contrast microscope (Figure 3A). Purified PINHE is added to a final concentration of 640 AU per milligram of protein. At different time intervals samples are taken to be photographed under a microscope. After ten minutes, a deformation and swelling of the cells is observed, which increases progressively over time until cell lysis occurs. After 24 hours, only remnants of cellular envelopes, called ghosts, can be observed (Figure 3B).

Example 4 PINHE diuretic and natriuretic effect in rats

A group of 6 wistar rats was divided into two, three for the control group and another three to be treated. This group would be given intraperitoneally a single injection of purified PINHE at a concentration of 125 AU per gram of animal weight. The control group was treated with saline. Animals were introduced into individual metabolic cages. After administration of the PINHE, the weight of the animal and the intake of food and water were monitored at 24, 48 and 72 hours after administration. Also the volume of urine was collected and measured. To control the effect of PINHE on natriuresis, the concentration of sodium in the urine of the rats was determined. At 24 hours an increase in the volume of urine of the order of 89-98% and an increase in the concentration of sodium in the urine of the order of 39-43% of the rats treated with PINHE with respect to the control rats were observed, which indicates a clearly diuretic and natriuretic effect of PINHE. These effects are compatible with a PINHE inhibitory activity on NHE. The mechanism could be a decrease in the rate of tubular reabsorption. The indicated effects reverse spontaneously within 48-72 hours of treatment.

Example 5 NHE inhibition of 293HEK cells (primary cell line of human embryonic kidney)

293HEK cells were grown in the middle of DMEM culture containing 4 g of glucose and 3.7 g of sodium bicarbonate and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin 1000 U / mi, Streptomycin 1000 amphotericin 2.5 µg / ml and gentamicin 501.tg/ml. The culture was carried out at 37 ° C and in an atmosphere of 5% CO2. The cells were grown to 80% confluence, then they collected by incubation with trypsin 0.25-0.12% and EDTA 0.01-0.03% and centrifugation at 300g for 10 minutes and the precipitate is resuspended in DMEM medium without bicarbonate, containing 10% serum Fetal bovine (FBS) and 25 mM Hepes. A count of the number of living cells using a hemocytometer and a dye vital (trypan blue dye). The cell suspension was stored on ice. Until the rehearsal. The inhibitory effect of PINHE on NHE of 293HEK cells were performed by an assay with a cytometer of flow, measuring intracellular pH recovery after a acidification with 1M propionic acid (pH 7-7.5) in presence or absence (control) of PINHE. The essay contained: 1x105 cells / ml, 10% FBS, 2 µg / ml BCECF (2 ', 7'-bis (carboxyethyl) -5,6-carboxyfluorescein) and up to 1-2 ml of DMEM medium (Dulbecco's Modified Eagle Medium) with glucose and without bicarbonate, 10% FBS and 25mM of Hepes It was incubated at 37 ° C for 10 minutes and in the tubes corresponding PINHE was added to the desired concentrations between 0 and 3000 U / ml and incubated until pH stabilization intracellular (5-30 minutes).

It was measured in a cytometer (EPICS Beckman-Coulter XL-MCL) BCECF fluorescence - measured as a quotient between the emissions of green (FL1) and orange (FL3) fluorescence - and then acidified by adding propionic acid neutralized with NaOH until pH 7-7.5 of a stock solution until you get a desired intracellular acidification from 7.2 to 6.4, then the BCECF fluorescence measurement was continued. The relationship between BCECF fluorescence and internal cell pH 293HEK was performed in parallel experiments balancing the pH external (using different pH in the test solution) with the Internal pH, for which 2 µg / ml was added to the test solution of nigericin to permeabilize the cells to the protons of the extracellular medium An example of the relationship between BCECF fluorescence - FL3 / FL1 ratio - of the cells and their pH internal is shown in Figure 4. When acidification occurs with propionic a sharp decrease in the internal pH of the cells from equilibrium values 7.3-7.6 to 7.1-6.4. At this time the cell starts up its intracellular pH regulation mechanisms to restore the pH balance. However, the absence of bicarbonate in the test medium disables the bicarbonate / chloride exchanger (CO3H - / Cl -) and also the presence of Hepes in the medium inhibits the NBS cotransporter so the pH recovery intracellular that occurs after acidification should almost exclusively to the NHE exchanger. In Figure 5 it is shows an example of acidification of 293HEK cells in the absence (control) and in the presence of PINHE (1000 U / ml). Acidification with sodium propionate produced an acidification of the cytosol of the cells from a pH in equilibrium of 7.4 to a value of 7.04. Immediately the cell begins to restore the intracellular pH as shown in Figure 5, both in the control case as in the presence of PINHE. From the experimental values an initial recovery speed estimate was made of intracellular pH based on a mathematical adjustment of the experimental data to a curve and obtaining its derivative at zero time, which allows to estimate initial velocity values of intracellular pH recovery of 0.37exp (-2) Δp / s for the control and of 0.11 exp (-2) Δp / s in the case of presence of PINHE. The results show that the PINHE, after the cytosol acidification, inhibits pH recovery intracellular due to the Na + / H + exchanger.

<110> SUPERIOR INVESTIGATION COUNCIL SCIENTISTS

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<120> NEW INHIBITING PEPTIDE OF EXCHANGER OF Na + / H + (PINHE) AND ITS APPLICATIONS

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<130> PINHE

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<160> 1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<170> PatentIn version 3.1

         \ vskip0.400000 \ baselineskip
      

<210> 1

         \ vskip0.400000 \ baselineskip
      

<211> 28

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Haloferax gibbonsii alicante SPH7

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 1

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

\ sa {Ser Trp Ile Asp Ser Ala Ser Thr Ala Ala Leu Gly Thr Asn Pro}

\ sac {Val Thr Met Ser Ala Pro Gly Gly Thr Val Asn Ile Asp}

Claims (10)

1. A Na + / H + exchanger inhibitor peptide characterized by an amino acid sequence belonging to the following group: the amino acid sequence identified as SEQ ID NO 1 or a fragment thereof; and an amino acid sequence analogous to the sequence defined in a). 2. A peptide according to claim 1 characterized in that it is obtained, among other microorganisms, from the "Alicante SPH7" strain of Haloferax gibbonsii haloarchaea (CECT 4547). 3. A peptide according to claim 1 characterized in that it is obtained by genetic engineering procedures that allow the expression of said peptide, by artificial synthesis or by mixed procedures. 4. A composition characterized in that it comprises a peptide according to any one of claims 1 to 3. 5. A composition according to claim 4, characterized in that it is a pharmaceutical composition comprising a therapeutically effective amount of a peptide according to any one of claims 1 to 3 and at least one pharmaceutically acceptable excipient. 6. Use of a pharmaceutical composition according to claims 4 to 5 in the preparation of a medicament for the treatment or prophylaxis of diseases and alterations pathological in mammals, preferably human, caused by a hyperactivity or unwanted activity of the NHE, such as, among others, cardiovascular diseases (e.g. hypertension, myocardial infarction, arrhythmias, angina pectoris, etc.), brain diseases (e.g. cerebral infarction, cerebral edema, embolisms, etc.), kidney diseases (e.g. renal failure), metabolic diseases (diabetes mellitus, hyperlipidemias, etc.), diseases with hyperproduction of hydrochloric acid from the gastric mucosa, diseases with transport disorders epithelial, diseases with alterations of cell volume, pathological proliferative processes (e.g. cancer, tumors, fibrosis, etc.), and alterations associated with excessive activity of immune system cells (e.g. inflammation, hypersensitivity). 7. Use of a pharmaceutical composition according to claims 4 to 5 in the preparation of a medicament for the treatment or prophylaxis of diseases and alterations pathologies in mammals, preferably humans, that occur with ischemia and post-ischemic reperfusion injury due to the hyperactivity of NHE, among others,

to)

ischemic lesions caused by vascular alterations (both primary and secondary) in tissues belonging to the cardiocirculatory systems (heart and blood and lymphatic vessels), respiratory, digestive, genito-urinary, nervous (central and peripheral), musculoskeletal, hematic, endocrine and skin; as well as in general all organs attached to the systems above mentioned.

b)

ischemic reperfusion processes post-infarction,

C)

ischemic processes after transplantation of organs and / or tissues,

d)

ischemia or hypoxia that takes place during cardiac and vascular surgery,

and)

tissue lesions caused by Ischemia in physiological situations such as physical exercise, pregnancy, childbirth, menstruation, etc.

F)

ischemia or hypoxia as a result of trauma including crush syndrome and destruction of members.

g)

Any pathology that involves tissue hypo-perfusion (hemorrhagic shock, cardiac, thermal, neurogenic, anaphylactic, etc.).

8. Use of a pharmaceutical composition according to claims 4 to 5 in the preparation of a medicament that can be used as a contraceptive in male mammals, including humans 9. Use of a pharmaceutical composition according to claims 4 to 5 in the preparation of an antibiotic for the treatment of infections in mammals, preferably human, among others, caused by microorganisms such as Helicobacter pylori . 10. Use of effective amounts of the peptide inhibitor according to one of claims 1 to 3 to modulate the expression of the Na + / H + exchanger in plants that had an increased overexpression (constitutive or not) of Na + / H + exchanger or the like.