WO2012104462A1 - Heptapeptides and the use thereof for controlling hypertension - Google Patents
Heptapeptides and the use thereof for controlling hypertension Download PDFInfo
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- WO2012104462A1 WO2012104462A1 PCT/ES2012/070055 ES2012070055W WO2012104462A1 WO 2012104462 A1 WO2012104462 A1 WO 2012104462A1 ES 2012070055 W ES2012070055 W ES 2012070055W WO 2012104462 A1 WO2012104462 A1 WO 2012104462A1
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- heptapeptide
- amino acid
- pharmaceutical composition
- peptides
- heptapeptides
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/15—Peptidyl-dipeptidases (3.4.15)
- C12Y304/15001—Peptidyl-dipeptidase A (3.4.15.1)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8103—Exopeptidase (E.C. 3.4.11-19) inhibitors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
Definitions
- the present invention relates to heptapeptides that are characterized as inhibitors of angiotensin I converting enzyme (RCT). It also refers to the use of heptapeptides for the preparation of a medicament for the reduction of hypertension by inhibition of the activity of ACE.
- RCT angiotensin I converting enzyme
- Hypertension is characterized in that the blood pressure is higher than desirable for health. Hypertensive individuals have a higher risk of cardiovascular and cerebrovascular complications, resulting in an increase in mortality prematurely in these subjects (Giles TD 2009. J Clin Hypertens (Greenwich) 1 1 (1 1): 61 1 -4). The reversal of hypertension through changes in lifestyle or drug treatments reduces the risk of heart attack (30-40%) and other coronary problems (20%) and seems to also reduce the incidence of other vascular disorders.
- renin-angiotensin-aldosterone (SRA) system Since alterations in the renin-angiotensin-aldosterone (SRA) system have been associated with hypertension, antihypertensive medications have been developed that use different components of this system as targets, such as reninna inhibitors, angiotensin converting enzyme inhibitors , angiotensin receptor blockers or aldosterone receptor antagonists (Williams B 2009. J Hypertens 27 (3): S19-26).
- the angiotensin converting enzyme is a peptidase present in vascular endothelial cells in the lung, kidney and brain that converts angiotensin I into the vasoconstrictor peptide angiotensin li, thus contributing to increasing blood pressure.
- ACE inhibition for the treatment of hypertension is currently performed using medications, such as captopril whose active substance is hydrochlorothiazide, but they cause effects. Side effects such as dry cough and angioedema.
- medications such as captopril whose active substance is hydrochlorothiazide
- Side effects such as dry cough and angioedema.
- studies have been conducted for the inhibition of ACE with peptides of different sequence and length than the peptides of the present invention.
- Peptides derived from plant and animal proteins, such as milk, soy or fish, have been used for ACE inhibition.
- Such peptides can be produced by enzymatic hydrolysis from precursor proteins during food processing and digestion. These peptides can be incorporated into functional foods for the development of health-beneficial products and currently there are various products on the market or in development that contain these bioactive peptides with ACE inhibitory activity. However, clinical trials conducted so far with several of these peptides have not demonstrated a clear beneficial effect in all hypertensive patients ⁇ Engberink MF et al 2008. Hypertension 51 (2): 399-405 and De Leo et al 2009. Curr Pharm Des 15 (31): 3622-43).
- Tryptophan (W), tyrosine (Y), phenylalanine (F), proline (P), isoleucine (I), alanine (A), leucine (L) have been described as important amino acids for ACE activity inhibition and methionine (M) located last in the carboxy terminal end, while the important amino acids in the penultimate positions are valine (V), isoleucine (I), alanine (A), arginine (R), tyrosine (Y) and phenylalanine (F) ⁇ Cheung HS et al 1980.
- the present invention relates to heptapeptides with antihypertensive effect and the use of said heptapeptides for the preparation of a pharmaceutical composition to decrease hypertension.
- the peptides of the invention are inhibitors of angiotensin I converting enzyme (RCT).
- RCT angiotensin I converting enzyme
- the ACE inhibitory activity of the peptides of the invention was compared with other peptides described above. It was shown that the antihypertensive activity of heptapeptides is superior to those described above.
- a first aspect of the invention relates to a heptapeptide consisting of the sequence SEQ ID NO: 1, where the first amino acid is an arginine (R) the second amino acid is a lysine (K), the third amino acid is a tryptophan (W), the fourth amino acid is a histidine (H) or a leucine (L), the fifth amino acid is a phenylalanine (F), the sixth amino acid is a leucine ⁇ ! _) Or a histidine (H) and the seventh amino acid It is a tryptophan (W).
- heptapeptides of the invention coincide in amino acid sequence in the positions described but vary in the fourth and sixth positions counting from the amino terminal end.
- a preferred embodiment of the first aspect of the invention relates to a heptapeptide consisting of the sequence SEQ ID NO: 2 where the fourth position amino acid is a histidine (H) and the sixth position amino acid is a leucine (L).
- another preferred embodiment refers to the heptapeptide consisting of the sequence SEQ ID NO: 3 where the fourth position amino acid is a leucine (L) and the sixth position amino acid is a histidine (H).
- the variation variable
- place of variation the amino acid that replaces it
- the present invention also relates to both dextrogyric steroisomers (D) and levogyric stereoisomers (L), whereby another preferred embodiment of the first aspect of the invention refers to the heptapeptide whose steroisomerism is D and another preferred embodiment refers to the heptapeptide whose stereoisomery is L.
- stereoisomer in the present invention refers to an optical isomer or enantiomer, that is, in the present invention a heptapeptide of the same molecular form and amino acid sequence but of different structural formula.
- the stereoisomers D and the stereoisomers L differ in the property of diverting the plane of the polarized light in a different direction, with the stereoisomers D being those which deflect it to the right (dextrogyric isomer) and the isomers L (levogyric isomer) which turn left.
- the enantiomers that form the proteins are L-isomers but to increase the stability and resistance to degradation by proteases present in biological fluids, in the present invention D-isomers have also been generated.
- the amino terminal and carboxyl terminal ends of the heptapeptides may or may not be modified.
- the amino terminal end may be acetylated and the carboxyl terminal end may be amidated following the procedures known to the person skilled in the art, and said modification can be performed in solid phase chemical synthesis.
- Such modifications favor the stability of the peptides against degradation by proteases but do not affect their biological activity.
- another preferred embodiment of the first aspect of the invention relates to a heptapeptide where the amino terminal end is acetylated and the carboxyl end is amidated.
- another preferred embodiment of the first aspect of the invention relates to a heptapeptide consisting of SEQ ID NO: 4.
- Another preferred embodiment refers to a heptapeptide consisting of SEQ ID NO: 5.
- modification MOD_RES
- acetylation refers to acetylation and “amidation” refers to amidation.
- heptapeptide refers to a peptide consisting of seven amino acids linked by peptide bond. From now on we will refer to the "heptapeptides of the invention” as any of the peptides described in the first aspect of the invention. In this description we will refer to the "natural heptapeptides of the invention” such as the isomeric heptapeptides L since they are those that occur in nature.
- inhibitor primarily refers to the fact that the heptapeptide inhibits (decreases) the activity of ACE and therefore reduces hypertension.
- An antihypertensive agent is therefore an agent that decreases hypertension.
- the angiotensin I converting enzyme (ECA, peptidyl dipeptidase A, BC036375.2, EC 3.4.15.1, "angiotensin converting enzyme” or ACE) refers to the enzyme that uses angiotensin I as a substrate converting it into angiotensin II (which is a vasoconstrictor molecule) and bradykinin (which is a vasodilator molecule), which it converts into a non-functional peptide.
- the activity of this enzyme increases blood pressure and therefore contributes to hypertension in humans.
- Hypertension as described in the present invention is a disease of diverse etiology characterized by high blood pressure in the systemic circulation (diastolic ⁇ 90 mm Hg; systolic ⁇ 140 mm Hg;).
- the peptides of the invention can be obtained by chemical synthesis or by strategies derived from biotechnology.
- the heptapeptide of the invention could be produced from a polypeptide comprising the heptapeptide of the invention, for example after fragmentation thereof.
- a second aspect of the invention relates to a polypeptide comprising the heptapeptide of the invention.
- the natural heptapeptide of the invention, or the polypeptide comprising it could be produced from a nucleic acid that encodes it.
- a third aspect of the invention relates to a nucleic acid comprising the nucleotide sequence encoding the natural heptapeptides of the invention.
- the peptide sequence would be encoded by a DNA fragment (deoxyribonucleic acid or DNA) according to the laws of the genetic code. All these procedures are common for all those experts in the area of knowledge of the present invention.
- DNA genetic constructs encoding sequences of the natural peptides of the invention are also part of the invention.
- Said genetic construction of DNA would direct the intracellular transcription of the heptapeptide sequence, and comprises at least one of the following types of sequences: a) DNA sequence comprising the nucleotide sequence encoding the natural heptapeptide of the invention for its intracellular transcription and translation; b) a gene expression cassette comprising the DNA sequence defined in a) operably linked to transcription control and optionally translation elements; c) DNA sequence corresponding to a gene expression system or vector comprising the natural heptapeptide coding sequence of the invention defined in a) or the expression cassette defined in b), operably linked to at least one promoter that directs the transcription of said sequence, and with other sequences to regulate its transcription such as, for example, start and end signals, or polyadenylation signal, where preferably the vector is a plasmid.
- a sixth aspect of the invention relates to the host cell comprising the nucleic acid, the expression cassette or the vector encoding sequences of the natural heptapeptide of the invention, where the cell is preferably a mammalian cell (not belonging to the group of human embryonic or germ cells), insect, fungus (including yeasts) or bacteria.
- the introduction of said gene constructs can be carried out with the methods known in the state of the art.
- a seventh aspect of the invention relates to the production process of the natural heptapeptide of the invention which comprises culturing the host cells described in the sixth aspect of the invention and the subsequent purification of the natural peptide of the invention.
- the heptapeptides of the invention can also be synthesized by chemical synthesis, preferably on a solid phase. In this case, both L and D enantiomers can be produced.
- the synthesis procedure allows the N-terminal end of the peptides to be acetylated and the C-terminal end amidated, which would not significantly affect the ACE inhibitory activity of said peptides.
- one aspect of the present invention refers to the use of the heptapeptide of the invention for the preparation of a pharmaceutical composition.
- Another aspect of the invention relates to the use of the heptapeptide of the invention for the preparation of a pharmaceutical composition for the prevention or treatment of arterial hypertension.
- composition refers to any substance used for prevention, relief, treatment or cure of diseases. In the context of the present invention it refers to a composition comprising at least the heptapeptides of the invention for the treatment of arterial hypertension, that is, the decrease in hypertension.
- the pharmaceutical composition of the invention can be used both alone and in combination with other compositions for the treatment of hypertension.
- Another aspect of the present invention relates to the pharmaceutical composition comprising the heptapeptide of the invention.
- a preferred embodiment of this aspect of the invention relates to the pharmaceutical composition which further comprises a pharmaceutically acceptable excipient and / or at least one vehicle.
- Another even more preferred embodiment relates to the pharmaceutical composition which also also comprises at least one other active ingredient.
- excipient refers to a substance that aids in the absorption of the pharmaceutical composition of the invention, stabilizes said pharmaceutical composition or aids in its preparation in the sense of giving it consistency or providing flavors that make it more pleasant.
- the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, protection function of the pharmaceutical composition such as to isolate it from air and / or moisture. , filling function of a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
- a “pharmacologically acceptable vehicle” refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants.
- the carrier can be an inert substance or action analogous to any of the compounds of the present invention.
- the function of the vehicle is to facilitate the incorporation of the expression product of the invention as well as other compounds, to allow a better dosage and administration or to give consistency and form to the pharmaceutical composition.
- the presentation form is liquid, the vehicle is the diluent.
- pharmaceutically acceptable refers to the compound referred to being allowed and evaluated so as not to cause damage to the organisms to which it is administered.
- the heptapeptides of the invention can be consumed as such, forming part of pharmaceutical compositions or food products.
- an even more preferred embodiment of the last aspect of the invention refers to the pharmaceutical composition that is presented in a form adapted to oral, parenteral or intradermal administration.
- the heptapeptide of the invention it is necessary that the amount of said peptide present in the pharmaceutical composition or in the food products containing them be sufficient to exert such action.
- the composition provided by this invention can be provided by any route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen.
- the amount of the heptapeptide of the invention in said compositions is administered at a therapeutically effective concentration.
- the term "therapeutically effective concentration” refers to the concentration of the heptapeptides of the invention calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said heptapeptides and the therapeutic effect to be achieved.
- Pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
- FIG. 1 Contraction of rabbit carotid artery segments.
- A effect of potassium chloride (KCI) to check the viability of the segment and the reproducibility of angiotensin-mediated vasoconstriction I (Ang I).
- B effect of the PIECA50L heptapeptide on contraction induced by angiotensin I. Isometric tension was measured in grams (g) and time in minutes (min).
- FIG. 1 Decreased systolic blood pressure (SBP) obtained in spontaneously hypertensive rats (SHR).
- SBP spontaneously hypertensive rats
- the decrease in SBP in SHR ⁇ "spontaneous hypertensive rats') is shown after oral administration of 1 ml of the saline control (O), 50 mg / Kg of captopril (#), 10 mg / Kg of PIECA32L ( ⁇ ), 10 mg / Kg PIECA50L ( ⁇ ), 10 mg / Kg PIECA52L ( ⁇ ).
- the data represent the mean ⁇ SEM for a minimum of 5 animals and were subjected to ANOVA one way followed by Dunnett test. * , ** , significantly different with respect to the control at P ⁇ 0.05 and P ⁇ 0.01, respectively.
- the peptides analyzed and characterized in the present invention were chemically synthesized on a solid phase following standard procedures using the N- (9-fluorenyl) methoxycarbonyl group (Fmoc) for the protection of the amino group of the constituent amino acids (Felds GB et al. 1990. International Journal of Peptide and Protein Research 34: 161-214).
- the amino-terminal end of the peptides was acetylated (Ac) and the carboxy-terminal end amidated (NH 2 ).
- the peptides were purified by RP-HPLC (reverse phase high performance liquid chromatography) and their identity was confirmed by MALDI-TOF mass spectrometry. "matrix-assisted laser desorption / ionization time-of-flighf). All these procedures are common for all those experts in the area of knowledge of the present invention.
- PIECA 32L C SEQ ID NO: 6 Ac-RKWHFW-NH 2 b a PIECA50D unmodified, PIECA52D unmodified and PIECA 50D are, respectively, the dextrorome isomers (D) of unmodified PIECA50L, unmodified PIECA52L and PIECA 50L. Since they have the same amino acid sequence, they also refer to the amino acid sequences of their respective L. isomers.
- PIECA refers to "ACE activity inhibiting peptide” (it can also refer to the English term “PACEI”, of "ACE inhibitor peptide”).
- EXAMPLE 2 ACE inhibitory potency on the natural substrate Angiotensin I and Bradykinin
- the inhibitory capacity of the peptides was determined by measuring by HPLC (high performance liquid chromatography ') Angiotensin II or the Bradykinin fragment 1 -5 resulting from the hydrolysis of the natural substrate Angiotensin I or Bradykinin, (Calbiochem Co,) respectively, based on the method proposed in the literature (Centeno et al 2006.
- EXAMPLE 4 In vivo tests of the antihypertensive effect after oral administration.
- the antihypertensive effect of the peptides was evaluated by administering them orally at a dose of 10 mg / kg to spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive (WKY).
- Figure 2 shows the changes in systolic blood pressure (APAS) in SHR rats at different times after oral administration. It also includes the decrease in SBP observed after administration of 50 mg / kg of captopril (Sigma Chemical Co.) or saline solution.
- APAS systolic blood pressure
- the systolic blood pressure (SBP) of the control SHR rats, measured by the tail-cuff method, was 201 ⁇ 2 mm Hg (n 59).
- SBP systolic blood pressure
- the oral administration of the peptides described PIECA 50L, PIECA 52L and PIECA 32L at 10 mg / Kg induced a significant reduction in SBP whose evolution over time is shown in Figure 2.
- Figure 2 the lack of effect of the saline solution and the antihypertensive effect of captopril (50 mg / Kg).
- EXAMPLE 5 In vivo tests of the antihypertensive effect after intravenous administration. In order to assess the use in vivo of the heptapeptides of the invention, these peptides were injected intravenously into spontaneously hypertensive rats.
- the assay was carried out by sowing the cells in 96-well plates (25 x 10 3 cells / well) and after 24 h they were placed in Contact with increasing concentrations of peptide After 24 hours of exposure, the cytotoxic effects were measured using the MTT reduction test After washing the cells with PBS buffer (phosphate buffered saline), 100 ⁇ / well of the reagent was added MTT and incubated for 2 h After this time, the supernatant was discarded and the cells were washed with the same buffer. The dye was extracted with DMSO and the OD540 was measured in a microplate reader. The percentage inhibition of reduction MTT po The medium of the succinic enzyme dehydrogenase was calculated in relation to the control without peptide (100% viability).
- the power of the peptide to reduce cell viability is summarized in Table 5. Although there were significant differences depending on the cell type, the IC50 values were in the mimeolar range, from 0.41 ⁇ 0.07 mM for primary hepatocytes to 1.38 ⁇ 0.06 mM on HeptG2 cells. It should be noted that these cytotoxic concentrations were 1000 times higher than the peptide concentrations necessary to inhibit ECA activity.
- IC50 defined as the Peptide concentration capable of reducing cell viability by 50%. It was calculated for each trial by non-linear regression of the experimental data. The results are expressed as the mean ⁇ SEM for three repetitions.
- PIECA 50L (SEQ ID. NO 4) 0.41 ⁇ 0.07 1 .31 ⁇ 0.13 1 .38 ⁇ 0.06
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Abstract
The present invention relates to angiotensin-converting-enzyme (ACE) inhibitor heptapeptides. The invention also relates to the process for producing the peptides of the invention by means of biotechnological strategies and therefore, furthermore, relates to the nucleic acid, the expression cassette and the vector that encodes said peptides, and also to the host cell that comprises said peptides. Moreover, the invention also relates to the use of the peptides for preventing and/or treating hypertension and also to the pharmaceutical composition that comprises said peptides.
Description
HEPTAPÉPTIDOS Y SU USO PARA EL CONTROL DE LA HIPERTENSIÓN HEPTAPEPTIDES AND ITS USE FOR HYPERTENSION CONTROL
La presente invención se refiere a heptapéptídos que se caracterizan por ser inhibidores de la enzima conversora de angiotensina I (ECA). También se refiere al uso de los heptapéptídos para la preparación de un medicamento para la disminución de la hipertensión mediante inhibición de la actividad de la ECA. The present invention relates to heptapeptides that are characterized as inhibitors of angiotensin I converting enzyme (RCT). It also refers to the use of heptapeptides for the preparation of a medicament for the reduction of hypertension by inhibition of the activity of ACE.
ESTADO DE LA TÉCNICA ANTERIOR STATE OF THE PREVIOUS TECHNIQUE
La hipertensión se caracteriza porque la presión sanguínea es superior a la deseable para la salud. Los individuos hipertensos presentan mayor riesgo de complicaciones cardiovasculares y cerebrovasculares, lo que produce un aumento de la mortalidad de forma prematura en dichos sujetos (Giles TD 2009. J Clin Hypertens (Greenwich) 1 1 (1 1 ):61 1 -4). La reversión de la hipertensión medíante cambios en el estilo de vida o tratamientos farmacológicos consigue reducir el riesgo de padecer infarto (30-40%) y otros problemas coronarios (20%) y parece que también reduce la incidencia de otros desordenes vasculares. Dado que se han relacionado alteraciones en el sistema renina-angiotensina-aldosterona (SRA) con la hipertensión, se han desarrollado medicamentos antihípertensivos que utilizan como diana diferentes componentes de este sistema, como por ejemplo inhibidores de reniña, inhibidores de la enzima conversora de angiotensina, bloqueantes de los receptores de angiotensina o antagonistas del receptor de aldosterona (Williams B 2009. J Hypertens 27(3):S19-26). Hypertension is characterized in that the blood pressure is higher than desirable for health. Hypertensive individuals have a higher risk of cardiovascular and cerebrovascular complications, resulting in an increase in mortality prematurely in these subjects (Giles TD 2009. J Clin Hypertens (Greenwich) 1 1 (1 1): 61 1 -4). The reversal of hypertension through changes in lifestyle or drug treatments reduces the risk of heart attack (30-40%) and other coronary problems (20%) and seems to also reduce the incidence of other vascular disorders. Since alterations in the renin-angiotensin-aldosterone (SRA) system have been associated with hypertension, antihypertensive medications have been developed that use different components of this system as targets, such as reninna inhibitors, angiotensin converting enzyme inhibitors , angiotensin receptor blockers or aldosterone receptor antagonists (Williams B 2009. J Hypertens 27 (3): S19-26).
La enzima conversora de angiotensina (ECA) es una peptidasa presente en las células endoteliales vasculares en pulmón, riñon y cerebro que convierte la angiotensina I en el péptido vasoconstrictor angiotensina li, contribuyendo así a aumentar la presión arterial. La inhibición de la ECA para el tratamiento de la hipertensión se realiza en la actualidad utilizando medicamentos, como el captopril cuyo principio activo es hídroclorotiazida, pero provocan efectos
secundarios como la tos seca y el angioedema. Como alternativa, se han realizado estudios para la inhibición de la ECA con péptidos de distinta secuencia y longitud a la de los péptidos de la presente invención. Se han utilizado péptidos derivados de proteínas de plantas y anímales, tales como leche, soja o pescado, para la inhibición de la ECA. Dichos péptidos pueden ser producidos por hidrólisis enzimátíca desde las proteínas precursoras durante el procesamiento de los alimentos y la digestión. Estos péptidos pueden ser incorporados en alimentos funcionales para el desarrollo de productos beneficiosos para la salud y en la actualidad existen en el mercado o en desarrollo diversos productos que contienen estos péptidos bioactivos con actividad inhibitoria de la ECA. Sin embargo, los ensayos clínicos realizados hasta el momento con varios de estos péptidos no han demostrado un claro efecto beneficioso en todos los pacientes hipertensos {Engberink MF et al 2008. Hypertension 51 (2):399-405 y De Leo et al 2009. Curr Pharm Des 15(31 ):3622-43). The angiotensin converting enzyme (RCT) is a peptidase present in vascular endothelial cells in the lung, kidney and brain that converts angiotensin I into the vasoconstrictor peptide angiotensin li, thus contributing to increasing blood pressure. ACE inhibition for the treatment of hypertension is currently performed using medications, such as captopril whose active substance is hydrochlorothiazide, but they cause effects. Side effects such as dry cough and angioedema. Alternatively, studies have been conducted for the inhibition of ACE with peptides of different sequence and length than the peptides of the present invention. Peptides derived from plant and animal proteins, such as milk, soy or fish, have been used for ACE inhibition. Such peptides can be produced by enzymatic hydrolysis from precursor proteins during food processing and digestion. These peptides can be incorporated into functional foods for the development of health-beneficial products and currently there are various products on the market or in development that contain these bioactive peptides with ACE inhibitory activity. However, clinical trials conducted so far with several of these peptides have not demonstrated a clear beneficial effect in all hypertensive patients {Engberink MF et al 2008. Hypertension 51 (2): 399-405 and De Leo et al 2009. Curr Pharm Des 15 (31): 3622-43).
Por otra parte, también se han descrito péptidos desarrollados de novo para la disminución de la hipertensión. Se ha descrito la importancia de la localización de diferentes aminoácidos en el extremo carboxiio terminal de los péptidos para la inhibición de la actividad de la ECA. En relación a dipéptídos, se ha descrito que el dipéptido valina-triptófano es el que mayor actividad inhibitoria presenta (Cheung HS et al 1980. J Bíol Chem 255(25):401 -7). En péptidos de seis aminoácidos se ha descrito que los tres aminoácidos en el extremo carboxiio terminal influyen en la inhibición de la actividad de la ECA, siendo las secuencias terminales histidina-fenilalanina-triptófano (HFW) y leucina- fenilalanina-triptófano (LFW) las preferidas entre los péptidos estudiados {Centeno et al 2006. J Agrie Food Chem 54(15):5323-9). Se han descrito como aminoácidos importantes para la inhibición de la actividad de la ECA el triptófano (W), tirosina (Y), fenilalanina (F), prolina (P), isoleucina (I), alanina (A), leucina (L) y metionina (M) situados en última posición en el extremo carboxiio terminal, mientras que los aminoácidos importantes en la penúltima
posición son valina (V), isoleucina (I), alanina (A), arginina (R), tirosina (Y) y fenílalanina (F) {Cheung HS et al 1980. J Biol Chem 255(25) :401 -7). En una comparación entre péptidos derivados de la lactoferricina B se comprobó que al comparar la actividad inhibitoria de la ECA entre un heptapéptido y un hexapéptido cuya secuencia estaba contenida en la del primero, ambos de distinta secuencias a los de la invención, el heptapéptido presentaba menor actividad inhibitoria de la ECA que el hexapéptido, demostrándose que un aumento de la longitud de un péptido con actividad inhibitoria de la ECA no significa un mayor aumento de dicha actividad (Ruiz-Giménez P et al 2010. 58(1 1 ): 6721 -27). On the other hand, de novo developed peptides for the reduction of hypertension have also been described. The importance of the localization of different amino acids at the carboxy-terminal end of the peptides for the inhibition of ACE activity has been described. In relation to dipeptides, it has been described that the valine-tryptophan dipeptide has the highest inhibitory activity (Cheung HS et al 1980. J Bíol Chem 255 (25): 401-7). In peptides of six amino acids it has been described that the three amino acids at the carboxy terminal end influence the inhibition of ACE activity, the terminal sequences being histidine-phenylalanine-tryptophan (HFW) and leucine-phenylalanine-tryptophan (LFW). preferred among the peptides studied {Centeno et al 2006. J Agrie Food Chem 54 (15): 5323-9). Tryptophan (W), tyrosine (Y), phenylalanine (F), proline (P), isoleucine (I), alanine (A), leucine (L) have been described as important amino acids for ACE activity inhibition and methionine (M) located last in the carboxy terminal end, while the important amino acids in the penultimate positions are valine (V), isoleucine (I), alanine (A), arginine (R), tyrosine (Y) and phenylalanine (F) {Cheung HS et al 1980. J Biol Chem 255 (25): 401-7) . In a comparison between peptides derived from lactoferricin B it was found that when comparing the ACE inhibitory activity between a heptapeptide and a hexapeptide whose sequence was contained in that of the first, both of different sequences to those of the invention, the heptapeptide presented less ACE inhibitory activity than the hexapeptide, showing that an increase in the length of a peptide with ACE inhibitory activity does not mean a greater increase in said activity (Ruiz-Giménez P et al 2010. 58 (1 1): 6721 - 27).
Dado que los compuestos utilizados en la actualidad producen efectos secundarios y los ensayos clínicos realizados hasta la fecha con péptidos inhibidores de la actividad ECA no demuestran un claro efecto antihipertensivo, se hace necesaria una mejora en los medicamentos para controlar la hipertensión. Since the compounds currently used produce side effects and clinical trials conducted to date with peptides inhibiting ACE activity do not demonstrate a clear antihypertensive effect, an improvement in medications is needed to control hypertension.
EXPLICACIÓN DE LA INVENCIÓN La presente invención trata de heptapéptidos con efecto antihipertensivo y del uso de dichos heptapéptidos para la elaboración de una composición farmacéutica para disminuir la hipertensión. Los péptidos de la invención son inhibidores de la enzima conversora de angiotensina I (ECA). Con el fin de evaluar el potencial antihipertensivo de los péptidos de la invención, se comparó la actividad inhibitoria de la ECA de los péptidos de la invención con otros péptidos descritos anteriormente. Se demostró que la actividad antihipertensiva de los heptapéptidos es superior a los anteriormente descritos. La diferencia fundamental de los péptidos de la invención con respecto a péptidos descritos anteriormente es que los aminoácidos presentes en el extremo carboxilo terminal de los péptidos de la invención varían con respecto a los aminoácidos que se consideran importantes para la actividad
inhibitoria de la ECA en el estado de la técnica. De este modo, los heptapéptidos de la invención presentan un extremo carboxilo terminal diferente a los descritos previamente y esto resulta en un aumento de la actividad inhibitoria de la ECA. EXPLANATION OF THE INVENTION The present invention relates to heptapeptides with antihypertensive effect and the use of said heptapeptides for the preparation of a pharmaceutical composition to decrease hypertension. The peptides of the invention are inhibitors of angiotensin I converting enzyme (RCT). In order to evaluate the antihypertensive potential of the peptides of the invention, the ACE inhibitory activity of the peptides of the invention was compared with other peptides described above. It was shown that the antihypertensive activity of heptapeptides is superior to those described above. The fundamental difference of the peptides of the invention with respect to peptides described above is that the amino acids present at the carboxyl end of the peptides of the invention vary with respect to the amino acids considered important for the activity. ACE inhibitor in the state of the art. Thus, the heptapeptides of the invention have a different carboxyl terminus than those previously described and this results in an increase in ACE inhibitory activity.
Así, un primer aspecto de la invención se refiere a un heptapéptido que consiste en la secuencia SEQ ID NO: 1 , donde el primer aminoácido es una arginina (R) el segundo aminoácido es una lisina (K), el tercer aminoácido es un triptófano (W), el cuarto aminoácido es una histidina (H) o una leucína (L), el quinto aminoácido es una fenilalanina (F), el sexto aminoácido es una leucina {!_) o una histidina (H) y el séptimo aminoácido es un triptófano (W). Thus, a first aspect of the invention relates to a heptapeptide consisting of the sequence SEQ ID NO: 1, where the first amino acid is an arginine (R) the second amino acid is a lysine (K), the third amino acid is a tryptophan (W), the fourth amino acid is a histidine (H) or a leucine (L), the fifth amino acid is a phenylalanine (F), the sixth amino acid is a leucine {! _) Or a histidine (H) and the seventh amino acid It is a tryptophan (W).
Los heptapéptidos de la invención coinciden en secuencia de aminoácidos en las posiciones descritas pero varían en las posiciones cuarta y sexta contando desde el extremo amino terminal. Por este motivo, una realización preferida del primer aspecto de la invención se refiere a un heptapéptido que consiste en la secuencia SEQ ID NO: 2 donde el aminoácido de la cuarta posición es una histidina (H) y el aminoácido de la sexta posición es una leucina (L). Por otro lado, otra realización preferida se refiere al heptapéptido que consiste en la secuencia SEQ ID NO: 3 donde el aminoácido de la cuarta posición es una leucína (L) y el aminoácido de la sexta posición es una histidina (H). En el listado de secuencias se indica la variación ("variant"), el lugar de variación y cuál es el aminoácido que lo reemplaza ("replace"). La presente invención también se refiere tanto a los esteroisómeros dextrógiros (D) como los esteroisómeros levógiros (L), por lo que otra realización preferida del primer aspecto de la invención se refiere al heptapéptido cuya esteroisomería es D y otra realización preferida se refiere al heptapéptido cuya esteroisomería es L. The heptapeptides of the invention coincide in amino acid sequence in the positions described but vary in the fourth and sixth positions counting from the amino terminal end. For this reason, a preferred embodiment of the first aspect of the invention relates to a heptapeptide consisting of the sequence SEQ ID NO: 2 where the fourth position amino acid is a histidine (H) and the sixth position amino acid is a leucine (L). On the other hand, another preferred embodiment refers to the heptapeptide consisting of the sequence SEQ ID NO: 3 where the fourth position amino acid is a leucine (L) and the sixth position amino acid is a histidine (H). In the sequence listing, the variation ("variant"), the place of variation and the amino acid that replaces it ("replace") are indicated. The present invention also relates to both dextrogyric steroisomers (D) and levogyric stereoisomers (L), whereby another preferred embodiment of the first aspect of the invention refers to the heptapeptide whose steroisomerism is D and another preferred embodiment refers to the heptapeptide whose stereoisomery is L.
El término "esteroisómero" en la presente invención se refiere a un isómero óptico o enantiomero, es decir, en la presente invención un heptapéptido de la
misma forma molecular y secuencia de aminoácidos pero de diferente fórmula estructural. Los esteroisómeros D y los esteroisómeros L se diferencian en la propiedad de desviar el plano de la luz polarizada en diferente dirección, siendo los esteroisómeros D los que la desvían hacia la derecha (isómero dextrógiro) y los isómeros L (isómero levógiro) los que la desvían hacia la izquierda. En la naturaleza los enantiómeros que forman las proteínas son isómeros L pero para aumentar la estabilidad y resistencia a la degradación por proteasas presentes en los fluidos biológicos, en la presente invención se han generado también isómeros D. The term "stereoisomer" in the present invention refers to an optical isomer or enantiomer, that is, in the present invention a heptapeptide of the same molecular form and amino acid sequence but of different structural formula. The stereoisomers D and the stereoisomers L differ in the property of diverting the plane of the polarized light in a different direction, with the stereoisomers D being those which deflect it to the right (dextrogyric isomer) and the isomers L (levogyric isomer) which turn left. In nature, the enantiomers that form the proteins are L-isomers but to increase the stability and resistance to degradation by proteases present in biological fluids, in the present invention D-isomers have also been generated.
En la presente invención los extremos amino terminal y carboxilo terminal de los heptapéptidos pueden o no estar modificados. Por ejemplo el extremo amino terminal puede estar acetilado y el extremo carboxilo terminal puede estar amidado siguiendo los procedimientos conocidos por el experto en la materia, y dicha modificación se puede realizar en la síntesis química en fase sólida. Dichas modificaciones favorecen la estabilidad de los péptidos frente a la degradación por proteasas pero no afectan a la actividad biológica de los mismos. Por estos motivos otra realización preferida del primer aspecto de la invención se refiere a un heptapéptido donde el extremo amino terminal está acetilado y el extremo carboxilo terminal está amidado. Por este motivo, otra realización preferida del primer aspecto de la invención se refiere a un heptapéptido que consiste en la SEQ ID NO: 4. Otra realización preferida se refiere a un heptapéptido que consiste en la SEQ ID NO: 5. En el listado de secuencias se indica la modificación (MOD_RES), el lugar de modificación y cuál es el dicha modificación ("acetylation" se refiere a acetilación y "amidation" se refiere a amidación). In the present invention the amino terminal and carboxyl terminal ends of the heptapeptides may or may not be modified. For example, the amino terminal end may be acetylated and the carboxyl terminal end may be amidated following the procedures known to the person skilled in the art, and said modification can be performed in solid phase chemical synthesis. Such modifications favor the stability of the peptides against degradation by proteases but do not affect their biological activity. For these reasons another preferred embodiment of the first aspect of the invention relates to a heptapeptide where the amino terminal end is acetylated and the carboxyl end is amidated. For this reason, another preferred embodiment of the first aspect of the invention relates to a heptapeptide consisting of SEQ ID NO: 4. Another preferred embodiment refers to a heptapeptide consisting of SEQ ID NO: 5. In the list of sequences indicate the modification (MOD_RES), the place of modification and what is said modification ("acetylation" refers to acetylation and "amidation" refers to amidation).
El término "heptapéptido" se refiere a un péptido formado por siete aminoácidos unidos por enlace peptídico. A partir de ahora nos referiremos a los "heptapéptidos de la invención" como cualquiera de los péptidos descritos en el primer aspecto de la invención. En la presente descripción nos referiremos a
los "heptapéptidos naturales de la invención" como los heptapéptidos de isomería L ya que son los que se presentan en la naturaleza. The term "heptapeptide" refers to a peptide consisting of seven amino acids linked by peptide bond. From now on we will refer to the "heptapeptides of the invention" as any of the peptides described in the first aspect of the invention. In this description we will refer to the "natural heptapeptides of the invention" such as the isomeric heptapeptides L since they are those that occur in nature.
El término "inhibición" como se usa aquí, se refiere principalmente a que el heptapéptido inhibe (disminuye) la actividad de la ECA y por lo tanto reduce la hipertensión. Un agente antihipertensivo es por lo tanto un agente que disminuye la hipertensión. The term "inhibition" as used herein, primarily refers to the fact that the heptapeptide inhibits (decreases) the activity of ACE and therefore reduces hypertension. An antihypertensive agent is therefore an agent that decreases hypertension.
La enzima conversora de angiotensina I (ECA, peptidyl-dipeptidase A, BC036375.2, EC 3.4.15.1 , "angiotensin converting enzyme" o ACE) se refiere a la enzima que utiliza como sustrato la angiotensina I convírtiéndola en angiotensina II (que es una molécula vasoconstrictora) y la bradiquinina (que es una molécula vasodilatadora), a la cual convierte en un péptido no funcional. La actividad de esta enzima aumenta la presión arterial y por lo tanto contribuye a la hipertensión en humanos. Existen tres isoformas principales de la ECA, somática, testicular o germinal y plasmática o soluble, siendo la primera de ellas la principal productora de angiotensina II. The angiotensin I converting enzyme (ECA, peptidyl dipeptidase A, BC036375.2, EC 3.4.15.1, "angiotensin converting enzyme" or ACE) refers to the enzyme that uses angiotensin I as a substrate converting it into angiotensin II (which is a vasoconstrictor molecule) and bradykinin (which is a vasodilator molecule), which it converts into a non-functional peptide. The activity of this enzyme increases blood pressure and therefore contributes to hypertension in humans. There are three main isoforms of RCT, somatic, testicular or germinal and plasma or soluble, the first being the main producer of angiotensin II.
La hipertensión tal y como se describe en la presente invención es una enfermedad de etiología diversa que se caracteriza por una elevada presión sanguínea en la circulación sistémíca (diastólica≥ 90 mm Hg; sístólíca≥ 140 mm Hg;). Hypertension as described in the present invention is a disease of diverse etiology characterized by high blood pressure in the systemic circulation (diastolic ≥ 90 mm Hg; systolic ≥ 140 mm Hg;).
Los péptidos de la invención pueden obtenerse mediante síntesis química o mediante estrategias derivadas de la biotecnología. The peptides of the invention can be obtained by chemical synthesis or by strategies derived from biotechnology.
Medíante estrategias biotecnológicas conocidas por el experto en la materia, se podría producir el heptapéptido de la invención a partir de un polipéptido que comprendiera el heptapéptido de la invención, por ejemplo tras fragmentación del mismo. Por este motivo, un segundo aspecto de la invención se refiere a un polipéptido que comprende el heptapéptido de la invención.
También mediante estrategias biotecnológicas ampliamente conocidas en el estado de la técnica, el heptapéptído natural de la invención, o el polípéptido que lo comprendiera, podría ser producido a partir de un ácido nucleico que lo codificara. Por este motivo, un tercer aspecto de la invención se refiere a un ácido nucleico que comprende la secuencia de nucleótídos que codifica para los heptapéptidos naturales de la invención. Through biotechnological strategies known to the person skilled in the art, the heptapeptide of the invention could be produced from a polypeptide comprising the heptapeptide of the invention, for example after fragmentation thereof. For this reason, a second aspect of the invention relates to a polypeptide comprising the heptapeptide of the invention. Also by means of biotechnological strategies widely known in the state of the art, the natural heptapeptide of the invention, or the polypeptide comprising it, could be produced from a nucleic acid that encodes it. For this reason, a third aspect of the invention relates to a nucleic acid comprising the nucleotide sequence encoding the natural heptapeptides of the invention.
En el supuesto de la producción mediante biotecnología, la secuencia del péptído sería codificada por un fragmento de DNA (ácido desoxirribonucleico o ADN) de acuerdo a las leyes del código genético. Todos estos procedimientos son habituales para toda aquella persona experta en el área de conocimiento de la presente invención. In the case of biotechnology production, the peptide sequence would be encoded by a DNA fragment (deoxyribonucleic acid or DNA) according to the laws of the genetic code. All these procedures are common for all those experts in the area of knowledge of the present invention.
Por lo tanto también forman parte de la invención las construcciones genéticas de DNA que codifican secuencias de los péptidos naturales de la invención. Dicha construcción genética de DNA dirigiría la transcripción intracelular de la secuencia del heptapéptído, y comprende, al menos, uno de los siguientes tipos de secuencias: a) secuencia de DNA que comprende la secuencia de nucleotidos codificante para el heptapéptído natural de la invención para su transcripción y traducción intracelular; b) un cassette de expresión géníca que comprende la secuencia de DNA definida en a) unido operativamente a elementos de control de la transcripción y opcionalmente de traducción; c) secuencia de DNA correspondiente a un sistema o vector de expresión génica que comprende la secuencia codificante del heptapéptído natural de la invención definida en a) o el cassette de expresión definido en b), operativamente enlazados con, al menos, un promotor que dirija la transcripción de dicha secuencia, y con otras secuencias para regular su transcripción tales como, por ejemplo, señales de inicio y terminación, o señal de poliadenilacíón, donde preferentemente el vector es un plásmido. Esto queda reflejado en el tercer, cuarto y quinto aspecto de la presente invención.
Un sexto aspecto de la invención se refiere a la célula hospedadora que comprende el ácido nucleico, el cassette de expresión o el vector que codifican secuencias del heptapéptido natural de la invención, donde la célula preferentemente es una célula de mamífero (no perteneciente al grupo de las células embrionarias o germinales humanas), de insecto, de hongo (entre los que se incluyen las levaduras) o bacteria. La introducción de dichas construcciones génicas se puede realizar con los métodos conocidos en el estado de la técnica. Un séptimo aspecto de la invención se refiere al proceso de producción del heptapéptido natural de la invención que comprende cultivar las células hospedadoras descritas en ei sexto aspecto de la invención y la posterior purificación del péptido natural de la invención. Los heptapéptidos de la invención se pueden sintetizar también mediante síntesis química, preferiblemente sobre fase sólida. En este caso se pueden producir tanto enantiómeros L como D. Ei procedimiento de síntesis permite que el extremo N-terminal de los péptidos se pueda encontrar acetilado y el extremo C-terminal amidado, lo que no afectaría significativamente la actividad inhibitoria de la ECA de dichos péptidos. Therefore, DNA genetic constructs encoding sequences of the natural peptides of the invention are also part of the invention. Said genetic construction of DNA would direct the intracellular transcription of the heptapeptide sequence, and comprises at least one of the following types of sequences: a) DNA sequence comprising the nucleotide sequence encoding the natural heptapeptide of the invention for its intracellular transcription and translation; b) a gene expression cassette comprising the DNA sequence defined in a) operably linked to transcription control and optionally translation elements; c) DNA sequence corresponding to a gene expression system or vector comprising the natural heptapeptide coding sequence of the invention defined in a) or the expression cassette defined in b), operably linked to at least one promoter that directs the transcription of said sequence, and with other sequences to regulate its transcription such as, for example, start and end signals, or polyadenylation signal, where preferably the vector is a plasmid. This is reflected in the third, fourth and fifth aspects of the present invention. A sixth aspect of the invention relates to the host cell comprising the nucleic acid, the expression cassette or the vector encoding sequences of the natural heptapeptide of the invention, where the cell is preferably a mammalian cell (not belonging to the group of human embryonic or germ cells), insect, fungus (including yeasts) or bacteria. The introduction of said gene constructs can be carried out with the methods known in the state of the art. A seventh aspect of the invention relates to the production process of the natural heptapeptide of the invention which comprises culturing the host cells described in the sixth aspect of the invention and the subsequent purification of the natural peptide of the invention. The heptapeptides of the invention can also be synthesized by chemical synthesis, preferably on a solid phase. In this case, both L and D enantiomers can be produced. The synthesis procedure allows the N-terminal end of the peptides to be acetylated and the C-terminal end amidated, which would not significantly affect the ACE inhibitory activity of said peptides.
En la presente invención se han realizado estudios in vitro, ex vivo e in vivo en animales espontáneamente hipertensos con los péptidos de la invención y se han comparado ios resultados con esteroisómeros de los mismos y con un péptido más corto de seis aminoácidos ya conocido. Los resultados obtenidos demuestran que ambos poseen actividad inhibidora de la ECA in vitro, inhibición de la contracción arterial inducida por Angiotensina I y efecto antihipertensivo en ratas espontáneamente hipertensas (SHR). Además, en la presente invención se demuestra que no cualquier secuencia peptídica produce el mismo efecto antihipertensivo y que los esteroisómeros no presentan ios mismos efectos. Comparativamente en los ensayos in vivo con administración oral, el uso de uno de los heptapéptidos produjo una mayor disminución de la
presión arterial que el uso del hexapéptido analizado en el ensayo, así como también en comparación con un compuesto antihipertensivo conocido (captopril); mientras que el uso del otro heptapéptido produjo resultados similares a los observados con el hexapéptido. Además, también se han realizado ensayos in vivo en anímales normotensos no observándose hipotensión en los mismos. Ensayos de citotoxicidad con el heptapéptido de mayor actividad antihipertensiva in vivo han revelado que presentaba una baja toxicidad en varios tipos celulares. Por todo lo aquí descrito un aspecto de la presente invención se refiere al uso del heptapéptido de la invención para la elaboración de una composición farmacéutica. Otro aspecto de la invención se refiere al uso del heptapéptido de la invención para la elaboración de una composición farmacéutica para la prevención o el tratamiento de la hipertensión arterial. In the present invention, in vitro, ex vivo and in vivo studies have been carried out in spontaneously hypertensive animals with the peptides of the invention and the results have been compared with stereoisomers thereof and with a shorter known six amino acid peptide. The results show that both have ACE inhibitory activity in vitro, inhibition of arterial contraction induced by Angiotensin I and antihypertensive effect in spontaneously hypertensive rats (SHR). Furthermore, it is demonstrated in the present invention that not any peptide sequence produces the same antihypertensive effect and that the stereoisomers do not have the same effects. Comparatively in in vivo trials with oral administration, the use of one of the heptapeptides produced a greater decrease in the blood pressure than the use of the hexapeptide analyzed in the assay, as well as in comparison with a known antihypertensive compound (captopril); while the use of the other heptapeptide produced results similar to those observed with the hexapeptide. In addition, in vivo tests have also been performed in normotensive animals, with no hypotension observed in them. Cytotoxicity tests with the heptapeptide of greater antihypertensive activity in vivo have revealed that it presented a low toxicity in several cell types. For all that is described herein, one aspect of the present invention refers to the use of the heptapeptide of the invention for the preparation of a pharmaceutical composition. Another aspect of the invention relates to the use of the heptapeptide of the invention for the preparation of a pharmaceutical composition for the prevention or treatment of arterial hypertension.
El término "composición farmacéutica" hace referencia a cualquier sustancia usada para prevención, alivio, tratamiento o curación de enfermedades. En el contexto de la presente invención se refiere a una composición que comprenda al menos los heptapéptidos de la invención para el tratamiento de la hipertensión arterial, es decir la disminución de la hipertensión. The term "pharmaceutical composition" refers to any substance used for prevention, relief, treatment or cure of diseases. In the context of the present invention it refers to a composition comprising at least the heptapeptides of the invention for the treatment of arterial hypertension, that is, the decrease in hypertension.
La composición farmacéutica de la invención puede utilizarse tanto sola como en combinación con otras composiciones para el tratamiento de la hipertensión. Otro aspecto de la presente invención se refiere a la composición farmacéutica que comprende el heptapéptido de la invención. Una realización preferida de este aspecto de la invención se refiere a la composición farmacéutica que además comprende un excipiente y/o al menos un vehículo farmacéuticamente aceptables. Otra realización aún más preferida se refiere a la composición farmacéutica que además también comprende al menos otro principio activo.
El término "excipiente" hace referencia a una sustancia que ayuda a la absorción de la composición farmacéutica de la invención, estabiliza dicha composición farmacéutica o ayuda a su preparación en el sentido de darle consistencia o aportar sabores que lo hagan más agradable. Así pues, los excipientes podrían tener la función de mantener los ingredientes unidos como por ejemplo almidones, azúcares o celulosas, función de endulzar, función de colorante, función de protección de la composición farmacéutica como por ejemplo para aislarla del aire y/o la humedad, función de relleno de una pastilla, cápsula o cualquier otra forma de presentación como por ejemplo el fosfato de calcio dibásico, función desintegradora para facilitar la disolución de los componentes y su absorción en el intestino, sin excluir otro tipo de excipientes no mencionados en este párrafo. The pharmaceutical composition of the invention can be used both alone and in combination with other compositions for the treatment of hypertension. Another aspect of the present invention relates to the pharmaceutical composition comprising the heptapeptide of the invention. A preferred embodiment of this aspect of the invention relates to the pharmaceutical composition which further comprises a pharmaceutically acceptable excipient and / or at least one vehicle. Another even more preferred embodiment relates to the pharmaceutical composition which also also comprises at least one other active ingredient. The term "excipient" refers to a substance that aids in the absorption of the pharmaceutical composition of the invention, stabilizes said pharmaceutical composition or aids in its preparation in the sense of giving it consistency or providing flavors that make it more pleasant. Thus, the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, protection function of the pharmaceutical composition such as to isolate it from air and / or moisture. , filling function of a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
Un "vehículo farmacológicamente aceptable" se refiere a aquellas sustancias, o combinación de sustancias, conocidas en el sector farmacéutico, utilizadas en la elaboración de formas farmacéuticas de administración e incluye, pero sin limitarse, sólidos, líquidos, disolventes o tensoactivos. El vehículo puede ser una sustancia inerte o de acción análoga a cualquiera de los compuestos de la presente invención. La función del vehículo es facilitar la incorporación del producto de expresión de la invención así como también de otros compuestos, permitir una mejor dosificación y administración o dar consistencia y forma a la composición farmacéutica. Cuando la forma de presentación es líquida, el vehículo es el diluyente. El término "farmacológicamente aceptable" se refiere a que el compuesto al que hace referencia esté permitido y evaluado de modo que no cause daño a los organismos a los que se administra. A "pharmacologically acceptable vehicle" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants. The carrier can be an inert substance or action analogous to any of the compounds of the present invention. The function of the vehicle is to facilitate the incorporation of the expression product of the invention as well as other compounds, to allow a better dosage and administration or to give consistency and form to the pharmaceutical composition. When the presentation form is liquid, the vehicle is the diluent. The term "pharmacologically acceptable" refers to the compound referred to being allowed and evaluated so as not to cause damage to the organisms to which it is administered.
Los heptapéptidos de la invención pueden consumirse como tales, formando parte de composiciones farmacéuticas o de productos alimenticios. Por lo que otra realización aún más preferida del último aspecto de la invención se refiere a la composición farmacéutica que se presenta en una forma adaptada a la administración por vía oral, parenteral o intradérmíca.
Para que el heptapéptido de la invención sea capaz de inhibir la actividad de la ECA y por lo tanto disminuir la hipertensión, es necesario que la cantidad de dicho péptído presente en la composición farmacéutica o en los productos alimenticios que los contengan sea la suficiente para ejercer dicha acción. Por este motivo, la composición proporcionada por esta invención puede ser facilitada por cualquier vía de administración, para lo cual dicha composición se formulará en la forma farmacéutica adecuada a la vía de administración elegida. La cantidad del heptapéptido de la invención en dichas composiciones se administra a una concentración terapéuticamente efectiva. The heptapeptides of the invention can be consumed as such, forming part of pharmaceutical compositions or food products. Thus, an even more preferred embodiment of the last aspect of the invention refers to the pharmaceutical composition that is presented in a form adapted to oral, parenteral or intradermal administration. In order for the heptapeptide of the invention to be able to inhibit the activity of ACE and therefore decrease hypertension, it is necessary that the amount of said peptide present in the pharmaceutical composition or in the food products containing them be sufficient to exert such action. For this reason, the composition provided by this invention can be provided by any route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen. The amount of the heptapeptide of the invention in said compositions is administered at a therapeutically effective concentration.
En el sentido utilizado en esta descripción, la expresión "concentración terapéuticamente efectiva" se refiere a la concentración de los heptapéptidos de la invención calculada para producir el efecto deseado y, en general, vendrá determinada, entre otras causas, por las características propias de dichos heptapéptidos y el efecto terapéutico a conseguir. Los adyuvantes y vehículos farmacéuticamente aceptables que pueden ser utilizados en dichas composiciones son los vehículos conocidos por los técnicos en la materia. In the sense used in this description, the term "therapeutically effective concentration" refers to the concentration of the heptapeptides of the invention calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said heptapeptides and the therapeutic effect to be achieved. Pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Las siguientes figuras y ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCION DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Figura 1. Contracción de segmentos de arteria carótida de conejo. A, efecto del cloruro potásico (KCI) para comprobar la viabilidad del segmento y la reproducibilidad de la vasoconstricción mediada por angíotensina I (Ang I). B, efecto del heptapéptido PIECA50L sobre la contracción inducida por
angiotensina I. La tensión isométrica se midió en gramos (g) y el tiempo en minutos (min). Figure 1. Contraction of rabbit carotid artery segments. A, effect of potassium chloride (KCI) to check the viability of the segment and the reproducibility of angiotensin-mediated vasoconstriction I (Ang I). B, effect of the PIECA50L heptapeptide on contraction induced by angiotensin I. Isometric tension was measured in grams (g) and time in minutes (min).
Figura 2. Disminución de ia presión arterial sistóiica (PAS) obtenida en ratas espontáneamente hipertensas (SHR). Se muestra la disminución de la PAS en SHR {"spontaneous hypertensive rats') tras la administración oral de 1 ml del control de solución salina (O), 50 mg/Kg de captopril (#),10 mg/Kg de PIECA32L (▼), 10 mg/Kg PIECA50L (Δ), 10 mg/Kg PIECA52L (□). Los datos representan la medía ± SEM para un mínimo de 5 animales y fueron sometidos a un análisis ANOVA de una vía seguido de un test Dunnett. *, **, significativamente diferente con respecto al control a P< 0.05 y P< 0.01 , respectivamente. APAS, cambio de la PAS, al ser un cambio negativo se traduce en disminución de la PAS. Milímetros de mercurio, mm Hg. Figura 3. Registro de ia presión arterial medida en la arteria femoral de ratas espontáneamente hipertensas. Se muestran los cambios inducidos por la inyección intravenosa de A, solución salina (salino); B, PIECA50L (1 mg/Kg); y C, PIECA 50D (1 mg/Kg). ABP, presión arterial {"arterial blood pressure"), Milímetros de mercurio, mm Hg. Figure 2. Decreased systolic blood pressure (SBP) obtained in spontaneously hypertensive rats (SHR). The decrease in SBP in SHR {"spontaneous hypertensive rats') is shown after oral administration of 1 ml of the saline control (O), 50 mg / Kg of captopril (#), 10 mg / Kg of PIECA32L (▼ ), 10 mg / Kg PIECA50L (Δ), 10 mg / Kg PIECA52L (□). the data represent the mean ± SEM for a minimum of 5 animals and were subjected to ANOVA one way followed by Dunnett test. * , ** , significantly different with respect to the control at P <0.05 and P <0.01, respectively. APAS, change of the PAS, being a negative change translates into decrease in the PAS. Millimeters of mercury, mm Hg. Figure 3 Record of blood pressure measured in the femoral artery of spontaneously hypertensive rats, changes induced by intravenous injection of A, saline (saline), B, PIECA50L (1 mg / kg), and C, PIECA 50D ( 1 mg / kg). ABP, blood pressure {"arterial blood pressure"), Millimeters of mercury, mm Hg.
EJEMPLOS ILUSTRATIVOS DE LA INVENCIÓN ILLUSTRATIVE EXAMPLES OF THE INVENTION
Los siguientes ejemplos específicos que se proporcionan en este documento de patente sirven para ilustrar la naturaleza de la presente invención. Estos ejemplos se incluyen solamente con fines ilustrativos y no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ilustran la invención sin limitar el campo de aplicación de la misma.
EJEMPLO 1 : Síntesis de péptidos. The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. EXAMPLE 1: Synthesis of peptides.
Los péptidos analizados y caracterizados en la presente invención se sintetizaron químicamente sobre fase sólida siguiendo procedimientos habituales que utilizan el grupo N-(9-fluorenyl) methoxycarbonyl (Fmoc) para la protección del grupo -amino de los aminoácidos constituyentes (Fíelds G B et al.1990. International Journal of Peptide and Protein Research 34:161 -214). El extremo amino-terminal de los péptidos se encontraba acetilado (Ac) y el extremo carboxilo-terminal amidado (NH2). Después de la síntesis, los péptidos se purificaron mediante RP-HPLC (del inglés "reverse phase-high performance liquid chromatography", cromatografía líquida de alta resolución de fase inversa) y su identidad se confirmó mediante espectrometría de masas MALDI- TOF (del inglés " matrix-assisted láser desorption/ionization time-of-flighf). Todos estos procedimientos son habituales para toda aquella persona experta en el área de conocimiento de la presente invención. The peptides analyzed and characterized in the present invention were chemically synthesized on a solid phase following standard procedures using the N- (9-fluorenyl) methoxycarbonyl group (Fmoc) for the protection of the amino group of the constituent amino acids (Felds GB et al. 1990. International Journal of Peptide and Protein Research 34: 161-214). The amino-terminal end of the peptides was acetylated (Ac) and the carboxy-terminal end amidated (NH 2 ). After the synthesis, the peptides were purified by RP-HPLC (reverse phase high performance liquid chromatography) and their identity was confirmed by MALDI-TOF mass spectrometry. "matrix-assisted laser desorption / ionization time-of-flighf). All these procedures are common for all those experts in the area of knowledge of the present invention.
Las modificaciones que se producen en los extremos de los péptidos mostrados en este ejemplo de la invención no son esenciales para la actividad inhibitoria de la ECA y se pueden introducir medíante procesos habituales en el proceso de síntesis química. En la tabla 1 se muestran los péptidos de la invención sin modificar y modificados de dicha manera. The modifications that occur at the ends of the peptides shown in this example of the invention are not essential for the ACE inhibitory activity and customary processes can be introduced through the chemical synthesis process. Table 1 shows the peptides of the invention unmodified and modified in that manner.
Tabla 1 . Péptidos de la invención. Table 1 . Peptides of the invention.
Nomenclatura SEQ ID NO: Secuencia Nomenclature SEQ ID NO: Sequence
PIECA 50L sin modificar SEQ ID NO: 2 RKWHFLW PIECA 50L unmodified SEQ ID NO: 2 RKWHFLW
PIECA 50Da sin modificar SEQ ID NO: 2 RKWHFLW PIECA 50D a unmodified SEQ ID NO: 2 RKWHFLW
PIECA 52L sin modificar SEQ ID NO: 3 RKWLFHW PIECA 52L unmodified SEQ ID NO: 3 RKWLFHW
PIECA 52Da sin modificar SEQ ID NO: 3 RKWLFHW PIECA 52D a unmodified SEQ ID NO: 3 RKWLFHW
PIECA 50L SEQ ID NO: 4 Ac-RKWHFLW-NH2 b PIECA 50L SEQ ID NO: 4 Ac-RKWHFLW-NH 2 b
PIECA 50Da SEQ ID NO: 4 Ac-RKWHFLW-NH2 b PIECA 50D to SEQ ID NO: 4 Ac-RKWHFLW-NH 2 b
PIECA 52L SEQ ID NO: 5 Ac-RKWLFHW-NH2 b PIECA 52L SEQ ID NO: 5 Ac-RKWLFHW-NH 2 b
PIECA 32LC SEQ ID NO: 6 Ac-RKWHFW-NH2 b
a PIECA50D sin modificar, PIECA52D sin modificar y PIECA 50D son, respectivamente los isómeros dextrógiros (D) de PIECA50L sin modificar, PIECA52L sin modificar y PIECA 50L. Dado que tienen la misma secuencia de aminoácidos, se refieren también a las secuencias de aminoácidos de sus respectivos isómeros L. PIECA 32L C SEQ ID NO: 6 Ac-RKWHFW-NH 2 b a PIECA50D unmodified, PIECA52D unmodified and PIECA 50D are, respectively, the dextrorome isomers (D) of unmodified PIECA50L, unmodified PIECA52L and PIECA 50L. Since they have the same amino acid sequence, they also refer to the amino acid sequences of their respective L. isomers.
b heptapéptidos acetilados en su extremo amino-terminal (Ac-) y amidados en su extremo carboxilo-terminal (NH2). b heptapeptide acetylated at its amino terminus (Ac-) and amidated carboxyl terminus (NH 2).
c hexapéptido descrito en Centeno et al. 2006. J Agrie Food Chem 54(15):5323- 9. c hexapeptide described in Centeno et al. 2006. J Agrie Food Chem 54 (15): 5323-9.
PIECA se refiere a "péptido inhibidor de la actividad de la ECA" (también se puede referir al término en inglés "PACEI", de "peptide ACE inhibitor"). PIECA refers to "ACE activity inhibiting peptide" (it can also refer to the English term "PACEI", of "ACE inhibitor peptide").
EJEMPLO 2: Potencia inhibitoria de ECA sobre el sustrato natural Angiotensina I y Bradiquinina EXAMPLE 2: ACE inhibitory potency on the natural substrate Angiotensin I and Bradykinin
La capacidad inhibidora de los péptidos se determinó midiendo por HPLC (del inglés "high performance liquid chromatography') la Angiotensina II o el fragmento Bradiquinina 1 -5 resultante de la hidrólisis del sustrato natural Angiotensina I o Bradiquinina, (Calbiochem Co,) respectivamente, basándose en el método propuesto en la literatura (Centeno et al 2006. J Agrie Food Chem 54(15):5323-9). Brevemente, el método consistió en poner en contacto 75 μΙ de ECA (20 mU/ml en tampón Tris-HCI 200 mM pH 8.3, 600 mM NaCI y 10 μΜ ZnC^) con 50 μΙ de angiotensina I (0.31 mM) o 20 μΙ de bradiquinina (0.94 mM) con diferentes concentraciones de los péptidos, todo ello se incubó a 37 °C durante 30 min. A partir de los resultados obtenidos con Angiotensina I, se calcularon los valores de IC50 (concentración de péptido a la cual se consigue una inhibición de la actividad enzimática del 50%) para ambos péptidos (tabla 2). Cabe destacar la potencia de ambos péptidos, cuyos valores de IC50 fueron inferiores a 10 μΜ. Los resultados obtenidos con bradiquinina pusieron de manifiesto que ninguno de los péptidos ensayados fue capaz de inhibir la hidrólisis de la bradiquinina (resultados no mostrados).
Tabla 2. Efecto inhibidor de los péptidos descritos en la presente invención expresado como IC50, que se define como la concentración de péptido capaz de inhibir el 50% de la actividad de ECA y calculado para cada ensayo por regresión no-lineal de los datos experimentales. Los resultados se expresan como la medía ± SEM (error estándar de la media) para un número de repeticiones (n). The inhibitory capacity of the peptides was determined by measuring by HPLC (high performance liquid chromatography ') Angiotensin II or the Bradykinin fragment 1 -5 resulting from the hydrolysis of the natural substrate Angiotensin I or Bradykinin, (Calbiochem Co,) respectively, based on the method proposed in the literature (Centeno et al 2006. J Agrie Food Chem 54 (15): 5323-9) Briefly, the method consisted of contacting 75 μΙ of ECA (20 mU / ml in Tris- buffer 200 mM HCI pH 8.3, 600 mM NaCI and 10 μΜ ZnC ^) with 50 μΙ of angiotensin I (0.31 mM) or 20 μΙ of bradykinin (0.94 mM) with different concentrations of the peptides, all of which was incubated at 37 ° C for 30 min From the results obtained with Angiotensin I, the IC 50 values (peptide concentration at which 50% inhibition of enzymatic activity is achieved) for both peptides were calculated (table 2). potency of both peptides, whose value IC50's were less than 10 μΜ. The results obtained with bradykinin showed that none of the peptides tested were able to inhibit the hydrolysis of bradykinin (results not shown). Table 2. Inhibitory effect of the peptides described in the present invention expressed as IC 50 , which is defined as the concentration of peptide capable of inhibiting 50% of ECA activity and calculated for each assay by non-linear regression of the data. Experimental The results are expressed as the mean ± SEM (standard error of the mean) for a number of repetitions (n).
Peptido IC50 (μΜ) IC 50 peptide (μΜ)
PIECA 50L (SEQ ID NO: 4) 8.2 ± 1 .0 (3) PIECA 50L (SEQ ID NO: 4) 8.2 ± 1 .0 (3)
PIECA 52L (SEQ ID NO: 5) 3.0 ± 0.2 (4) EJEMPLO 3: Ensayos ex vivo de la inhibición de la contracción de arterias aisladas. PIECA 52L (SEQ ID NO: 5) 3.0 ± 0.2 (4) EXAMPLE 3: Ex vivo tests for the inhibition of contraction of isolated arteries.
Se llevaron a cabo ensayos empleando segmentos de arteria carótida de conejo. Tras comprobar la viabilidad de dichos segmentos arteriales, se llevaron a cabo los ensayos cuyos resultados se resumen en la tabla 3. Los dos péptidos de secuencia PIECA50L y PIECA52L produjeron inhibiciones significativas de la contracción dependiente de ECA en comparación con el control sin péptido, más acusada en el caso de PIECA 50L. A modo de ejemplo ilustrativo, se muestra en la Figura 1 el efecto causado por el péptido PIECA 50L. Trials were conducted using rabbit carotid artery segments. After checking the viability of these arterial segments, the tests were carried out whose results are summarized in Table 3. The two sequence peptides PIECA50L and PIECA52L produced significant inhibitions of ECA-dependent contraction compared to the control without peptide, more charged in the case of PIECA 50L. As an illustrative example, the effect caused by the PIECA 50L peptide is shown in Figure 1.
Tabla 3. Efecto inhibidor de los péptidos descritos en la presente invención sobre la contracción arterial ECA-dependiente con Angiotensina I en arterias aisladas de conejo. Los resultados indican la contracción en respuesta a Angiotensina I (1 μΜ), en % respecto a una respuesta previa en el mismo segmento arterial y se expresan como la media ± SEM de los experimentos realizados en (n) segmentos arteriales. Los datos fueron sometidos a un análisis de ANOVA de una vía seguido de un test Student-Newman-Keuls. Los datos seguidos de letras distintas son diferentes entre sí (a) y (b) (P<0.01 ). *Significativamente diferente con respecto al control P<0.01 .
Peptido Contracción (%) Table 3. Inhibitory effect of the peptides described in the present invention on ECA-dependent arterial contraction with Angiotensin I in isolated rabbit arteries. The results indicate the contraction in response to Angiotensin I (1 μΜ), in% with respect to a previous response in the same arterial segment and are expressed as the mean ± SEM of the experiments performed in (n) arterial segments. The data were subjected to a one-way ANOVA analysis followed by a Student-Newman-Keuls test. The data followed by different letters are different from each other (a) and (b) (P <0.01). * Significantly different with respect to the control P <0.01. Contraction Peptide (%)
Control 87 ± 3 (33) Control 87 ± 3 (33)
PIECA 50L (SEQ ID NO: 4) 33 ± 5 (9)* (a) PIECA 50L (SEQ ID NO: 4) 33 ± 5 (9) * (a)
PIECA 52L (SEQ ID NO: 5) 62 ± 5 (9)* (b) PIECA 52L (SEQ ID NO: 5) 62 ± 5 (9) * (b)
EJEMPLO 4: Ensayos in vivo del efecto antihipertensivo tras administración oral. Se evaluó el efecto antihipertensor de los péptidos administrándolos vía oral a una dosis de 10 mg/Kg a ratas espontáneamente hipertensas (SHR) y normotensas Wistar-Kyoto (WKY). La Figura 2 muestra los cambios de la presión arterial sistólica (APAS) en ratas SHR a distintos tiempos tras su administración oral. Así mismo incluye la disminución de la PAS observada tras la administración de 50 mg/Kg de captopril (Sigma Chemical Co.) o solución salina. EXAMPLE 4: In vivo tests of the antihypertensive effect after oral administration. The antihypertensive effect of the peptides was evaluated by administering them orally at a dose of 10 mg / kg to spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive (WKY). Figure 2 shows the changes in systolic blood pressure (APAS) in SHR rats at different times after oral administration. It also includes the decrease in SBP observed after administration of 50 mg / kg of captopril (Sigma Chemical Co.) or saline solution.
La presión arterial sistólica (PAS) de las ratas SHR control, medida por el método del manguito en la cola ("tail-cuff"), fue de 201 ± 2 mm Hg (n = 59). La administración oral de los péptidos descritos PIECA 50L, PIECA 52L y PIECA 32L a 10 mg/Kg indujo una reducción significativa en la PAS cuya evolución en el tiempo se muestra en la Figura 2. En esta misma figura puede observarse la falta de efecto de la solución salina y el efecto antihipertensivo del captopril (50 mg/Kg). Para los péptidos PIECA 50L y PIECA 32L el efecto antihipertensivo máximo aparece transcurrida una hora después de su administración (-36.3 ± 3.3 mm Hg y -24.6 ± 5.3 mm Hg, respectivamente), mientras que para el péptido PIECA 52L se consigue el máximo efecto a las 2 horas (-18.6 ± 5.3 mm Hg). El efecto antihipertensivo de PIECA 50L se mantiene de forma significativa hasta tres horas después de su administración, mientras que el efecto de PIECA52L y de PIECA 32L se mantiene, respectivamente, hasta dos y una hora después de su administración. La administración oral de PIECA 50D a 10 mg/Kg no produjo cambios en la PAS en ratas SHR (no se muestran los datos).
En ratas normotensas WKY, la administración oral de PIECA 50L o PIECA 52L a 10 mg/Kg no produjo ningún cambio en la PAS. Estos resultados permiten descartar posibles efectos indeseables de los péptidos ensayados sobre la presión arterial de sujetos normotensos (no se muestran los datos). The systolic blood pressure (SBP) of the control SHR rats, measured by the tail-cuff method, was 201 ± 2 mm Hg (n = 59). The oral administration of the peptides described PIECA 50L, PIECA 52L and PIECA 32L at 10 mg / Kg induced a significant reduction in SBP whose evolution over time is shown in Figure 2. In this same figure the lack of effect of the saline solution and the antihypertensive effect of captopril (50 mg / Kg). For the PIECA 50L and PIECA 32L peptides the maximum antihypertensive effect appears after one hour after administration (-36.3 ± 3.3 mm Hg and -24.6 ± 5.3 mm Hg, respectively), while for the PIECA 52L peptide the maximum effect is achieved at 2 hours (-18.6 ± 5.3 mm Hg). The antihypertensive effect of PIECA 50L remains significantly up to three hours after administration, while the effect of PIECA52L and PIECA 32L is maintained, respectively, up to two and one hour after administration. Oral administration of PIECA 50D at 10 mg / Kg did not cause changes in SBP in SHR rats (data not shown). In normotensive WKY rats, oral administration of PIECA 50L or PIECA 52L at 10 mg / Kg did not produce any change in PAS. These results allow us to rule out possible undesirable effects of the peptides tested on the blood pressure of normotensive subjects (data not shown).
EJEMPLO 5: Ensayos in vivo del efecto antihipertensivo tras administración intravenosa. Con el objetivo de valorar el uso in vivo de los heptapéptidos de la invención, se procedió a inyectar por vía intravenosa dichos péptidos en ratas espontáneamente hipertensas. EXAMPLE 5: In vivo tests of the antihypertensive effect after intravenous administration. In order to assess the use in vivo of the heptapeptides of the invention, these peptides were injected intravenously into spontaneously hypertensive rats.
La inyección intravenosa de PIECA 50L a una dosis de 1 mg/Kg provocó una reducción aguda y transitoria de la presión arterial media (MABP), mientras que PIECA 52L a la misma concentración no produjo ningún cambio en la MABP. El esteroisómero D de PIECA 50L (PIECA 50D) (1 mg/Kg) provocó una reducción mantenida de la MABP. La magnitud de los distintos efectos antihipertensivos se resumen en la tabla 4, junto con el control negativo correspondiente (suero salino). En la Figura 3 se muestran, a modo de ejemplo, registros de los efectos causados por el péptido PIECA 50L y su esteroisómero D sobre la presión arterial (ABP). Intravenous injection of PIECA 50L at a dose of 1 mg / kg caused an acute and transient reduction in mean arterial pressure (MABP), while PIECA 52L at the same concentration did not cause any change in MABP. The stereoisomer D of PIECA 50L (PIECA 50D) (1 mg / kg) caused a sustained reduction in MABP. The magnitude of the different antihypertensive effects are summarized in Table 4, together with the corresponding negative control (saline serum). Figure 3 shows, by way of example, records of the effects caused by the PIECA 50L peptide and its stereoisomer D on blood pressure (ABP).
Tabla 4. Efecto de los péptidos descritos, administrados por vía intravenosa, sobre la presión arterial media (MABP) en ratas SHR. Los resultados indican el cambio de dicha presión arterial (ΔΜΑΒΡ) con respecto a la presión arterial anterior a la administración del péptido. Los datos son la media ± SEM para un número (n) de determinaciones y fueron sometidos a un análisis de ANOVA de una vía seguido de un test Student-Newman-Keuls. Los datos seguidos de letras distintas son diferentes entre sí, (a) y (b) (P<0.01 ). *Significativamente diferente con respecto al control P<0.01 .
Peptido ΔΜΑΒΡ (mm Hg) Table 4. Effect of the described peptides, administered intravenously, on the mean arterial pressure (MABP) in SHR rats. The results indicate the change in said blood pressure (ΔΜΑΒΡ) with respect to the blood pressure prior to the administration of the peptide. The data are the mean ± SEM for a number (n) of determinations and were subjected to a one-way ANOVA analysis followed by a Student-Newman-Keuls test. The data followed by different letters are different from each other, (a) and (b) (P <0.01). * Significantly different with respect to the control P <0.01. Peptide ΔΜΑΒΡ (mm Hg)
Control (salino) 5.9 ± 5.2 (5) Control (saline) 5.9 ± 5.2 (5)
PIECA 50L (SEQ ID NO 4) -76.1 ± 8.7 (8)* (a) PIECA 50L (SEQ ID NO 4) -76.1 ± 8.7 (8) * (a)
PIECA 50D (SEQ ID NO 4) -83.5 ± 6.7 (3)* (a) PIECA 50D (SEQ ID NO 4) -83.5 ± 6.7 (3) * (a)
PIECA 52L (SEQ ID NO 5) -1 .6 ± 2.1 (5) (b) PIECA 52L (SEQ ID NO 5) -1 .6 ± 2.1 (5) (b)
EJEMPLO 6: Ensayos de citotoxicidad EXAMPLE 6: Cytotoxicity tests
Para evaluar la posible toxicidad de PIECA 50L, se utilizaron tres tipos celulares diferentes (hepatocitos primarios de rata, células 3T3 (ATCC CL-173) y células HeptG2 (ATCC HB-8065) y la prueba de la sal de tetrazolio (MTT), siguiendo el protocolo descrito en Rodeiro et al. 2008. Chem Biol Interact 172:1 -10. El ensayo se llevó a cabo sembrando las células en placas de 96 pocilios (25 x 103 células/pocilio) y tras 24 h se pusieron en contacto con concentraciones crecientes de péptido. Después de 24 h de exposición, los efectos citotóxicos se midieron empleando el test de reducción del MTT. Tras un lavado de las células con tampón PBS (tampón salino fosfato), se adicionaron 100 μΙ/pocillo del reactivo MTT y se incubó durante 2 h. Transcurrido este tiempo, se descartó el sobrenadante y las células se lavaron con el mismo tampón. El colorante se extrajo con DMSO y se midió la DO540 en un lector de microplacas. El porcentaje de inhibición de la reducción del MTT por medio de la enzima succíníco deshidrogenasa se calculó en relación al control sin péptido (100 % de viabilidad). La potencia del péptido para reducir la viabilidad celular se resume en la tabla 5. Aunque había diferencias significativas dependiendo del tipo celular, los valores de IC50 se situaron en el rango mílimolar, desde 0.41 ± 0.07 mM para hepatocitos primarios hasta 1 .38 ± 0.06 mM sobre células HeptG2. Hay que destacar que estas concentraciones citotóxicas fueron 1000 veces superiores a las concentraciones de péptido necesarias para inhibir la actividad ECA. To assess the possible toxicity of PIECA 50L, three different cell types (primary rat hepatocytes, 3T3 cells (ATCC CL-173) and HeptG2 cells (ATCC HB-8065) and the tetrazolium salt test (MTT) were used, following the protocol described in Rodeiro et al. 2008. Chem Biol Interact 172: 1-10. The assay was carried out by sowing the cells in 96-well plates (25 x 10 3 cells / well) and after 24 h they were placed in Contact with increasing concentrations of peptide After 24 hours of exposure, the cytotoxic effects were measured using the MTT reduction test After washing the cells with PBS buffer (phosphate buffered saline), 100 μΙ / well of the reagent was added MTT and incubated for 2 h After this time, the supernatant was discarded and the cells were washed with the same buffer.The dye was extracted with DMSO and the OD540 was measured in a microplate reader.The percentage inhibition of reduction MTT po The medium of the succinic enzyme dehydrogenase was calculated in relation to the control without peptide (100% viability). The power of the peptide to reduce cell viability is summarized in Table 5. Although there were significant differences depending on the cell type, the IC50 values were in the mimeolar range, from 0.41 ± 0.07 mM for primary hepatocytes to 1.38 ± 0.06 mM on HeptG2 cells. It should be noted that these cytotoxic concentrations were 1000 times higher than the peptide concentrations necessary to inhibit ECA activity.
Tabla 5. Efecto del péptido PIECA 50L sobre la viabilidad de los cultivos celulares. El potencial citotóxico se expresa como IC50, definido como la
concentración de péptido capaz de reducir la viabilidad celular en un 50%. Se calculó para cada ensayo por regresión no-lineal de los datos experimentales. Los resultados se expresan como la media ± SEM para tres repeticiones. Table 5. Effect of the PIECA 50L peptide on the viability of cell cultures. The cytotoxic potential is expressed as IC50, defined as the Peptide concentration capable of reducing cell viability by 50%. It was calculated for each trial by non-linear regression of the experimental data. The results are expressed as the mean ± SEM for three repetitions.
1C50 (mM) 1C 50 (mM)
Hepatocitos Células Hepatocytes Cells
Péptido Células 3T3 3T3 Cell Peptide
primarios de rata HeptG2 HeptG2 rat primaries
PIECA 50L (SEQ ID. NO 4) 0.41 ± 0.07 1 .31 ± 0.13 1 .38 ± 0.06
PIECA 50L (SEQ ID. NO 4) 0.41 ± 0.07 1 .31 ± 0.13 1 .38 ± 0.06
Claims
1 . Heptapéptido que consiste en la secuencia SEQ ID NO: 1 donde el primer aminoácido es una arginina (R) el segundo aminoácido es una lisina (K), el tercer aminoácido es un triptófano (W), el cuarto aminoácido es una histidina (H) o una leucina (L), el quinto aminoácido es una fenilalanina (F), el sexto aminoácido es una leucina (L) o una histidina (H) y el séptimo aminoácido es un triptófano (W). one . Heptapeptide consisting of the sequence SEQ ID NO: 1 where the first amino acid is an arginine (R) the second amino acid is a lysine (K), the third amino acid is a tryptophan (W), the fourth amino acid is a histidine (H) or a leucine (L), the fifth amino acid is a phenylalanine (F), the sixth amino acid is a leucine (L) or a histidine (H) and the seventh amino acid is a tryptophan (W).
2. Heptapéptido según la reivindicación 1 que consiste en la secuencia SEQ ID NO: 2 donde el aminoácido de la cuarta posición es una histidina (H) y el aminoácido de la sexta posición es una leucina (L). 2. Heptapeptide according to claim 1 consisting of the sequence SEQ ID NO: 2 wherein the amino acid of the fourth position is a histidine (H) and the amino acid of the sixth position is a leucine (L).
3. Heptapéptido según la reivindicación 1 que consiste en la secuencia SEQ ID NO: 3 donde el aminoácido de la cuarta posición es una leucina (L) y el aminoácido de la sexta posición es una histidina (H). 3. Heptapeptide according to claim 1 consisting of the sequence SEQ ID NO: 3 wherein the fourth position amino acid is a leucine (L) and the sixth position amino acid is a histidine (H).
4. Heptapéptidos según cualquiera de las reivindicaciones 1 a 3 cuya esteroisomería es levógira (L). 4. Heptapeptides according to any one of claims 1 to 3 whose stereoisomery is levógira (L).
5. Heptapéptidos según cualquiera de las reivindicaciones 1 a 3 cuya esteroisomería es dextrógira (D). 5. Heptapeptides according to any one of claims 1 to 3 whose steroisomerism is dextrógira (D).
6. Heptapéptido según cualquiera de las reivindicaciones 1 a 5 donde el extremo amino terminal está acetilado y el extremo carboxilo terminal está amidado. 6. Heptapeptide according to any one of claims 1 to 5 wherein the amino terminal end is acetylated and the carboxyl terminal end is amidated.
7. Heptapéptido según la reivindicación 6 que consiste en la SEQ ID NO: 4. 7. Heptapeptide according to claim 6 consisting of SEQ ID NO: 4.
8. Heptapéptido según la reivindicación 6 que consiste en la SEQ ID NO: 5. 8. Heptapeptide according to claim 6 consisting of SEQ ID NO: 5.
9. Polipéptido que comprende el heptapéptido según cualquiera de las reivindicaciones 1 a 5. 9. Polypeptide comprising the heptapeptide according to any one of claims 1 to 5.
10. Ácido nucleico que comprende la secuencia de nucleótidos que codifica para el heptapéptido descrito según cualquiera de las reivindicaciones 1 a 4. 10. Nucleic acid comprising the nucleotide sequence encoding the heptapeptide described according to any one of claims 1 to 4.
1 1 . Cassette de expresión que comprende el ácido nucleico según la reivindicación 10. eleven . Expression cassette comprising the nucleic acid according to claim 10.
12. Vector recombinante que comprende el ácido nucleico según la reivindicación 10 o el cassette de expresión según la reivindicación 1 1 . 12. Recombinant vector comprising the nucleic acid according to claim 10 or the expression cassette according to claim 1 1.
13. Célula hospedadora que comprende el ácido nucléico según la reivindicación 10, o el cassette de expresión según la reivindicación 1 1 o el vector recombinante según la reivindicación 12, donde preferiblemente la célula hospedadora es una célula de mamífero, de insecto, hongo o bacteria. 13. Host cell comprising the nucleic acid according to claim 10, or the expression cassette according to claim 1 or the recombinant vector according to claim 12, wherein preferably the host cell is a mammalian, insect, fungus or bacterial cell .
14. Proceso para producir el heptapéptido descrito según cualquiera de las reivindicaciones 1 a 4, que comprende cultivar una célula hospedadora según la reivindicación 13. 14. Process for producing the heptapeptide described according to any one of claims 1 to 4, comprising culturing a host cell according to claim 13.
15. Uso del heptapéptido según cualquiera de las reivindicaciones 1 a 8 para la elaboración de una composición farmacéutica. 15. Use of the heptapeptide according to any one of claims 1 to 8 for the preparation of a pharmaceutical composition.
16. Uso del heptapéptido según cualquiera de las reivindicaciones 1 a 8 para la elaboración de una composición farmacéutica para la prevención y/o el tratamiento de la hipertensión arterial. 16. Use of the heptapeptide according to any of claims 1 to 8 for the preparation of a pharmaceutical composition for the prevention and / or treatment of arterial hypertension.
17. Composición farmacéutica que comprende el heptapéptido según cualquiera de las reivindicaciones 1 a 8. 17. Pharmaceutical composition comprising the heptapeptide according to any one of claims 1 to 8.
18. Composición farmacéutica según la reivindicación 17, que además comprende al menos un excipiente y/o al menos un vehículo farmacéuticamente aceptables. 18. Pharmaceutical composition according to claim 17, further comprising at least one pharmaceutically acceptable excipient and / or at least one vehicle.
19. Composición farmacéutica según cualquiera de las reivindicaciones 17 ó 18, donde la composición farmacéutica además comprende al menos otro principio activo. 19. Pharmaceutical composition according to any of claims 17 or 18, wherein the pharmaceutical composition further comprises at least one other active ingredient.
20. Composición farmacéutica según cualquiera de las reivindicaciones 17 a 19, donde la composición farmacéutica se presenta en una forma adaptada a la administración por vía oral, parenteral o intradérmíca. 20. Pharmaceutical composition according to any of claims 17 to 19, wherein the pharmaceutical composition is presented in a form adapted to oral, parenteral or intradermal administration.
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| ESP201130150 | 2011-02-04 | ||
| ES201130150A ES2387435B1 (en) | 2011-02-04 | 2011-02-04 | USE OF HEPTAPEPTIDES FOR HYPERTENSION CONTROL |
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| WO2012104462A1 true WO2012104462A1 (en) | 2012-08-09 |
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| PCT/ES2012/070055 WO2012104462A1 (en) | 2011-02-04 | 2012-01-31 | Heptapeptides and the use thereof for controlling hypertension |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004308A (en) * | 2019-12-30 | 2020-04-14 | 华南理工大学 | Heptapeptide for inhibiting angiotensin converting enzyme and application thereof |
| CN111087446A (en) * | 2019-12-30 | 2020-05-01 | 中新国际联合研究院 | Decapeptide for inhibiting angiotensin converting enzyme and application thereof |
| CN111116712A (en) * | 2019-12-30 | 2020-05-08 | 中新国际联合研究院 | Hexapeptide with ACE inhibitory activity and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2191516A1 (en) * | 2000-07-28 | 2003-09-01 | Consejo Superior Investigacion | Anti-fungal antibiotic of peptide kind is inhibitor of germination and growth of phytopathogenic fungi |
| ES2320057A1 (en) * | 2006-04-05 | 2009-05-18 | Consejo Superior De Investigaciones Cientificas | Use of hexapeptides for preparing angiotensin 1 converting enzyme medicinal products |
-
2011
- 2011-02-04 ES ES201130150A patent/ES2387435B1/en not_active Withdrawn - After Issue
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2012
- 2012-01-31 WO PCT/ES2012/070055 patent/WO2012104462A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2191516A1 (en) * | 2000-07-28 | 2003-09-01 | Consejo Superior Investigacion | Anti-fungal antibiotic of peptide kind is inhibitor of germination and growth of phytopathogenic fungi |
| ES2320057A1 (en) * | 2006-04-05 | 2009-05-18 | Consejo Superior De Investigaciones Cientificas | Use of hexapeptides for preparing angiotensin 1 converting enzyme medicinal products |
Non-Patent Citations (2)
| Title |
|---|
| CHEUNG, H.S. ET AL.: "Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 255, no. 2, 25 January 1980 (1980-01-25), pages 401 - 407, XP002388030 * |
| RUIZ-GIMENEZ, P. ET AL.: "Novel antihypertensive hexa- and heptapeptides with ACE-inhibiting properties: from the in vitro ACE assay to the spontaneously hypertensive rat", PEPTIDES, vol. 32, no. 7, July 2011 (2011-07-01), pages 1431 - 1438, XP028236675, DOI: doi:10.1016/j.peptides.2011.05.013 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004308A (en) * | 2019-12-30 | 2020-04-14 | 华南理工大学 | Heptapeptide for inhibiting angiotensin converting enzyme and application thereof |
| CN111087446A (en) * | 2019-12-30 | 2020-05-01 | 中新国际联合研究院 | Decapeptide for inhibiting angiotensin converting enzyme and application thereof |
| CN111116712A (en) * | 2019-12-30 | 2020-05-08 | 中新国际联合研究院 | Hexapeptide with ACE inhibitory activity and application thereof |
| CN111004308B (en) * | 2019-12-30 | 2021-06-08 | 华南理工大学 | Heptapeptide for inhibiting angiotensin converting enzyme and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2387435B1 (en) | 2013-07-31 |
| ES2387435A1 (en) | 2012-09-21 |
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