MXPA01001638A - Methods to increase blood flow to ischemic tissue - Google Patents

Abstract

The present invention provides methods and kits for increasing blood flow to ischemic tissue, comprising the administration of an effective amount of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists.

Description

METHODS TO INCREASE BLOOD FLOW IN THE ISCHEMIC TISSUE BACKGROUND OF THE INVENTION Collateral circulation to tissues can be stimulated in two independent routes: 1) the formation of new blood vessels through angiogenesis and 2) the improvement in function of pre-existing blood vessels (Fernández et al., J. Lab. Clin. Med. 105: 141-145 (1985)). Angiogenesis is the process in which new blood vessels are formed from existing capillaries. (U.S. Patent No. 5,318,957, incorporated herein by reference in its entirety). Angiogenesis plays an important role in which biological conditions such as embryogenic development, tumor growth, wound healing and chronic inflammatory diseases diverge widely. (Folkman et al., Science 235: 442-447 (1987)). The capillaries are composed almost entirely of endothelial cells. Angiogenesis comprises a cascade of events, including the secretion of protease by endothelial cells, degradation of the base membrane, migration through the surrounding matrix, proliferation, alignment, differentiation between structures such as tubes and the synthesis of a new base membrane (U.S. Patent No. 5,318,957). Various angiogenic agents with different properties and mechanisms of action are well known in the art, including the basic and acidic fibroblast growth factor, the alpha and beta growth transformation factor, the tumor necrosis factor, the growth factor derived from platelets, vascular endothelial growth factor, angiogenin and haptoglobin (U.S. Patent No. 5,318, 957). However, the therapeutic applicability of some of these compounds is limited by Felfea its potent pleiotropic effects in several cell types. Therefore, there is a need in the art for angiogenic agents with more general applicability. Very few therapies have been shown to increase collateral circulation by improving the function of existing blood vessels. In an isolated case 5, Pat. of E.U.A. No. 4,296,100 describes experiments in animals wherein bovine fibroblast growth factor (FGF) was injected into the heart to distribute the desired amount of FGF in the area of the heart to be treated. This treatment was given as close as possible to the heart attack to control the damage, possibly through improved collateral circulation, although the exact mechanism is unknown. The experiments showed that a treatment in time can reduce the size of the infarct (area that will heal or continue to be permanently damaged) in the test animal by one quarter the size of the control hearts (untreated). Histological studies did not show a significant increase in capillary areas in hearts as a result of such treatment with FGF. 15 Congestive heart failure is the leading cause of cardiovascular mortality (Tyagi, J. Cell Biochem 65: 388-394 (1997)) and is caused by cardiac remodeling leading to widening of the heart after myocardial infarction (Pfeffer, Am. J. Cardiol. 68: 17D- 25D (1991)) There is a strong correlation between the morbidity of congestive heart failure and the degree of post-infarction remodeling. as a 35% increase in the volume of the heart in the months following the infarction leads to an increase in mortality by a factor of 3-4 (Hammermeister et al., Circulation 59: 421-435 (1979)). Therefore, therapies that reduce cardiac remodeling after a myocardial infarction are needed to prevent the development of congestive heart disease. 25 The loss of blood flow in ischemic tissue is the main factor to cause cardiac remodeling. Studies show that there is an important t -li¿ ^ fc ^ ¿- ^^ '^. .í? Kiat .x. ... cardiac remodeling after myocardial infarction if the infarct-related artery is totally blocked (Kim and Braunwald, Circulation 88: 2426-2436 (1993)). However, cardiac remodeling can be reduced if blood flow can be restored in the ischemic tissue. In addition, if the anterior blood flow is restored, cardiac remodeling can be further attenuated. (Hochman et al., Circulation 75: 299-306 (1987), Bonaduce et al., J. Am. Coll. Cardiol. 16: 1561-1568 (1990) .Therefore, treatments aimed at the rapid restoration of Blood flow after myocardial infarction can reduce cardiac remodeling and the development of congestive heart disease Chemical therapies are currently one of the most useful treatments for the restoration of blood flow in ischemic tissue after myocardial infarction. Thrombolytics are based on clearing the clogged coronary artery to restore blood flow (US Pat. No. 5,589,173) In addition to clearing the clogged coronary artery, the presence of a collateral blood supply can decrease the effect of cardiac remodeling. Therefore, chemical therapies aimed at stimulating collateral circulation could reduce cardiac remodeling after myocardial infarction. , there are currently few materials available to stimulate collateral blood flow. Based on the above, there is a need for the development of methods to increase blood flow in the heart after a myocardial infarction.

SUMMARY OF THE INVENTION The present invention provides improved methods and equipment for increasing blood flow in ischemic tissue comprising the administration of angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogs of the MhMÉMüHi ^ - *** "* - * thereof, angiotensin II (All) All analogues, fragments All or analogs thereof or AT2 receptor agonists All type 2.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the effect of the analogues All in the blood flow in conjunction with the Apligraf treatment. Figure 2 is a graph showing the effect of the All analogs on endothelial cell production in conjunction with the Apligraf treatment. Figure 3 is a graph showing the effect of the All analogues on blood flow after a thermal injury.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES All references, patents and patent applications are incorporated herein in their entirety as a reference. As used herein, the term "ischemia" refers to any condition associated with inadequate flow of oxygenated blood in a part of the body. Ischemia occurs at any time when the blood flow in a tissue is reduced below its critical level. This reduction in blood flow can result from the following non-limiting conditions: (i) blockage of a vessel by a plunger (blood clot); (ii) blockage of a vessel due to arteriesclerosis; (iii) the breaking of a blood vessel (a bleeding stroke); (iv) blockage of a blood vessel due to vasos- stenosis such as occurs during vasospasm and possibly during transient ischemic attacks (TIA) and after subarachnoid hemorrhage. Additional conditions in which ischemia occurs, include (i) during myocardial infarction (when the heart stops, the blood flow to organs slows causing ischemia); (ii) trauma and (i¡¡) during neurosurgery and ^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ g & ^^^^^^^^^ heart (blood flow needs to be reduced or stopped to achieve the goals of surgery). As used herein, the term "ischemic tissue" refers to any tissue that is receiving an inadequate flow of oxygenated blood. As used in this document, "increased blood flow" refers to such increases mediated by the formation of new blood vessels through angiogenesis or improvement in the function of pre-existing blood vessels. Within this application, unless stated otherwise, the techniques used can be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press ), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego CA), "Guide to Protein Purification" in Methods in Enzymology (MP Deutscher, ed. (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al., 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2 a. Ed. (R. Freshney, 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, NJ), and the Ambion Catalog 1998 (Ambion, Austin, TX). The Patent of E.U.A. No. 5,015,629 to DiZerega (the entire disclosure of which was incorporated herein by reference) discloses a method for increasing the healing rate of tissue from a wound, comprising the application to said tissue of angiotensin II (All) in a amount that is sufficient for said increase. The application of All to the wound tissue significantly increases the healing rate of the wound, leading to a faster re-epithelialization and tissue repair. The term All refers to an octapeptide present in humans and other species ^ g ^^^? ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^ ^^^^^^^^^^ having the sequence Asp-Arg-Val-Tyr-lle-His-Pro-Phe [SEQ ID NO: 1]. The biological formation of angiotensin is initiated by the action of renin in the angiotensinogen of the plasma substrate (Clouston et al., Genomics 2: 240-248 (1988); Kageyama et al, Biochemistry 23: 3603-3609; Ohkubo et al. , Proc. Nati, Acad. Sci. 80: 2196-2200 (1983), each reference is incorporated in its entirety in this document). The substance formed is a decapeptide called angiotensin I (Al) which is converted to All by the conversion of the enzyme angiotensin (ACE) which removes the C-terminal residues His-Leu from Al [SEQ ID NO: 37]. All is a known precursor agent and is commercially available. Studies have shown that All increases mitogenesis and chemotaxis in cultured cells that are involved in wound repair and also increases their release of extracellular growth factors and matrices (diZerega, U.S. Patent No. 5,015,629; Dzau et al. al., J. Mol. Cell, Cardiol.21: S7 (Supp III) 1989, Berk et al., Hypertension 13: 305-14 (1989), Kawahara et al., BBRC 150: 52-9 (1988); Naftilan, et al., J. Clin Invest. 83: 1419-23 (1989); Taubman et al., J. Biol. Chem 264: 526-530 (1989); Nakahara, et al., BBRC 184: 811 -8 (1992), Stouffer and Owens, Circ. Res. 70: 820 (1992), Wolf, et al., Am. J. Pathol., 140: 95-107 (1992), Bell and Madri, Am. Pathol., 137: 7-12 (1990) In addition, the All proved to be angiogenic in rabbit eye cornea and chorioallantic chicken membrane models (Fernandez, et al., J. Lab. Clin. Med. 105: 141. (1985), LeNoble, et al., Eur. J. Pharmacol., 195: 305-6 (1991). In addition, it has been shown that All and the angiotensin III analogs and the fragments and the All 2 type 2 receptor agonists thereof are effective in tissue repair. (U.S. Patent Nos. 5,629,292; 5,716,935; International Applications Nos. WO 94/10502, WO 94/10503, WO 95/14764, WO 95/08565, WO 95/08337, WO 96/09747, WO 96/39164, WO 97/23461, WO 98/26437, all references incorporated in their entirety in this document).

Angiotensin II is also known as a potent stimulator of angiogenesis (Fernandez et al., J. Lab. Clin.Med. 105: 141-145 (1985)) and has been shown to activate collateral circulation via preformed blood vessels. in rat kidneys (Fernandez et al., Am. J. Physiol. 243: H869-H875 (1982)). The hypothesis has been formulated that the effect of All in a given cell type is dependent, in part, on the cellular expressions of the All receptor subtypes (Shanugam et al., Am. J. Physiol. 268: F922-F930 (1995), Helin et al., Annals of Medicine 29: 23-29 (1997), Bedecs et al., Biochem J. 325: 449-454 (1997)). These studies have shown that the expression of the All receptor subtype is a dynamic process that changes during development in at least some cell types (Id). The activity of All is typically modulated by one or both of the All AT1 and AT2 receivers. However, All has recently shown that it stimulates the proliferation of primary human keratinocytes via a non-AT1, non-AT2 receptor. (Steckelings et al., Biochem. Biophys., Res. Commun. 229: 329-333 (1996)). These results underscore the specific nature of the cell type of All activity (ie, based on the expression of the receptor). Other data suggest that the All Al 1 (1-7) fragment acts through a receptor that is distinct from the AT1 and AT2 receptors that modulates All activity. (Ferrario et al., J. Am. Soc. Nephrol., 9: 1716-1722 (1998), lyer et al., Hypertension 31: 699-705 (1988), Freeman et al., Hypertension 28: 104 (1996).; Ambuhl et al., Brain Res. Bull. 35: 289 (1994).

Therefore, Al 1 activity (1 -7), in a particular cell type can not be predicted based solely on the effect of All in the same cell type. However, based on the above, it is unknown whether the use of angiotensinogen, angiotensin I (Al), Al analogs, Al fragments and analogs thereof, All analogues, All fragments or analogs thereof or the agonists All-AT2 receptors of type 2 (AT2) would be effective in stimulating blood flow in ischemic tissue. Identification of analogues and All fragments that stimulate the Blood flow with side effects less than the All could be extremely beneficial. A selective peptide agonist for the AT2 receptor has been defined (All has 100 times higher affinity for AT2 than for AT1). This peptide is p-aminophenylalanine6-AII ["p-NH2-Phe) 6-AII"], Asp-Arg-Val-Tyr-lle-Xaa-Pro-Phe [SEQ ID NO. 36] where Xaa is p-NH2-Phe (Speth and Kim, BBRC 169: 997-1006 (1990) .This peptide gave comparable binding characteristics to the AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol 256: 93-97 (1994), Bryson, et al., Eur. J. Pharmacol 225: 119-127 (1992) The effects of the All receptor and the All receptor antagonists have been examined in two experimental models of vascular damage and repair that follow that both subtypes of the All receptor (AT1 and AT2) play a role in wound healing (Janiak et al., Hypertension 20: 737-45 (1992); Prescott, et al. ., Am. J. Pathol., 139: 1291-1296 (1991), Kauffman et al., Life Sci. 49: 223-228 (1991), Viswanathan, et al., Peptides 13: 783-786 (1992); Kimura, et al., BBRC 187: 1083-1090 (1992) Many studies have focused on AII (1-7) (residues All 1-7) or other All fragments to evaluate their activity. 7) produces some, but not the full range of effects produced by Al I. Pfeilschifter, et al., Eur. J. Pharmacol. 225: 57-62 (1992); JaiswaI, et al., Hypertension 19 (Supp.ll): ll-49-ll-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266: 506-510 (1993); JaiswaI, et al., J. Pharmacol. Exper. Ther. 265: 664-673 (1991); JaiswaI, et al., Hypertension 17: 1115-1120 (1991); Portsi, et a., Br. J. Pharmacol. 111: 652-654 (1994). As defined above, a preferred class of AT2 agonists to be used in accordance with the present invention comprises angiotensinogen, angiotensin I (Al), Al analogs, Al fragments and analogs thereof, All, All analogs, All fragments or analogs thereof. themselves or All AT2 receptor agonists of type 2 having p-NH- ?? b ?? ^ ^? ^ ^ Jj ^ Phe in a position corresponding to a position 6 of All. In addition to the peptide agents, various non-peptide agents (eg, peptidomimetics) that have the requisite activity of the AT2 agonist are further contemplated for use in accordance with the present invention. The active analogs All, the All fragments and analogs thereof of particular interest according to the present invention comprise a sequence consisting of at least three contiguous amino acids of the groups R1-R8 in the sequence of the general formula I R1- R2-R3-R4-R5-R6-R7-R8 in which R1 and R2 together form a group of formula X-RA-RB-, wherein X is H or one of the three peptide groups, or is absent , RA is appropriately selected from H, Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc, RB is appropriately selected from Arg, Lys, Ala, Orn, Citron, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Lie, Gly, Pro, Aib, Acpc and Tyr; R4 is selected from the group consisting of Tyr, Tyr (PO3) 2, Thr, Ser, Ala, Ser, homoSer and azaTyr; R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and . - (, 22.:%^. "R8 is selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding sequences that include R4 as a terminal group Tyr, or is absent, wherein the active agent is not All 5 Compounds that fall within the category of AT2 agonists useful in the practice of the invention include the All analogues set forth above subject to the restriction that R6 is p-NH2-Phe. for RA and RB are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys Particularly preferred embodiments of this class include the following: There or AII (2-8), Arg-Val-Tyr-lle His-Pro-Phe [SEQ ID NO: 2]; AII (3-8), also known as desl-AIII or AIV, Val-Tyr-lle-His-Pro-Phe [SEQ ID NO: 3]; AII (1-7), Asp-Arg-Val-Tyr-lle-His-Pro [SEQ ID NO: 4]; AII (2-7). Arg-Val-Tyr-lle-His-Pro [SEQ ID NO: 5]; AII (3-7), Val-Tyr-lle-His-Pro [SEQ ID NO: 6]; AII (5-8), lle-His-Pro-Phe [SEQ ID NO: 7]; All (1-6), Asp-Arg-Val-Tyr-lle-His [SEQ ID NO: 8]; AII (1-5), Asp-Arg-Val-Tyr-lle [SEQ ID NO: 9]; 15 AII (1-4), Asp-Arg-Val-Tyr [SEQ ID NO: 10] and AII (1-3), Asp-Arg-Val [SEQ ID NO: 11]. Other preferred embodiments include: Arg-norLeu-Tyr-lle-His-Pro-Phe [SEQ ID NO: 12] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 13]. Another also preferred embodiment comprised within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-lle-His-Pro-Phe [SEQ ID NO: 31]. AII (6-8), His-Pro-Phe [SEQ ID NO: 14] and 20 AII (4-8), Tyr-lle-His-Pro-Phe [SEQ ID NO: 15], were also tested and found that they are not effective. In a particularly preferred embodiment, the active compounds of the present invention are selected from those comprising the following general formula: R1-Arg-R2-R3-R4-His-Pro-R5, wherein R1 is Asp or is absent; ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ lie, Pro, Lys, NorLeu and Leu; R3 is selected from the group consisting of Ala, Tyr and Tyr (PO3) 2 and R4 is selected from the group consisting of Val, Ala, Lie, Norleu and Leu; R5 is Phe, lie or absent. In a more particularly preferred embodiment, the active compound is selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 19; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42. Another class of compounds of particular interest according to the present invention are those of the general formula II R2-R3-R4-R5-R6-R7-R8 wherein R2 is selected from the group consisting of H, Arg, Lys, Ala , Orn, Citron, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Lie, Gly, Pro, Aib, Acpc and Tyr; R4 is selected from the group consisting of Tyr, Tyr (PO3) 2, Thr, Ser, homoSer, Ala and azaTyr; R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and R8 is selected from the group consisting of Phe, Phe (Br), lie and Tyr. A particularly preferred subclass of the compounds of the general formula II has the formula R2-R3-Tyr-R5-His-Pro-Phe [SEQ ID NO: 16] wherein R2, R3 and R5 are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-lle-His-Pro-Phe [SEQ ID NO: 2]. ^ ^^^^^^^^^^^^^ * 2 ^^^^^^^^^^^^^^^^^^^^^^^^ J ^ ^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ = g ^ * Other preferred compounds include peptides that have the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO: 18]. The Al I fragment (4-8) was ineffective in the repeated tests; this is believed due to the tyrosine exposed in the N-terminus. In the above formulas, the standard three-letter abbreviations for amino acid residues are used. In the absence of an indication to the contrary, the L form of the amino acid is tried. Other waste is abbreviated as follows: TABLE 1 Abbreviation for Amino Acids It has been suggested that the All and its analogues adopt a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974).) In general, it is believed that the neutral side chains in position R3, R5 and R7 can be involved in maintaining the appropriate distance between the active groups in positions R4, R6 and R8 basically responsible for binding to receptors and / or intrinsic activity.The hydrophobic side chains in positions R3, R5 and R8 can also play an important role in the complete conformation of the peptide and / or contribute to the formation of a hypothetical hydrophobic cavity.

Appropriate side chains at the amino acid at the R2 position can contribute to the affinity of the compounds for target receptors and / or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R2. For purposes of the present invention, it is considered that R3 can be involved in the formation of linear and non-linear hydrogen bonds with R5 (in the gamma model) or R6 (in the beta spin model). R3 would also participate in the first turn in an antiparallel beta structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it seems that the beta and gamma branches are equally effective in this position. In addition, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R3 can be appropriately selected from Val, Ala, Leu, norLeu, Lie, Gly, Pro, Aib, Acpc and Tyr. It has also been found that Lys is effective in the R3 position. With respect to R4, conformational analyzes have suggested that the side chain in this position (as well as in R3 and R5) contributes to a hydrophobic cluster that is believed to be essential for receptor occupancy and stimulation. Thus, R4 preferably is selected from Tyr, Thr, Tyr (PO3) 2, homoSer, Ser and azaTyr. In this position, Tyr is particularly preferred to form a hydrogen bond with the receptor site capable of accepting a hydrogen of the phenolic hydroxyl (Regoli, et al. (1974), supra). It has been found that Ala is effective in the R4 position. In the R5 position, an amino acid with an aliphatic or alicyclic β chain is particularly desirable. Therefore, while Gly is appropriate in the R5 position, it is preferred that the amino acid in this position be selected from lie, Ala, Leu, norLeu, Gly and Val. In angiotensinogen, Al, Al analogs, Al fragments and analogs thereof, All analogs, fragments and analogs of fragments of particular interest in accordance With the present invention, R6 is His, Arg or 6-NH2-Phe. The unique properties of the imidazole ring of histidine (ie, ionization at physiological pH, the ability to act as a donor or proton acceptor, aromatic character) are considered to contribute to its particular utility as R6. For example, conformational models suggest that 5 His can participate in hydrogen bond formation (in the beta model) or in the second antiparallel structure spin by influencing the orientation of R7. Similarly, it is considered that R7 should be Pro to provide the most desirable orientation of R8. In the R8 position, a hydrophobic ring and a terminal anionic carboxyl are particularly useful in binding the analogs of interest to the receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention. R8 can also be appropriately lie. Analogs of particular interest include the following: TABLE 2 15 Analogs Angiotensin II The polypeptides of the present invention can be synthesized by any of the conventional methods including but not limited to those methods set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chemical Co., Rockford, Ill. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York (1965) for the synthesis of the solution. The descriptions of the previous treaties are incorporated in this document as a reference. In general, these methods involve the sequential addition of protected amino acids to a chain of growth peptides (U.S. Patent No. 5,693,616, incorporated herein by reference in its entirety). Normally, the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is subsequently bound to an inert solid support or used in solution and the next amino acid in the sequence, also appropriately protected, is added under conditions treatable for the formation of the amide bond. After all the desired amino acids have been linked in the proper sequence, the protecting groups and any solid support are removed to produce the natural polypeptide. The polypeptide is desalted and purified, preferably chromatographically to produce the final product. Preferably, the peptides are synthesized in accordance with standard solid phase methods, such as those that can be performed in a synthesizer.

^ ^ ^ B ^ 2 ^^^^^^^ Peptides from Applied Biosystems Model 430A (Applied Biosystems, Foster City, Calif.), In accordance with the manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase or liquid phase methods are well known to those skilled in the art. In one aspect, the present invention provides methods and equipment for increasing blood flow in ischemic tissue comprising administration of angiotensinogen, angiotensin I (Al), Al analogs, Al fragments and analogs thereof, angiotensin II (All), Analogs All, All or analogous fragments thereof or All AT2 type 2 receptor agonists (referred to herein as "active agents"). In accordance with the invention, an area of ischemic tissue is treated in vivo to maintain the viability of that area for a sustained period of time to rescue the area. An effective dose of the active agents is applied in ischemic tissue, preferably after ischemia, although it may also be applied when there is an indication of impending ischemia. Ischemic tissue is appropriately any tissue that can benefit by increased blood flow in the tissue to reverse or prevent the adverse effects of ischemia in this tissue. In the preferred modalities, the tissue is selected from the skin and the heart. In a preferred additional embodiment, the active agents can be used to stimulate blood flow in tissue grafts, either artificial or transplanted. The active agents can be administered by any appropriate route, including oral, parental, by inhalation spray, rectally or topically in unit dose formulations containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes the subcutaneous, intravenous, intraarterial, intramuscular, Intrasternal, intracardiac, intratendinous, intraspinal, intracranial, intrathoracic, infusion or intraperitoneal techniques. Therefore, angiotensinogen can be administered by gene therapy techniques. The active agents of the invention can be manufactured in a solid form (including granules, powders or suppositories) or in a liquid form (ie, solutions, suspensions or emulsions). The compounds of the invention can be applied in a variety of solutions. Appropriate solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the peptide and are not harmful to the proposed application. In this regard, the compounds of the present invention are very stable but are hydrolyzed by strong acids and bases. The compounds of the present invention are soluble in organic solvents and in aqueous solutions at a pH of 5-8. The active agents can be subjected to conventional pharmaceutical operations such as sterilization and / or they can contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, regulators, etc. The active agents of the invention can be used alone or in combination of active agents or can be used in combination with other agents that increase blood flow in the ischemic tissue either via the production of new blood vessels (including, but not limited to All, growth factor of the basic and acidic fibroblast, alpha and beta factor of growth transformation, tumor necrosis factor, platelet-derived growth factor, vascular endothelial growth factor, angiogenin and haptoglobin) or by blood flow in the ischemic tissue a through pre-existing blood vessels, including but not limited to the fibroblast growth factor.

For administration, active agents are ordinarily combined with one or more of the appropriate adjuvants for the indicated route of administration. The compounds can be mixed with lactose, sucrose, starch powder, cellulose esters or alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of sulfuric and phosphoric acids, acacia, gelatin, sodium alginate. , polyvinylpyrrolidine and / or polyvinyl alcohol and are tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention can be dissolved in saline, water, polyethylene glycol, propylene glycol, colloidal solutions of carboxymethyl cellulose, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum and / or several stabilizers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include a retarding material such as glyceryl monostearate or glyceryl distearate alone or with a wax or other materials well known in the art. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (ie, liniments, lotions, ointments, creams or pastes) and appropriate drops for administration to the eye, ear or nose . The dose regimen for increasing blood flow in ischemic tissue with the active agents of the invention is based on a variety of factors, including the type of damage, the age, the weight, the sex, the medical condition of the individual, the severity of the condition, the route of administration and the particular compound used. Therefore, the dosage regimen can vary widely, but can be determined routinely by a physician using standard methods. The dose levels in the order of between 0.1 ng / kg and 10 mg / kg by weight of the active agent per body weight are useful for all the methods of use described herein.

"% G &; * fe¡;» »fe¿ *» f ¿¡l ^ £? X The treatment regimen will also vary depending on the condition of the subject, based on a variety of factors, including the type of damage, age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration and the particular compound used. For example, active agents are administered to a patient as soon as possible after a myocardial infarction for more than 30 days. The therapy is administered 1 to 6 times per day in the doses described above. In a preferred embodiment, the active agent is administered subcutaneously. An appropriate subcutaneous dose of the active agent is preferably between about 0.1 ng / kg and about 10 mg / kg administered twice a day for a sufficient time to increase blood flow in the ischemic tissue. In a more preferred embodiment, the concentration of the active agent is between about 100 ng / kg per body weight and about 10.0 mg / kg per body weight. In a more preferred embodiment, the concentration of the active agent is between about 10 μg / kg per body weight and about 10.0 mg / kg per body weight. This dose regimen maximizes the therapeutic benefits of the subject of the invention while minimizing the amount of the antagonist needed. Such an application minimizes costs as well as possible deleterious side effects. For subcutaneous administration, the active ingredient may comprise from 0.0001% to 10% w / w, that is, from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w / w, but preferably not more than 5% w / w and more preferably from 0.1% to 1% of the formulation. In a preferred embodiment, a catheter is placed in the coronary artery of a subject between almost immediately after approximately 24 hours after myocardial infarction and by injecting an effective amount of the active agent into the subject's heart. The concentration of the active agent injected is between about 100 ng / kg body weight and about 10.0 mg / kg body weight, as described above. The injection may also be repeated as needed to stimulate increased blood flow in the existing collateral circulation. The injections can also be by other routes, including but not limited to by a catheter such as arterial angiography, intra-coronary injection or in a cardioplegic solution via the aortic route. Increased collateral blood flow was determined by standard procedures. For example, collateral flow can be quantified during maximal vasodilation induced with Cromonar and expressed as a ratio of normal / ischemic zone (IZ / NZ). (U.S. Patent No. 5,244,460 incorporated herein by reference in its entirety). Alternatively, collateral blood flow can be quantified using radioactive microspheres using a reference flow technique (Dole et al., Am. J. Physiol.; 243: H371-H378). In another preferred embodiment of the present invention, the active agent is administered topically. The appropriate topical dose and the concentration of the active ingredient in the formulation are as described for subcutaneous administration. In a preferred additional embodiment of all aspects of the invention, the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEC ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO : 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 , SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEC ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO : 41y SEC ID NO: 42. In a further aspect, the present invention provides equipment for increasing blood flow in ischemic tissue, wherein the equipment comprises ^ s ^. G ^^ jH ^ gg ^,? I -? B? I - an effective amount of the active agent for increasing blood flow in the ischemic tissue and instructions for using the effective amount of the active agent as a therapeutic. In a preferred embodiment, the kit further comprises a pharmaceutically acceptable carrier, such as those adjuvants described above. In another preferred embodiment, the kit comprises a means for releasing the active agent in a patient. Such devices include, but are not limited to, injections, micellar or matrix solutions, dressings, wound preparations, aerosols, lipid foams, transdermal patches, topical administration agents, polyethylene glycol polymers, carboxymethyl cellulose preparations, crystalloid preparations (eg. say, saline solution, Ringer's lactate solution, stabilized phosphate saline solution, etc.), viscoelastic, polyethylene glycols and polypropylene glycols. The means for the release may contain the effective amount of the active agent or may be separated from the compounds, which are subsequently applied to the means for release at the time of use. The present invention can be better understood with reference to the accompanying examples which are for illustrative purposes only and should not limit the scope of the invention.

Example 1: Grafting of Live Skin Equivalent 20 Swiss male mice (22-24 g) were anesthetized with an intramuscular injection of Ketaset / Rompun and an incision was made in the skin with a total thickness of 1 cm x 1 cm in their dorsal surface. The live skin equivalent (LSE) was placed in the defect and trimmed with micro-scissors so that the graft will not be observed between the edges of the mouse skin and the LSE. Mice were divided into 6 groups based on the peptide tested: (0 [LRS-Dextrose] or 1.0 mg / ml All or analogues All, All (1-7) [SEQ ID NO: 4]; Ala4AIII [SEQ ID NO: 18]; Pro3AII [SEQ ID NO: 31] ] and NedAII [SEQ ID NO: 42], see Table 1) and the LSE was soaked for 15 minutes in Ringers solution lactated with 5% dextrose with or without peptides before placement. After the graft was placed, the dorsal surface of the mouse was covered with gauzes embedded in petrolatum followed by two adhesive bands. After recovery from anesthesia, the mice were returned to their 5 individual cages and observed daily until euthanasia was applied. The mice received intramuscular analgesia during the first three days after surgery. No mice lost their bandages before necropsy or the removal of the bandages on the 7th. Day. The mice were necropsied on the 7th day. At necropsy, the degree of graft taking and the appearance of the grafted tissue was observed before placement of the biopsy in 10% stabilized formalin in preparation for paraffin embedding and division for hematoxylin and staining with eosin. The number of endothelial cells and vascular channels were determined microscopically and the data are presented in Figures 1 and 2. These data demonstrate that All and the All analogue treated with LSE increased angiogenesis (formation of the vascular channel) and the endothelial cell production in the graft tissue compared to the untreated LSE.

Table 1. Designation for the Analogs / Fragments used in Examples 1-3 1GD Ala4-AII (1-7) DRVAIHP SEQ ID NO: 38 2GD Pro3-AII (1-7) DRPYIHP SEQ ID NO: 39 5GD Lys3-AII (1-7) DRKYIHP SEQ ID NO: 40 9GD: NorLeu3-AII (1-7) DR (nor) YIHYP SEQ ID NO: 41 All (1-7) DRVYIHP SEQ ID NO: 4 All DRVYIHPF SEQ ID NO: 1 Ala4-AIII RVAHPF SEQ ID NO: 18 Pro3-AII DRVYIHPF SEQ ID NO : 31 lled-AII DRVYIHPI SEQ ID NO: 42 Example 2: Thermal Lesion Model in Partial Thickness Animals Thirty-six Hartley male guinea pigs weighing approximately 500 5 grams were purchased at the Charles Rivers Laboratory. The guinea pigs were housed in a light cycle: darkness of 12:12 hours, the cycle in the Vivario USC. Water and food were available ad libitum.

Surgical Procedures 10 The guinea pigs were anesthetized by intramuscular injection of 14 mg / kg Rompun and 130 mg / kg Cetamine. Subsequently the hair was removed from the dorsal surface through shaving with an animal shearing machine after treatment with a thioglycollate depilatory. After hair removal, the area was betadine carved twice followed by 70% ethanol. Two burns are produced in each guinea pig with an 18 mm solid brass rod, which was heated in a water bath at 75 ° O One end of the brass rod was placed on the back of the guinea pig for 50 seconds . This procedure was repeated with different brass bars for each animal in each burn. Each burn was treated with CMC with a low viscosity of 10% with and without 1 mg / ml of All, analogue All or fragment (0 [LRS-Dextrose] or 1.0 mg / ml of All or analogues All, All (1-7) [SEQ ID NO: 4], Ala4AIII [SEQ ID NO: 18 ]; Pro3AII [SEQ ID NO: 31] or MedAII [SEQ ID NO: 42j) in stabilized phosphate 0.05 mol / L, pH 7.3 and individually bandaged with Hilltop Chamber and covered with Tegaderm. The bandages were checked and changed daily for the first 5 days and every day until the necropsy on the 7th. days after the injury. The guinea pigs were given 20 μg / kg of bupronex (buprenorphine hydrochloride) intramuscularly for pain on the day of the injury and the first 3 days after the injury. * Validation with Chinese Ink Injection 5 Initial studies were carried out, in which the depth of the burn was evaluated with intra-aortic injection of Chinese ink. One or two days after the start of the thermal injury, the guinea pigs were anesthetized with Cetamine and broke intramuscularly. The ascending thoracic aorta was cannulated and 60 ml of Chinese ink was injected through the cannula under 500 mm Hg. This procedure was used to ensure the filling of the obvious vessels. After the Chinese Ink was injected, the animals were sacrificed and the sites of injury were cut and placed in 10% stabilized formalin and prepared for histological evaluation. The histological sections of these preparations were examined to evaluate the depth of the necrosis and capillary ischemia. 15 Histological Evaluation In the 7th. day after the thermal injury, the guinea pigs were euthanized, the burned areas were cut en bloc and the tissues were placed in 10% stabilized formaldehyde solution overnight. The tissues are placed in paraffin and sections of 5 μm were prepared. Sections were processed for immunohistochemical analysis with a major antibody to the ciclin (MIB-1) (AMAC, Westbrook, MA) followed by recognition of the primary antibody with a DAKO equipment (DAKO, Santa Barbara, CA) used as described below . Sections placed in paraffin were baked overnight at 60 ° W Deparaffinization was performed by four 5-minute incubations in "fresh" xylene followed by two 5-minute incubations with 100% ethanol, two incubations . íí ^^^^ g? ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ minutes with 95% ethanol and a five minute incubation with H2O. The endogenous peroxidase activity was subsequently tempered with 0.3% H2O2 in H2O. The recovery of the antigen was carried out following the protocol of Shi et al. with minor modifications. In summary, the plates were placed in plastic containers 5 coplin in 5% urea in Tris stabilizer 0.1 mol / L (pH 9.0). Subsequently the plates were placed in a microwave oven and placed at maximum temperature for 3 minutes. The antigen recovery solution was refilled with distilled water when the evaporation was excessive and was put in the microwave again for 5 minutes at medium power (50%). Subsequently, the plates were allowed to cool for 10 minutes and were placed in phosphate stabilized saline (PBS) for 5 minutes. Immunohistochemical staining was performed with the conjugate method of proxidase-blotin-avidin with some modifications. The plates were placed in a PBS bath for 5 minutes and then rested in humidified incubation chambers. Blocking was performed for the non-specific binding of the antibody by incubation of the sections in 5% horse serum in PBS. The solution was decanted and replaced with the main antibody solution. The main antibody, MIB-1, was used at a 1: 100 dilution in PBS and incubated for 60 minutes at room temperature. The platens were washed with PBS for 5 minutes and subsequently incubated with the biotinylated horse anti-mouse secondary antibody. After a wash of 5 minutes in PBS the biotin-avidin complex was applied to the platens at a dilution of 1: 100 in PBS at room temperature and incubated for 1 hour. After a final rinse in PBS, the sections were incubated in 0.06% 3,3'-diaminobenzidine in PBS with 0.03% hydrogen peroxide for 5 minutes. After a counter-stain was performed on Harris eosin-hematoxylin After modification, the sections were dehydrated and covered by sliding with Permount.

With an Olympus AH-2 Vanox-S dissection microscope and a magnification power of 100x, each section of the biopsy specimen was separated into its areas at the burned edge or the actual burned areas. The area of the burn was cut and serially divided into three and five sections in a thickness of 4 to 5 mm. The total area of the burn and the edge of the burn was placed and examined histologically. In each section, four to six consecutive average power fields (mpf, 100x) were evaluated. The cells that were stained with the MIB-1 antibody were of a different brown color. All stained cells located in the hair follicles of the biopsy sections were counted. To count the cells stained with MIB-1, each section on the stage was separated into fields of individual average power (mpf). Subsequently, each field was determined in a section on the edge of the burn or a part of the area of the burn itself. An edge was indicated by a positive spotting showing brown epithelial cells along the edge of the section. An area burned was indicated by the absence of stained coffee cells along the edge. The brown cells located in each hair follicle were counted once under mpf amplification. A mark was established to move to the next mpf and the stage subsequently moved to the next adjacent field. All animals survived the burn procedures and no evidence of significant discomfort was shown throughout the study interval. The depth of the burns was determined by the partial thickness of the depth to the total thickness by (1) the analysis of evidence in the vessels with intra-aortic injection of Chinese ink and (2) the appearance of the cells in the hair follicles by the microscopic analysis of sections stained with hematoxylin-eosin. This analysis revealed that most of the pre-existing blood vessels and cells were destroyed at the site of the burn, although the lesions did not spread through the panniculus.

Figure 3 demonstrates the effect of All and the same analogues All used in Example 1 on angiogenesis after thermal injury. These data demonstrate that the treatment or burn areas with the All and the All analogs produces an increased blood flow response compared to All. Example 3. Effect of Angiotensin Peptides on the Formation of Collateral Circulation in Ischemic Myocardial Tissue The mice underwent a surgical procedure under intramuscular anesthesia (Cetamine and Rompum) after shaving with the animal shearing machine 10 and preparation with betadine and isopropyl alcohol. A median sternotomy was performed. After the exposure of the pericardial sac, a third incision was made in the pericardium. After visualization of the epicardial surface, two left coronary arteries, the left circumflex and the left anterior descending arteries were exposed and ligated by 4-0 Vicry suture. Vehicle 15 (10% hydron, 60% ethanol and 1% polyethylene glycol) was injected with or without peptide (1 mg / ml, 0.05 ml) into the distal of the cardiac muscle at the site of coronary occlusion. The sternum was subsequently closed with Silk 2-0. The muscle and skin were subsequently closed with 3-0 Dexon suture. Starting 7 days after the surgery, the animals were euthanized and the necropsies were performed. The number of blood vessels present at the infarct site 20 was determined totally and by microscopic evaluation. The presence of a blood vessel was determined to be defined as a channel aligned with the endothelial cells that contained red blood cells (indicating that the vessels have a blood source). All the peptides (All, AII (1-7), 1GD, 2GD, 5GD and 9GD: see table 2) tested increase the vascularization of the infarct site. The increases ranged from approximately 4 times to approximately 14 times, increase in the formation of * í ** ¿í £ &* *, ^^ ^ lgi ^^. *. < ** & amp; amp; amp; & - .. .. ^ *. . .u * .¿ **? a .. collateral circulation after ligating the artery. The data collected from the microscopic examination of the multiple tissue sections are in Table 2.

Table 2. Effect of Angiotensin Peptides in the Formation of Collateral Circulation in Ischemic Myocardial Tissue The methods and equipment of the present invention by increasing blood flow in the ischemic tissue, significantly improve the usefulness of the currently available treatments for ischemia and particularly benefit the treatments for the skin and cardiac ischemia and significantly benefit the tissue by increasing blood flow in tissue grafts. It is understood that the invention is not limited to the exact details of operation or to the exact compounds, compositions, methods, procedures or modalities shown and described, as obvious and equivalent modifications that will be apparent to those skilled in the art and the invention is therefore limited only by the full scope of the claims that accompany it.

J -... AMÍ- »- ^ £ g¡i asm ^ ÉM ^ o ^ | it i? ^ |? LIST OF SEQUENCES < 110 > Rodgers, Kathleen diZerega, Gere < 120 > Methods to Increase Blood Flow in Ischemic Tissue < 130 > 98364B < 140 > To be assigned < 141 > 1999-08-13 < 160 > 42 < 170 > Patent See 20 < 210 > 1 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption All < 400 > 1 Asp Arg Val Tyr He His Pro Phe 1 5 < 210 > 2 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence All (2-8) < 400 > 2 Arg Val Tyr lie His Pro Phe 1 5 < 210 > 3 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 220 > Description of the Artificial Sequence: All (3-8) < 400 > 3 Val Tyr lie His Pro Phe 1 5 < 210 > 4 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: All (1-7) < 400 > 4 Asp Arg Val Tyr lie His Pro fifteen < 210 > 5 < 21 1 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: All (2-7) < 400 > 5 Arg Val Tyr He His Pro 1 5 < 210 > 6 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: There (3-7) < 400 > 6 Val Tyr He His Pro 1 5 < 210 > 7 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: All (5-8) < 400 > 7 He His Pro Phe 1 < 210 > 8 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence. All (1-6) < 400 > 8 Asp Arg Val Tyr He His 1 5 < 210 > 9 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: All (1-5) < 400 > 9 Asp Arg Val Tyr lie 1 5 < 210 > 10 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > * < 223 > Description of the Artificial Sequence: All (1-4) < 210 > 11 < 211 > 3 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: All (1-3) < 400 > 11 Asp Arg Val 1 < 210 > 12 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Analog All < 220 > < 221 > MOD_RES < 222 > (2) < 223 > NLE < 400 > 12 Arg Xaa Tyr He His Pro Phe fifteen < 210 > 13 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Analog Artificial Sequence All < 220 > < 221 > MOD_RES < 222 > (4) < 223 > NLE < 400 > 13 Arg Val Tyr Xaa His Pro Phe 1 5 < 210 > 14 < 211 > 3 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence All (6-8) < 400 > 14 His Pro Phe 1 < 210 > 15 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence All (4-8) < 400 > 15 Tyr He His Pro Phe 1 5 < 210 > 16 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > . ., .. p. * s? * < j * ¿*. < 223 > Description of the Artificial Sequence- Analog Class All < 220 > < 221 > UNCERTAIN < 222 > (1) < 223 > Xaa in position 1 can be Arg, Lys, Ala, Orn, Ser, MeGly, D-Arg or D-Lys < 220 > < 221 > UNCERTAIN < 222 > (2) < 223 > Xaa in position 2 can be Val, Ala, Leu, NIe, He, Gly, Pro, Aib, Acp or Tyr. < 220 > < 221 > UNCERTAIN < 222 > (4) < 223 > Xaa in position 4 can be lie, Ala, Leu, NIe, Val or Gly. < 400 > 16 Xaa Xaa Tyr Xaa His Pro Phe 1 5 < 210 > 17 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Analog All < 400 > 17 Arg Val Tyr Gly His Pro Phe 1 5 < 210 > 18 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Analog All < 400 > 18 Arg Val Tyr Ala His Pro Phe 1 5 < 210 > 19 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 1 All < 400 > 19 Asp Arg Val Tyr Val His Pro Phe 1 5 < 210 > 20 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 2 All < 400 > 20 Asn Arg Val Tyr Val His Pro Phe 1 5 < 210 > 21 < 211 > 11 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 3 All < 400 > 21 Wing Pro Gly Asp Arg He Tyr Val His Pro Phe 1 5 10 < 210 > 22 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 4 All < 400 > 22 Glu Arg Val Tyr He His Pro Phe 1 5 < 210 > 23 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 5 All < 400 > 23 Asp Lys Val Tyr He His Pro Phe 1 5 < 210 > 24 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analogous 6 All < 400 > 24 Asp Arg Ala Tyr He His Pro Phe 1 5 < 210 > 25 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analogous 7 All < 400 > 25 ^^^^^^^^^^^^^ ^^^^^^^^^^^^^ Asp Arg Val Thr He His Pro Phe fifteen < 210 > 26 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 8 All < 400 > 26 Asp Arg Val Tyr Leu His Pro Phe 1 5 < 210 > 27 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 9 All < 400 > 27 Asp Arg Val Tyr He Arg Pro Phe fifteen < 210 > 28 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 10 All < 400 > 28 Asp Arg Val Tyr He His Wing Phe fifteen < 210 > 29 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analogous 11 A, jfgf < 400 > 29 Asp Arg Val Tyr He His Pro Tyr fifteen < 210 > 30 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analogous 12 All < 400 > 30 Pro Arg Val Tyr lie His Pro Phe fifteen < 210 > 31 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 13 All < 400 > 31 Asp Arg Pro Tyr He His Pro Phe fifteen < 210 > 32 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analogous 14 All < 220 > < 221 > MOD_RES < 222 > (4) < 223 > PHOSPHORYLATION < 400 > 32 Asp Arg Val Tyr He His Pro Phe 1 5 < 210 > 33 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analogous 15 All < 220 > < 221 > MOD_RES < 222 > (3) < 223 > NIe < 400 > 33 Asp Arg Xaa Tyr He His Pro Phe 1 5 < 210 > 34 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 16 All < 220 > < 221 > MOD_RES < 222 > (5) < 223 > NIe < 400 > 34 Asp Arg Val Tyr Xaa His Pro Phe 1 5 - '- - • * • * - * - ^ A ^ ^ £ ^^^^. ^ Aa ^ .g ^^. What ... .. ^ < 210 > 35 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: analog 17 All < 220 > < 221 > MOD_RES < 222 > (4) < 223 > homo Being < 400 > 35 Asp Arg Val Ser Tyr He His Pro Phe 1 5 < 210 > 36 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: p-aminophenylalanine 6 All < 221 > MOD_RES < 222 > (6) < 223 > p-aminophenylalanine < 400 > 36 Asp Arg Val Tyr He Xaa Pro Phe 1 5 < 210 > 37 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: angiotensin I ? f? -? ti? Hrfrr '-i - - - - - • tttr-tnv'ínr r i n. ¡R, ^^^ ^ b ^^^ l? ¿¿¿¿? ¿? ¿? ¿? 400 > 37 Asp Arg Val Tyr He His Pro Phe His Leu 1 5 10 < 210 > 38 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: 1 GD: Ala4-AII (1-7) < 400 > 38 Asp Arg Val Ala He His Pro. fifteen < 210 > 39 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: 2GD: Pro3-AII (1-7) < 400 > 39 Asp Arg Pro Tyr He His Pro. fifteen < 210 > 40 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: 5GD: Lys3-AII (1-7) < 400 > 40 Asp Arg Lys Tyr He His Pro. fifteen «G-. -iMt-a .. "j? 8 &<210> 41 <211> 7 <212> PRT <213> Artificial Sequence < 220 > < 221 > MOD_RES < 222 > (3) < 223 > NIe < 220 > < 223 > Description of the Artificial Sequence: 9GD: norLeu3-AII (1-7) < 400 > 41 Asp Arg Xaa Tyr He His Pro .1 5 < 210 > 42 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Ile8-All < 400 > 42 Asp Arg Val Tyr He His Pro Phe .1 5 ^^^ ¡^^^^^^^^^^^^^^^^^^ 2 ^^^^^^^^^ 2 ^^

Claims (15)

1. A method for increasing blood flow in ischemic tissue comprising administering an effective amount to increase blood flow in the ischemic tissue of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of the groups R1-R8 in the sequence of the general formula I R1-R2-R3-R4-R5-R6-R7-R8 in which R1 and R2 together form a group of formula X-RA-RB-, wherein X is H or one of the three peptide groups, or is absent, RA is appropriately selected from H, Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc, 15 RB is appropriately selected from Arg, Lys, Ala, Orn, Citron, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Lie, Gly, Pro, Aib, Acpc and Tyr; R4 is selected from the group consisting of Tyr, Tyr (PO3) 2, Thr, Ser, Ala, 20 Ser, homoSer and azaTyr; R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and R8 is selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding sequences that include R4 as a terminal group Tyr, or j ^^ jls ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^ S¡t & absent, and where the active agent is not All.

2. The method according to claim 1, wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO: 2, SEQ ID NO:

3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, 10 SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEC ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO : 41y SEC ID NO: 423. The method according to claim 1, wherein the ischemic tissue 15 is selected from the skin, the heart and the tissue grafts.

4. The method according to claim 1, further comprising administering an amount effective to increase blood flow in the ischemic tissue of at least one compound selected from the group consisting of All, factor 20 of the growth of the basic fibroblast, factor of the acid fibroblast growth, alpha and beta growth transformation factor, tumor necrosis factor, platelet-derived growth factor, vascular endothelial growth factor, angiogenin and haptoglobin

5. A device for increasing blood flow in ischemic tissue comprising: *** & áß & - 2. * $ **, 2 .... ^ < . . "> ^. ^^ asafc. 2 '.-.. ^" - • ^ t-2? l '& . (a) an amount effective to increase blood flow in the ischemic tissue of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of the groups R1-R8 in the sequence of the general formula I 5 R1- R -R3-R4-R5-R7-R7-Rβ in which R1 and R2 together form a group of formula X-RA-RB-, wherein X is H or one of the three peptide groups, or is absent , RA is appropriately selected from H, Asp, Glu, Asn, Acpc (1- 10-aminocyclopentane carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc, RB is appropriately selected from Arg, Lys, Ala, Orn, Citron, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Lie, Gly, Pro, Aib, Acpc and Tyr; R4 is selected from the group consisting of Tyr, Tyr (PO3) 2, Thr, Ser, Ala, Ser, homoSer and azaTyr; R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R6 is His, Arg or 6-NH2-Phe; R7 is Pro or Ala and R8 is selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding the sequences that include R4 as a terminal group Tyr, or is absent, wherein the active agent is not All and (b) instructions for using the effective amount of the active agent to increase blood flow in the ischemic tissue. Métóí

6. The kit according to claim 5, wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO. : 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16 , SEC ID 5 NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO. : 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 , SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42

7. The equipment according to claim 5, further comprising means for the release of the active agent.

8. The kit according to claim 5, further comprising an amount effective to increase blood flow in the ischemic tissue of at least one compound selected from the group consisting of All, basic fibroblast growth factor, fibroblast growth factor acid, alpha and beta growth transformation factor, tumor necrosis factor, platelet-derived growth factor, vascular endothelial growth factor, angiogenin and haptoglobin.

9. A method for increasing blood flow in ischemic tissue comprising administering an effective amount to increase blood flow in the ischemic tissue of at least one active agent comprising a selected sequence of the general formula: R1- Arg-R2-R3-R4-His-Pro-R5, wherein R1 is Asp or is absent; R2 is selected from the group consisting of Val, Ala, Lie, Pro, Lys, NorLeu and Leu; R3 is selected from the group consisting of Ala, Tyr and Tyr (PO3) 2 and R4 is selected from the group consisting of Val, Ala, Lie, Norleu and Leu; R5 is Phe, lie or is absent and where the compound is not All.

10. The method according to claim 9, wherein the active agent is selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 19; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

11. The method according to claim 9, further comprising administering an amount effective to increase blood flow in the ischemic tissue of at least one compound selected from the group consisting of All, growth factor of the basic fibroblast, growth factor of the acid fibroblast, alpha and beta growth transformation factor, tumor necrosis factor, platelet-derived growth factor, vascular endothelial growth factor, angiogenin and haptoglobin.

12. A device for increasing blood flow in ischemic tissue comprising: (a) an amount effective to increase blood flow in the ischemic tissue of at least one active agent comprising a selected sequence of the general formula: R1-Arg-R2 -R3-R4-His-Pro-R5, wherein R1 is Asp or absent; R2 is selected from the group consisting of Val, Ala, He, Pro, Lys, NorLeu and Leu; R3 is selected from the group consisting of Ala, Tyr and Tyr (PO3) 2 and R4 is selected from the group consisting of Val, Ala, Lie, Norleu and Leu; R5 is Phe, lie or absent where the compound is not All and (b) instructions for using the effective amount of the active agent to increase blood flow in the ischemic tissue.

13. The kit according to claim 12, wherein the active agent is selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 19; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

14. The equipment according to claim 12, further comprising means for the release of the active agent.

15. The kit according to claim 12, further comprising an amount effective to increase blood flow in the ischemic tissue of at least one compound selected from the group consisting of All, basic fibroblast growth factor, acid fibroblast growth factor , alpha and beta growth transformation factor, tumor necrosis factor, platelet-derived growth factor, vascular endothelial growth factor, angiogenin and haptoglobin. ^^^ is? * í '-, ja.