MXPA99005562A - Use of peptides for improving the incorporation of skin grafts - Google Patents

Abstract

Peptide compounds are used in the preparation of medicaments for promoting incorporation of a skin graft into underlying tissue. Compositions useful in connection with the invented use include:angiotensin II (AII), AII analogs, AII fragments and analogs thereof, angiotensinogen and analogs thereof, and angiotensinogen fragments and analogs thereof.

Description

USE OF PEPTIDES TO IMPROVE THE INCORPORATION OF LEATHER GRAFTS FIELD OF THE INVENTION This invention relates in general to the fields of biochemistry and medicine. More particularly, the present invention relates to compositions and methods for use in accelerating the growth or healing of skin grafts. • BACKGROUND OF THE INVENTION Wounds (ie, lacerations or openings) in mammalian tissue result in tissue rupture and coagulation of the microvasculature in the wound face. The healing of said tissue represents a cellular response controlled, ordered, to the injury. All soft tissue wounds, regardless of P size, heal in a similar way. Tissue growth and healing are biological systems in which cell proliferation and angiogenesis occur in the presence of an oxygen gradient. The sequential morphological and structural changes that occur during tissue healing have been characterized in great detail and in some cases have been quantified (Hunt et al., "Coagulation and macrophage stimulation of angiogenesis and wound healing," in The Surgical Wound, pp. 1-18, ed. F. Dineen &G. Hildrick-Smith (Lea &Febiger, Philadelphia: 1981)).

The cell morphology consists of three distinct zones. The central avascular space of the wound is deficient in oxygen, acidotic and hypercarbic, and has high lactate levels. Adjacent to the wound space is a gradient zone of local anemia (ischemia) that is populated by dividing fibroblasts. Behind the leader zone is an area of active collagen synthesis characterized by mature fibroblasts and numerous newly formed capillaries (ie, neovascularization). While this growth of new blood vessels (angiogenesis) is necessary for the healing of wounded tissue, angiogenic agents are generally unable to satisfy the much-felt need to provide the additional biosynthetic effects of tissue healing. Despite the need for faster healing of wounds (ie, severe burns, surgical procedures, lacerations and other injuries), to date there has been only limited success in accelerating wound healing with pharmacological agents. U.S. Patent No. 5,015,629 to DiZeraga (the entire disclosure of which is incorporated herein by reference) discloses a method for increasing the healing rate of wounded tissue, which consists in the application to said tissue of angiotensin II ( All) in an amount that is sufficient for said increase. The application of angiotensin II to wounded tissue significantly increases the rate of wound healing, leading to faster re-epithelialization and tissue healing. The term "angiotensin II" refers to an octapeptide present in humans and other species having the Asp-Arg-Val-Tyr-lle-His-Pro-Phe sequence (Id. Of Sec. No.:1). Angiotensin II is a known pressor agent and is commercially available. Despite the utility of angiotensin II for accelerating wound healing, there is a need for additional agents that are useful in promoting wound healing. Moreover, it would be very advantageous to use an agent that is less potent than angiotensin II for inducing hypertension. A selective agonist peptide for the AT2 receptor (the peptide has 100 times higher affinity for AT2 than for AT1) has been identified. This peptide, which is called p-aminophenylalanine6-AII or "p-NH-Phe6-AH", has the sequence Asp-Arg-Val-Tyr-lle-Xaa-Pro-Phe (Seq. ID No. 2) wherein Xaa is p-NH2-Phe (Speth et al., Biochem Biophys., Res. Commun. 169: 997 (1990)). Discrimination of two angiotensin II receptor subtypes with an angiotensin II selective agonist analogue, p-aminophenylalanine6 angiotensin II. Biochem Biophys Res Commun 169: 997). This peptide gave comparable binding characteristics to the AT2 agonists in the experimental models tested (Catalioto et al, Eur J Pharmacol 256: 93 (1994), Bryson et al., EurJ Pharmacol 225: 119 (1992)). invention provide compositions and methods of use that do not suffer from the disadvantages of the compositions known to date.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the invention relates to a method for promoting the incorporation of a skin graft into the underlying tissue of a mammal. This method includes the steps of: (1) applying to any of the skin graft or underlying tissue an effective amount of graft incorporation promotion of a composition including angiotensin II and a pharmaceutically acceptable carrier; (2) contact the skin graft and the underlying tissue; and (3) securing the skin graft to the underlying tissue, thereby promoting the incorporation of said skin graft into the underlying tissue. According to one modality, the skin graft is an autologous graft. According to a preferred embodiment of the invention, the pharmaceutically acceptable carrier includes a pH regulated saline solution. When the carrier includes saline, the composition including angiotensin II can be applied in the application step by soaking the skin graft. Alternatively, the pharmaceutically acceptable carrier can include carboxymethyl cellulose. When the composition includes carboxymethyl cellulose and angiotensin II, it can be applied to the underlying tissue. According to another preferred embodiment, the composition including the regulated salt solution of pH and angiotensin II can be applied to the underlying tissue. According to still other preferred embodiments, the fixation step can be achieved by saturation, bandaging or by applying a biological glue. Another aspect of the invention relates to a method for promoting the incorporation of a skin graft into the underlying tissue of a mammal.

This method includes the steps of: (1) applying to either the skin graft or the underlying tissue an effective promoter amount of incorporation of a composition that includes a pharmaceutically acceptable carrier and a peptide consisting of at least three contiguous amino acids of groups R1 -R8 in the general formula R1 - R2 - R3 - R4 - R5 - R6 - R7 - R8 In which R and R2 form a group of the formula Where X is H or a peptide group of one to three and a peptide bond between RA and RB is unstable to aminopeptidase A cut; 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 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 Tyr group; (2) contact the skin graft and the underlying tissue; and (3) fix the skin graft to the underlying tissue, thereby promoting the incorporation of the skin graft into the underlying tissue. According to one modality the graft is an autologous graft. According to a preferred embodiment of the invention, the pharmaceutically acceptable carrier includes a pH regulated saline solution. When the vehicle includes pH regulated salt, the composition including the peptide can be applied in the application step by soaking the skin graft. Alternatively, the pharmaceutically acceptable carrier can include carboxymethyl cellulose. When the composition contains carboxymethyl cellulose and the peptide, it can be applied to the underlying tissue. According to another preferred embodiment, the composition including the pH regulated salt solution and the peptide can be applied to the underlying tissue. According to still other preferred embodiments, the fixation step can be achieved by saturation, bandaging or by applying a biological glue. Yet another aspect of the invention relates to a method for promoting the incorporation of a skin graft into the underlying tissue of a mammal. This method includes the steps of: (1) applying to either the skin graft or the underlying tissue an effective promoter amount of incorporation of a composition that includes a pharmaceutically acceptable carrier and a peptide having the general formula R2-R3-R4- R5 - R6 - R7 - R8 Where R2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, 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, 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 lie, Phe, Phe (br) and Tyr; (2) contact the skin graft and the underlying tissue; and (3) fix the skin graft to the underlying tissue, thereby promoting the incorporation of the skin graft into the underlying tissue. According to one modality the graft is an autologous graft. According to a preferred embodiment of the invention, the pharmaceutically acceptable carrier includes a pH regulated saline solution. When the vehicle includes pH regulated salt, the composition including the peptide can be applied in the application step by soaking the skin graft. Alternatively, the pharmaceutically acceptable carrier may include carboxymethyl cellulose. When the composition contains carboxymethyl cellulose and the peptide, it can be applied to the underlying tissue. According to another preferred embodiment, the composition including the regulated pH saline and the peptide can be applied to the underlying tissue. According to still other preferred embodiments, the fixation step can be achieved by saturation, bandaging or by applying a biological glue. .

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood with reference to the appended drawings, in which: Figure 1 illustrates the response percentage of the control in response to closed wound in relation to a control treated by vehicle using analogs 1 and 4. Figure 2 illustrates the response percentage of the control in response to closed wound in relation to a control treated by vehicle using analogs 2 and 3 in two different dosages. Figure 3 illustrates the percentage response of the control in response to closed wound in relation to a control treated by vehicle using analog 2. Figure 4 illustrates the percentage of response of the control in response to closed wound in relation to a treated control by vehicle using analogues 5-8. Figure 5 illustrates the response percentage of the control in response to wound closure in relation to a control treated by vehicle using analogues 9-12. Figure 6 illustrates the percentage of control response in granulation tissue formation using analogs 6-8. Figure 7 illustrates the percentage of control response in granulation tissue formation using 9-1 1 analogues.

Figure 8 illustrates the increase in percent wound closure in relation to a control treated by vehicle using There. Figure 9 illustrates the percentage response of the control in response to closed wound in relation to a control treated by vehicle using various analogues there. Figure 10 illustrates the response percentage of the control in response to wound closure in relation to a control treated by vehicle using various analogues there. Figure 11 illustrates the percent response of the control in the formation of granulation tissue using various analogues there. Figure 12 illustrates the percent response of control in the formation of granulation tissue using analogs there. Figure 13 illustrates the response percentage of the control in response to closed wound in relation to a control treated by vehicle using various All fragments. Figure 14 illustrates the percent response of the control in response to wound closure in relation to a control treated by vehicle using various All fragments. Figure 15 illustrates the formation of granulation tissue in control wounds of animals treated with various All fragments. Figure 16 illustrates the formation of granulation tissue in control wounds of animals treated with various All fragments. Figure 17 illustrates the formation of granulation tissue in animals treated with various All fragments.

Figure 18 illustrates the formation of granulation tissue in animals treated with various All fragments. Figure 19 illustrates the formation of granulation tissue in control wounds of animals treated with various All fragments. Figure 20 illustrates the formation of granulation tissue in animals treated with various All fragments. Figure 21 illustrates the percent response of the control in response to wound closure in relation to a control treated by vehicle using various All fragments. Figure 22 illustrates the percent response of the control in response to closed wound in relation to a control treated by vehicle using (p-NH2-Phe) 6-AII. Figure 23 illustrates the formation of granulation tissue in wounds treated by vehicle, or All, or p-NH2-Phe6-AII. Figure 24 illustrates the effect of an All-containing medicament on graft taking as measured by% crunching of the graft or necrosis several times after injury in a random flap model. Figure 25 illustrates the effect of an All-containing medicament on grafting as measured by% crunching of the graft or necrosis several times after injury in a random flap model.

Figure 26 illustrates the effect of various All concentrations on the percentage of fully viable flaps at 8 and 12 days after the injury. Figure 27 illustrates the effect of various All concentrations on the percentage of viability of flaps at 8 and 12 days after the injury. Figure 28 illustrates the effect of All on the number of endothelial cells at the interface of the graft per high energy field at 7 and 9 days after the intervention. Figure 29 illustrates the effect of All on the number of vascular channels containing red blood cells at the end of the graft per high-energy field at 7 and 9 days after the intervention. Figure 30 illustrates how the number of endothelial cells in the graft interface depended on the concentration of All in the soaking solution. Figure 31 illustrates how the number of vascular channels in the graft interface depended on the concentration of All in the soaking solution.

DETAILED DESCRIPTION OF THE PREFERRED MODALITY According to the present invention, the healing of skin grafts in mammals is promoted by the use of a composition consisting of an effective amount of at least one AT2 agonist, angiotensin I (Al) or analogs thereof, angiotensinogen or the like of the same, All, an analogue of active All, fragment of All or analog of the same. Additionally, precursors that are metabolized to these compounds are also contemplated to be used to promote the healing of skin grafts according to the present invention, and are therefore designed to fall within the scope of the invention. Agonists of the AT2 receptor subtype are beneficial in wound healing but do not exhibit many of the side effects of AI I, such as increased blood pressure and thirst. The active agent is generally administered in a matrical or miscellalar solution or cristloid composition and is effective to accelerate re-epithelialization and tissue healing even in very low concentrations. As defined hereinafter, a preferred class of AT2 agonists to be used in accordance with the present invention consists of Al or analogs thereof, angiotensinogen or analogs thereof, All, All analogs or active fragments thereof having p -NH2-Phe in a position corresponding to position 6 of All. In addition to the peptide agents, several non-peptidic agents (for example peptidomimetics) having the necessary AT2 agonist activity are additionally contemplated for use in accordance with the present invention. The active All analogs, All fragments and analogues thereof of particular interest according to the present invention are characterized by consisting of a sequence consisting of at least three contiguous amino acids of groups R1-R8 of the general formula R1-R2-R3 - R4 - R5 - R6 - R7 - R8 In which R1 and R2 form a group of the formula X - RA - RB - wherein X is H or a peptide group of one to three and a peptide bond between RA and RB is unstable to aminopeptidase A cut; 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 and azaTyr; R5 is selected from the group consisting of He, 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 Tyr group. Compounds falling within the category of AT2 agonists useful in the practice of the invention include the All analogs set forth above subject to the restriction that R6 is p-NH2-Phe. In a preferred embodiment, RA is suitably selected from Asp, Glu, Asn, Acpc, (1-aminocyclopentane) carboxylic acid, Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc. RB is suitably selected from Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg, and D-Lys. Particularly preferred combinations for RA and RB are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys. Particularly preferred embodiments of this class include the following: All, There or AII (2-8), Arg-Val-Tyr-lle_His-Pro-Phe (Seq. ID No. 3); AII (3-8), also known as desl-AIII or AlV, Val-Tyr-llr-His-Pro-Phe (Seq. ID No. 4); All (1-7), Asp-Arg-Val-Tyr-lle-His-Pro (Seq. ID No. 5); All (2-), Arg-Val-Tyr-lle-His-Pro- (Seq. ID No. 6); AII83-7), Val-Tyr-lle-His-Pro- (Seq. ID No. 7); AII (5-8), lle-His-Pro-Phe (Seq. ID No.8); AII (1-6), Asp-Arg-Val-Tyr-lle-His (Seq. ID Sec .: 9); AII (1-5), Asp-Arg-Val-Tyr-lle (Seq. ID No. 10); All (1-4), Asp-Arg-Val-Tyr (Seq. ID No. 11); and All (1-3); Asp-Arg-Val (Seq. ID No. 12). Other preferred embodiments include: Arg-norLeu-Tyr-lle-His-Pro-Phe (Seq. ID No .: 13) and Arg-Val-Tyr-norLeu-His-Pro-Phe (Seq. Id. .: 14). Yet another preferred embodiment included within the scope of the invention is a peptide having the Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe sequence (Id. Sec. No .: 32). AII (6-8), His-Pro-Phe (Seq. ID No. 15) and AII (4-8), Tyr-lah-His-Pro-Phe (Seq. Id. No .: 16 ) were also tested and found to be ineffective. Another class of compounds of particular interest according to the present invention are those having the general formula II R2-R3-R4-R5-R6-R7 -R8 Where R2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc, and Tyr; R4 is selected from the group consisting of Tyr, Tyr (PO3) 2, Thr, Ser, homoSer, and azaTyr; R5 is selected from the group consisting of He, 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 lie, Phe, Phe (br) and Tyr; A particularly preferred sub-class of the compounds of general formula II has the formula R2-R3-Tyr-R5-His-Pro-Phe (Seq. Id. No: 17) wherein R2, R3 and R5 are as defined above. Particularly preferred is angiotensin III (There) of the formula Arg-Val-Tyr-lle-His-Pro-Phe (Id. Sec. No: 3). Other preferred compounds include peptides having the structure Arg-Val-Tyr-Gly-His-Pro-Phe (Id. Of Sec. No: 18) and Arg-Val-Tyr-Ala-His-Pro-Phe (Id. Sec. No.19). The AII fragment (4-8) was ineffective in repeated tests; this is believed to be due to the tyrosine exposed on the N-terminus. In the above formulas, the standard three-letter abbreviations for the amino acid residues are used. In the absence of indication to the contrary, the L form of the amino acid is designed. Other wastes are abbreviated as follows: TABLE 1 Abbreviations for Amino Acids It has been suggested that All and its analogues take a turn either gamma or jía (Regoli, et al., Pharmacological Reviews 26:69 (1974)). In general, it is believed that the neutral side chains at positions R3, R5 and R7 can be involved in maintaining the proper distance between the active groups at positions R4, R6 and R8 responsible primarily for binding receptors and / or intrinsic activity. The hydrophobic side chains at positions R3, R5 and R8 can also play a very important role in the total conformation of the peptide and / or contribute to the formation of a hypothetical hydrophobic sac.

Suitable side chains on the amino acid at the R 2 position can contribute to the affinity of the compounds for target receptors and / or play a very important role in the formation of the peptide. For this reason, Arg and Lys are particularly preferred as R2. For objects of the present invention, it is believed that R3 may be involved in the formation of linear and non-linear hydrogen bonds with R5 (in the gamma gyro model) or R6 (in the beta gyro model). R3 could 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. Still, a single hydrogen bond may be sufficient to maintain a relatively stable conformation.Accordingly, R3 can be suitably selected from Val, Ala, Leu, He, Gly, Pro, Aib, Acpc and Tyr. , conformational analyzes have suggested that the side chain in this position (as well as in R3 and R5) contributes to a hydrophobic agglomeration that is believed to be essential for receptor occupancy and stimulation.Therefore, R4 is preferably selected from Tyr, Thr , Ser and Tyr In this position, Tyr is particularly referred to as being able to form a hydrogen bond with the receptor site capable of accepting hydrogen from the phenolic hydroxyl (R egoli et al., (1974), supra). In position R5, an amino acid with an aliphatic or alicyclic β chain is particularly desirable. Therefore, although Gly is suitable in the R5 position, it is preferred that the amino acid in this position be selected from lie, Ala, Leu and Val. In the All analogs, fragments and analogs of fragments of particular interest according to the present invention, R6 is His, Arg, or 6-NH2-Phe. The unique properties of histidine imidazole starch (eg, ionization at physiological pH, ability to act as a donor or proton acceptor, aromatic character) are believed to contribute to its particular utility as R6. For example, conformational models suggest that His can participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R7. Similarly, it is currently considered that R7 should be Pro in order to provide the most desirable orientation of R8. In the R8 position, a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding the analogs of interest to the receptors; therefore, Tyr and especially Phe are preferred for objects of the. present invention. Analogs of particular interest include the following: TABLE 2 Angiotensin II analogues Agiotensin II is one of the most potent vasoconstrictors known, which causes the constriction of small arteries that branch to form capillaries, for example arterioles. The biological formation of angiotensin is initiated by the action of renin on the angiotensinogen plasma substrate. The substance thus formed is a decapeptide called angiotensin I which is converted to angiotensin II by the angiotensin converting enzyme which removes the C-terminal His-Leu residues from angiotensin I. Studies have shown that the vasoactive product of the renin-angiotensin system, All increases the release of growth factors, mitogenesis, chemotaxis and the release of extracellular matrices of cultured cells that are involved in wound healing (Dzau et al., J. Mol.Cell Cardiol. 21 (Suppl. III): S7 (1989). ), Berk et al., Hypertension 13: 305 (1989), Kawahara et al., BBRC 150: 52 (1988), Naftilan et al., J. Clin. Invest. 83: 1419 (1989), Taubman et al., J. Biol. Chem. 264: 526 (1989), Nakahara and others BBRC 184: 811-8 (1992), Stouffer et al., Circ. Res. 70: 820 (1992), Wolf et al., Am. J. Pathol. (1992) Bel et al., Am. J. Pathol., 137: 7 (1990). In addition, All showed to be angiogenic in the rabbit eye cornea models and chicken chorioallantic mbrana (Fernández et al., J. Lab. Clin. Med. 105: 141 (1985); LeNoble et al., Eur. J. Pharmacol. 195: 305 (1991)). Therefore, All can accelerate wound healing through increased neovascularization, release factor growth, reepithelialization, and extracellular matrix production. Through an increase in the flow of blood and nutrients to an injured tissue, All can increase the speed of wound healing. All can also accelerate the healing of wounds through the generation of growth factors at the site of the injury. Exogenous addition of growth factors has been shown to accelerate wound healing through a variety of mechanisms (Grotendorst et al., J. Clin. Invest. 76: 2323 (1985); Mustoe et al., Science 237: 1333 (1987). ), Pierce et al., J. Med. Exp. 167: 974 (1988), J. Clin.Research 84: 640 (1989), Greehalg et al., Am. J. Phatol. 136: 1235 (1990)). Recent studies showed that All increased neointimal formation in the carotid artery and in the aorta after injury (Powell et al., Science, 245: 186 (1989), Powell et al., J. Cardiovasc. Pharmacol. (Suppl. 4): S42-9 (1991), Capron et al, J. Cardiovasc Pharmacol 18: 207 (1991), Osterriedes et al., Hypertension 18: Suppl II60-64 (1991), Daemen et al. Circ. Rev. 68: 450 ( 1991)). As a result of these observations, studies were conducted to determine the mechanism by which endogenous All can induce intimal hyperplasia. All showed to act as a mitogen for soft muscle cells, fibroblasts and endothelial cells (Schelling et al., J. Cell Physiol., 98: 503 (1979); Campbell-Boswell Exp. Mol. Phatol. 35: 265 (1981); Emmet. and others J. Cell, Biol. 103-171 (1986), Paquet et al., J. Hypertens., 8: 565 (1990), Dzau et al., supra.). All also increased the protein content and the size of soft muscle vascular cells (Berk et al., (1989), supra; Geisterfer et al., Cir. Res. 62: 749 (1988)). Studies showed that All increases the release of growth factors of various types, including PDGF, heparin-binding EGF and ß-transforming growth factor (TGFβ), and proto-oncogenes related to the growth of soft muscle cells, cells endothelial and cardiac fibroblasts (Kawahara et al., (1988), supra, Naftilan, AJJ Cardiovas, Pharmacol 20: S37 (1992), Naftilan et al., (1989), supra, Taubman et al., (1989), supra, Nakahara. and others, (1992), supra, Temizer et al., (1992), supra, Gibbons et al., J. Clin. Invest. 90: 456 (1992), Bell et al., J. Clin. Invest. 89: 315 ( 1992); Stouffer and others, (1992), supra. The hypertrophy of soft muscle vascular cells by All was mediated through PDGF (Berk et al., J. Cell, Physiol. 154: 368 (1993)). Therefore, it is conceivable that All acts to accelerate the healing of wounds by increasing the levels of those growth factors involved in the wounded tissue. Additionally, All was shown to stimulate collagen synthesis thus suggesting a role for this factor in the formation of the extracellular matrix (Wolf et al., Cell Reg.2: 219 (1991)).; Wolf et al., (1992), supra; Zhou and others. FASEB. J. 6: A1914 (1992)). Wound healing also involves the chemotaxis of the necessary cell types within the wound bed. All also showed that it induces the migration of endothelial cells and soft muscle cells in vitro (Bell et al., (1990), supra). Recent studies have also indicated that the expression of the All receptors is altered during the wound healing process (Viswanathan et al., Peptides 13: 783 (1992); Kimura et al., BBRC 187: 1083 (1992)). These changes, together with evidence of an increase in local production of All at the site of healing, suggest that All can play a key role in the wound healing procedure. All actions that may be involved in wound healing have been reviewed recently (Phillips and others, Angiotensin receptor stimulation of transforming growth factor-ß in mouse skin and wound healing.) In Angiotensin Receptors (ed JM Saavedra and PBMWM Timmermans), Plenum Press, New York, NY, pp 377-396 (1994)). In most of the studies reported, these effects have been shown to be mediated by the AT1 receptor. The effects of blood pressure (and most other effects, such as aldosterone secretion and increased thirst) of All are mediated by the type 1 receptor (AT1 receptor) (Wong, PC Angiotensin antagonist in models of hypertension.) In: Anqiotensin Receptors (JM Saavedra and PBMWM Timmermans), Plenum Press, New York, NY, pp 319-336 (1994), Mckenzie et al, J. Hypertension 12 (Suppl 9): S1 1-S16 (1994); Gupta et al., Hypertension 25: 443 (1995), Llorens-Cortes et al., Hypertension 24: 538 (1994), Wong et al., Eur. J. Pharmacol 220: 267 (1992)). This conclusion is based on blocking the action of All by receptor antagonists of specific subtype. The effects of All and All antagonists have been examined in two experimental models of vascular healing and injury. The studies have been mixed in relation to the contribution of AT2 to the hyperplasia of the vessels after injury by inflammation to the vasculature. In the rat carotid artery, the majority of the receptors are AT2 (Pratt et al., Hypertension 20: 432 (1992)). In contrast, neointimal cells of the injured thoracic aorta of rats express predominantly AT1 receptors. (Viswanathan et al., J. Clin. Invest. 90: 1707 (1992)). Treatment of rats with PD 123319 (AT2-specific antagonist) reduced intimal hyperplasia by 73% while lesions (AT1 specific antagonist) decreased the intimal area by 95% (Pratt et al. (1992), supra). In a similar model, CGP 42112 (an AT2 antagonist) instilled perivascularly for 14 days prevented neointimal formation, but low dose lesions were ineffective (Janiak et al., Hypertension 20: 737 (1992)). In other studies, patients at higher doses were found to be effective (Forney Prescott et al., Am. J. Phatol. 139: 1291 (1991), Kauffman et al., Life Sci 49: 223 (1991)): Therefore, it is It is conceivable that both subtypes of receptors may play a role in the formation of vascular lesions after global injury. During the experimental healing of wounds in young animals, the expression of the All receptors increases significantly in a localized band of tissue within the superficial dermis of the surrounding skin To the wound. Most of this increase is due to AT2 receptors (Viswanathan et al., Peptides 13: 783 (1992); Kimura et al., Biochem Biophys Res Commun 187: 1083 (1992)): These results, and the results described Then, they were obtained using procedures that used adult rats as experimental animals. The AT1 receptors were altered after of the formation of incisional wounds in adult rats. The experimental designs in these latter studies do not distinguish between the dermis and other portions of the wound. It has been observed that All and There have quite different biological activities in several aspects. For example, All showed a biphasic effect on evoked neuronal norepinephrine release (an initial decrease followed by a subsequent increase), while increasing the spontaneous release of norepinephrine after only 12 minutes; All showed a biphasic effect on both evoked and spontaneous neoronal norepinephrine release (Vatta et al., Can.J. Physiol.Pharmacol.70: 821 (1992)). Moreover, All and There show differential influences on the baroreceptor reflex: All improves reflex sensitivity, whereas It makes it worse (Brattstrom et al., Progress in Brain Research 91: 75 (1992)). Surprisingly, without considering these significant differences in biological activity between All and There, There and the particular analogs thereof are useful for accelerating wound healing. Many studies have focused on All (1-7) to evaluate its activity. Many of the effects of Al 1 (1 -7) are attributed to the action through the AT2 receptor. However, this is not consistent and depends on the tissue examined. At 1 (1 -7) it does not have many of the effects of All. At 1 (1 -7) it lacks activity pressor or has very mild effect (effective at 10,000-100,000 times the dose of All) on blood pressure depending on the model tested and the route of administration. In fact, AII (1-7) has a depressant effect on blood pressure that can be mediated through prostanoid synthesis. In addition, in contrast to the effects of All, Al 1 (1-7) does not cause release of catecholamine and aldosterone release and is not dipsogenic (Webb et al., Peptides 13: 499 (1992); Cheng et al. Am. Physiol. 266: H2247-H2255 (1994), Moriguchi et al., Am. J. Physiol. 267: R786-R791 (1994), Schiavone et al., J. Cardiovascular Pharmacol. 16 (Suppl 4): S19-S24 ( 1990), Ferrario et al, Hypertension 19 (Suppl Ill): lll-126-lll-133 (1991)). In one report, AII (1-7) is a weak pressor that requires almost 10,000 times more AII (1-7) than All to obtain a comparable response (Benter et al., Peptides 14: 679 (1993)). In this system, AII (1-7) had a long depressant effect that was dose dependent. All (3-7) had a pressor effect less than AII (1-7), but had no depressant effect. It is also noted that AII (1 -7), AII (2-7) and AII (3-7) can affect the dopamine system and memory (suggesting a psychoactive effect). In several systems, the actions of AII (1-7) are quite different from All. All stimulates the production of choline in rat mesangial cells through the AT1 receptor; AII (1-7) and AII (1-6) have very weak effects on this parameter (Pfeilschifter et al., Eur. J. Pharmacol. 225: 57 (1992)). In porcine aortic endothelial cells Al and AII (1-7) stimulated the release of prostaglandin E2 and 12 but All did not have this effect (JaiswaI et al., Hypertension 19 (Suppl ll): 1 1-49-11-55 (1992) ). All is able to stimulate the release of prostanoids in other types of cells and in intact blood vessels but not in human or porcine endothelial cells. The effect on endothelial cells was through a different receptor than AT1 and AT2. In preparations of rat glomeruli, All inhibited the formation of cAMP in response to histamine, serotonin and parathyroid hormone through the AT1 receptor (Edwards et al., J. Pharmacol.Experience Ther 266-506 (1993)). All (1-7) did not have this effect. In porcine vascular soft muscle cells and human astrocytes, All and AII (1-7) increase the release of prostaglandin; only angiotensin II increases the release of Ca2 + (JaiswaI et al., J. Pharmacol.

And Exp. Therapeutic 265: 664 (1993); (JaiswaI and others, Hypertension 17: 1 115 (1991)). At I (1-7) it dilates the rings of the porcine coronary arteries, perhaps through nitric oxide (Porsti et al., Br. J. Pharmacol. 111: 652 (1994)). This was not observed with All, There or All (3-8). This effect was not attenuated by AT1 or AT2 receptor antagonists. All causes depolarization of knotty ganglion isolated from rat; At 1 (1 -7) no. (Widdop et al., Clin.And Exper. Hyper-Theory and Practice A14: 597 (1992)). In fact, AII (1-7) may have new actions on brain function (Schiavone et al., J. Cardiovascular Pharmacol. 16 (Suppl 4): S19-S24 (1990)). There are activities that AII (1-7) shares with All, such as the release of vasopressin and the modulation of phospholipase A2 activity in nearby tubular cells (Andreatta-Van Leyen et al., Kidney International 44: 932 (1993); Moriguchi et al. , Am. J. Physiol. 267: R786-R791 (1994); Ferrario et al., Hypertension (Suppl. Ill.): Ill. 1266.lll.133 (1991)). These activities, however, are also not involved in wound healing. The effects of other All fragments have been studied in very few instances. Most neurons in the paraventricular nucleus are excited by AII (1-7), All and There, but AII (1-7) is weaker in this effect; in many neurons, AII (1-7) was inactive (Ambuhl et al., Regulatory Peptides 38: 1 11 (1992)). All injected into the lateral cerebral ventricle increased motility, stereotopy and learning of conditioned cancellation responses; AII (1-6) and AII (2-6) were not active in these psychotropic activities (Holy et al., Polish J. Pharmacol 45:31 (1993)). AII (4-8), AII (5-8) and AII (1-4) showed only a slight effect on water consumption when injected into the anterior diencephalon in the rat, and AII (1-7) was completely inactive . (Fitzsimmons J. Physiol. 214: 295 (1971)). The intracerebroventricular infusion of All (AII (4-8) and AII (5-8) fragments in the rat produced a minimal effect on blood pressure even when they occur at concentrations 1,000 times higher than that of All that increased blood pressure (Wright et al., Am. J. Physiol. 257: R1551 (1989)). In both of these studies, the fragments were injected directly into the brain; this is highly artificial and does not allow a systemic metabolism. According to the method of the invention, All, an active analog of All, All fragments or analogs thereof according to the present invention are applied to wound tissue consisting of a skin graft in sufficient amounts to increase the tissue healing speed. These compounds can significantly accelerate the healing rate at nanomolar levels in vivo. For any given active agent, the optimum concentration for a given formulation can quickly be determined empirically. In general, an amount of active agent suitable for use in accordance with the present invention is in the range of 0.001 μg to 10 mg per kilogram of body weight, or 1 ng to 100 μg / cm2 of wound area. The compounds of the invention can be applied in a variety of solutions. Suitable solutions for use in accordance with the present invention are sterile, dissolve sufficient amounts of the peptide, and are not harmful to the injured tissue. In this regard, the compounds of the 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.

Any type of application medium that allows the influx of active agents into the tissue for a period of time can be used. For example, an aqueous solution could be applied to a wounded tissue through a bandage or strip of gauze, or such a solution could be formulated so that a scheduled perfusion can be obtained (using, for example, liposomes, ointments, micelles, etc.). The methods for the production of these formulations with the compounds of the present invention are apparent to those of ordinary skill in the art. The particular concentration of active agent used is not critical, and the tissue repair effect is present even when the compounds are present in nanomolar quantities. Preferably, a matrical, micellar, or crystalloid solution is used with the active ingredient present at a concentration of at least 0.01 μg / ml. A particular matrical solution which has been used to advantage in the described examples is a semi-solid polyethylene glycol polymer sold under the trademark HYDRON by Hydro Med Sciences (New Brunswick, NJ). Another preferred solution is a micellar solution sold under the tradename PLURONICS F108 by BASF (Ludwingshafen, germany). Under ambient temperature conditions, this solution is a liquid, but when applied to warm tissue the solution forms a gel that allows the infusion of the active agent into the wounded tissue.During a period of several days. Other preferred formulations include carboxymethyl cellulose preparations (such as those used in the Examples herein), crystalloid preparations (eg, saline, Ringer's lactate solution, Ringer's lactate solution. With 5% dextrose, phosphate pH, etc.), viscoelastic, polyethylene glycols, polypropylene glycols, glues or tissue adhesives (such as fibrin glues, albumin glues, thrombogenic sealants or collagen) and wound coverings (eg, bandages, etc.). The healing effects of the components of the present invention can be provided in a variety of instances. The solution can be applied topically to the surface of wounded tissue in the treatment of ulcers, injuries, wounds, diabetic ulcers, burns, traumatisms, ulcers, stasis, periodontal conditions, lacerations and other conditions. In addition, an injured intraperitoneal tissue such as that resulting from invasive surgery can be treated with a composition according to the present invention to accelerate healing. For example, following the surgical removal of a section of the colon or other tissue, the surgical plane can be coated with an active agent solution before closing the surgical site in order to accelerate perfusion and healing of the internal capillaries. In addition, the localized healing rate can be increased by subdermal administration of active agent by injection or other means. The invention may be better understood with reference to the appended Examples, which are only intended for purposes of illustration and are not to be construed as limiting the scope of the invention, as defined in the appended claims. Several Examples presented below establish the general utility of the compositions described herein to accelerate wound healing. The subsequent Examples describe how the compositions described herein can be used to promote the healing of skin grafts. Example 1 describes the methods used to confirm that full-thickness wound healing was accelerated by the administration of a medicament consisting of All analogues.

EXAMPLE 1 THE ANGIOTENSIN II ANALOGS ACCELERATE THE SCREENING OF WOUNDS OF COMPLETE THICKNESS.

Male twelve-week-old Sprague Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). On the day of surgery, the rats received intramuscular ketamine / anesthesia rompum before preparation for surgery. The rats were shaved and carved with betadine. Four dermal wounds of total thickness of 2 x 2 cm were created on the dorsal surface of each rat. Following the excision of the skin, the size of the wound was defined on a glass slide to establish the baseline wound size. The drug was administered in 100 μl of a solution consisting of 10% HYDRON, 1% polyethylene glycol (PM400) and 60% ethanol. The test materials were administered in a random manner. All materials were tested at 3 μg / wound, with analogues 2 and 3 being also evaluated at 10 μg / wound. Control wounds were treated only with vehicle. After administration of the test materials, the rats were bandaged and allowed to recover from anesthesia. On days 2, 5, 6, 8 and 10 the area of each wound was measured under anesthesia of methoxyflurane (commercially available as METOFANE from Pittman-Moore, Mundelein, IL). The area of the wound was determined: (1) tracing the shape of the wound on graph paper (1 x 1 mm squares); (2) cutting the shape; (3) weighing the paper and comparing the weight with a paper cut of 2 x 2 cm; and (4) counting the numbers of pictures on the graph paper. As illustrated in the drawings, the wound closure of full thickness was accelerated substantially in relation to the controls when the test wounds were treated with analogs 1 (Fig. 1), 2 and 3 (Fig. 2) according to the general formula I. The results represented in the figures illustrate the percentage increase in wound closure in relation to a control treated with vehicle. Surprisingly, the administration of analog 4 (Sar-Arg-Val-Tyr-lle-His-Pro-Phe (Id. Sec. No: 37)) which is outside the scope of general formula I because RA is Sar , did not accelerate wound healing (Figure 1 ). It has been reported that this analog has complete affinity and activity for angiotensin II receptor, but is resistant to aminopeptidase cleavage (Mendelsohn et al., Proc. Nat. Acad. Sci. USA 81: 1575 (1984), Israel and others, Brain Res. 322: 341 (1984), Harding et al., Brain Res. 424: 299 (1987)). Figure 3 illustrates that the 10 μg dosage accelerated wound healing more effectively than the 3 μg dosage. Example 2 provides a schematic illustration for how each of the eight amino acid positions of the All molecule can be replaced by a different amino acid to result in a compound having therapeutic utility in accordance with the methods described above.

EXAMPLE 2 THE ANGIOTENSIN ANALOGS REPLACED IN EACH OF THE EIGHT POSITIONS KEEP THE WOUND HEALING ACTIVITY 12-week-old female Sprague Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). On the day of surgery, the rats received intramuscular ketamine / anesthesia rompum before preparation for surgery. The rats were shaved and carved with betadine. Two dermal wounds of total thickness of 1.5 x 1.5 cm were created on the dorsal surface of each rat. Following the excision of the skin, the size of the wound was defined on a glass slide to establish the baseline wound size. He medication was administered in 100 μl of a solution consisting of 10% of • HYDRON, 1% polyethylene glycol (PM 400) and 60% ethanol. The test materials were administered in a random manner with all materials being tested at 10μg / wound. Control wounds were treated only with vehicle. After the administration of the test materials, the rats were bandaged and allowed to recover from anesthesia. On days 2, 5, 7 and 9, the area of each wound was measured under methoxyflurane anesthesia. The area of the wound was determined by: (1) tracing the shape of the wound on graph paper (1 x 1 mm squares); (2) cutting the shape; (3) weighing the paper and comparing the weight with a paper cut of 1.5 x 1.5 cm; and (4) counting the numbers of pictures on the graph paper. In addition, on days 2, 5 and 7 the granulation tissue area was determined by the animals administered with the 5-10 analogues. As illustrated in Figures 4-7, the wound closure was substantially accelerated relative to the control wounds when the test wounds were treated with the analogs 4-1 1 according to the general formula I. Figures 4 and 5 Illustrate the percentage of control response in wound closure compared to a vehicle treated control. In all cases, the administration of one of the analogues accelerated wound closure after surgery. Figures 6 and 7 illustrate the percentage of control response in the formation of granulation tissue. Again, in each case the administration of one of the analogues accelerated the formation of granulation tissue compared to vehicle administration alone. These results show how All analogs having amino acid sequences according to the invention can be used to accelerate wound healing. Example 3 describes the methods that were used to show that there accelerated the healing of full-thickness wounds.

EXAMPLE 3 ANGIOTENSIN III ACCELERATES THE SCREENING OF DERMAL WOUNDS OF TOTAL THICKNESS Male twelve-week-old Sprague Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). On the day of surgery, the rats received intramuscular ketamine / anesthesia rompum before preparation for surgery. The rats were shaved and cut with betadine. ' Four dermal wounds of total thickness of 2 x 2 cm were created on the dorsal surface of the rat. Following the excision of the skin, the size of the wound was defined on a glass slide to establish the baseline wound size. The drug was administered in 100 μl of a solution consisting of 10% HYDRON, 1% polyethylene glycol (PM 400) and 60% ethanol. The test materials were administered in a random manner. There it was evaluated at 3 and 10μg / wound. Control wounds were treated only with vehicle. After the administration of the materials, the rats were bandaged and allowed to recover from anesthesia. On days 2, 5, 6, 8 and 10, the area of each wound was measured under methoxyflurane anesthesia (available commercially as METOFANE from Pittman-Moore, Mundelein, IL). The area of the wound was determined by: (1) tracing the shape of the wound on graph paper (1 x 1 mm squares); (2) cutting the shape; (3) weighing the paper and comparing the weight with a paper cut of 2 x 2 cm; and (4) counting the numbers of pictures on the graph paper.

As illustrated in FIG. 8, the wound closure was substantially accelerated relative to the control animals when the test animals were treated there, at both dosages of 3μg and 10μg. The results presented in figure 8 illustrate the increase in percentage in wound closure in relation to the control treated with vehicle. Example 4 illustrates the methods used to demonstrate that analogs there also accelerated the healing of full-thickness dermal wounds.

EXAMPLE 4 THE ANGIOTENSIN ANALOGS III ACCELERATE THE SCREENING OF DERMAL WOUNDS OF TOTAL THICKNESS 12-week-old female Sprague Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA), and prepared for surgery as described in example 3. Two dermal wounds of total thickness of 2 x 2 cm were created on the dorsal surface of each rat. Following the excision of the skin, the size of the wound was defined on a glass slide to establish the baseline wound size. The drug was administered in a solution of 100 μl of a solution consisting of 10% HYDRON, 1% polyethylene glycol (PM 400) and 60% ethanol. The test materials were administered in a random manner with all the materials being tested at 10 μg / wound. Control wounds were treated only with vehicle. After the administration of the materials, the rats were bandaged and allowed to recover from anesthesia. On days 2-3, 5, 7-8 and 9-10 the area of the skin wounds was measured (for analogues 1A and 2-8 as shown in table 3) under anesthesia of methoxyflurane. The area of the wound was determined by: (1) tracing the shape of the wound on graph paper (1 x 1 mm squares); (2) cutting the shape; (3) weighing the paper and comparing the weight with a paper cut of 1.5 x 1.5 cm; and (4) counting the numbers of pictures on the graph paper. In addition, on days 2-3, 5 and 8 the area of granulation tissue was determined similarly (for analogs 1 A, 1 B and 2-7). The analogs used in these procedures have the structures presented in Table 3.

TABLE 3 Angiotensin Hl Analogs As illustrated in FIGS. 9-12, the wound closure was substantially accelerated relative to the control wounds when the test wounds were treated with analogues A1-8 there according to general formula I. FIGS. 10 illustrate the percentage of control response in wound closure in relation to the control treated with vehicle; in all cases, the administration of one of the analogues accelerated the closure of the wound after surgery. Figures 11 and 12 illustrate the percentage of control response in granulation tissue formation. Again, in all cases the administration of one of the analogues accelerated the formation of granulation tissue as compared to vehicle administration alone. Therefore, these analogues were clearly effective in promoting the healing of full-thickness dermal wounds. Example 5 describes the methods used to demonstrate that All fragments were useful in accelerating the healing of full-thickness dermal wounds.

EXAMPLE 5 ANGIOTENSIN II FRAGMENTS APPEAL FOR SCALING OF DERMAL WOUNDS OF TOTAL THICKNESS Female Sprague Dawley rats weighing 175-200 grams were obtained from Simonsen Laboratories (Gilroy, CA). On the day of surgery, the rats received intramuscular ketamine / anesthesia rompum before preparation for surgery. The rats were shaved and carved with betadine. Two dermal wounds of total thickness of 1.5 x 1.5 cm were created on the dorsal surface of the rat. Following excision of the skin, the size of the wound was defined on a glass slide and the medicament was administered in 100 μl of 10% carboxymethylcellulose of low viscosity (Sigma). The test materials were tested at 100 μg / wound. Control wounds were treated only with vehicle. After the administration of the materials, the rats were bandaged and allowed to recover from anesthesia. On days 1-4 after surgery, the rats were treated with an additional 100μg of suitable peptide formulations. On days 2, 4, 7 and 9, the area of skin wounds was measured under methoxyflurane anesthesia. The area of the wound was determined by: (1) tracing the shape of the wound on graph paper (1 x 1 mm squares); (2) cutting the shape; (3) weighing the paper and comparing the weight with a paper cut of 1.5 x 1.5 cm; and (4) counting the numbers of pictures on the graph paper. In addition, on days 2, 4 and 7 the granulation tissue area was also determined. As illustrated in Figures 13-21, the wound closure was substantially accelerated relative to the control animals when the test animals were tested with all the fragments except Al I (6-8) and Al I (4-8). ). Figures 13, 14 and 21 illustrate the percentage of control response relative to a vehicle treated control using All fragments as defined herein. Figures 15-18 and 20-21 compare the percentage of control wounds per vehicle filled with granulation tissue with that of peptide treated wounds. Figures 15, 16 and 19 reflect the data from the control wounds to which figures 17, 18 and 20, respectively, should be compared. In the compounds of particular interest according to the present invention, R6 is pNH2-Phe; the literature suggests that this amino acid confers agonist activity. It is currently considered that R7 should be Pro in order to provide the most desirable orientation of R8. In the R8 position, a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding analogues of interest to the receptors; Therefore, Tyr and especially Phe are preferred for objects of the present invention. The agonists pNH2-Phe6-AII of AT2 were tested in a rat model for dermal healing and were found to give results comparable to All in a dose of 100 μg / day for 5 days. Both granulation tissue formation and wound healing were examined and found to be accelerated by pNH2-Phe6-AII. According to the method of the invention, at least one AT2 agonist is applied to the wounded tissue in sufficient quantities to increase tissue healing rate. These compounds can significantly accelerate the healing rate at nanomolar levels. in vivo For any given agonist (peptide or nopeptide), optimal levels for use in a given formulation can be quickly determined empirically. In general, an amount of active agent suitable for use in accordance with the present invention is in the range of 0.0001 μg to 10 mg per kilogram of body weight or 1 ng to 100 mg / cm2 of wound area. Example 6 describes the methods used to demonstrate that AT2 receptor agonists were useful in accelerating the healing of full-thickness dermal wounds.

EXAMPLE 6 AT2 RECEIVER AGONISTS ACCELERATE THE SCREENING OF DERMAL WOUNDS OF TOTAL THICKNESS Female Sprague Dawley rats weighing 175-200 grams were obtained from Simonsen Laboratories (Gilroy, CA). On the day of surgery, the rats received intramuscular ketamine / anesthesia rompum before preparation for surgery. The rats were shaved and carved with betadine. Two dermal wounds of total thickness of 1.5 x 1.5 cm were created on the dorsal surface of each rat. Following the excision of the skin, the size of the wound was defined on a glass slide to define the baseline wound size. The drug was administered in 100 μl of 10% low viscosity carboxymethylcellulose (Sigma). The test materials pNH2-Phe6-AII were administered in a random manner with all materials tested at 100 μg / wound. Control wounds were treated only with vehicle. After the administration of the materials, the rats were bandaged and allowed to recover from anesthesia. On days 1-4 after surgery, the rats were treated with an additional 100 μg of peptide formulation. On days 2, 4, 7 and 9, the area of skin wounds was measured under methoxyflurane anesthesia. The area of the wound was determined by: (1) tracing the shape of the wound on graph paper (1 x 1 mm squares); (2) cutting the shape; (3) weighing the paper and comparing the weight with a paper cut of 1.5 x 1.5 cm; and (4) counting the numbers of pictures on the graph paper. In addition, on days 2, 4 and 7 the granulation tissue area was similarly determined. As illustrated in Figures 22-23, wound closure and granulation tissue formation was substantially accelerated relative to the control animals when the test animals were brought with an AT2 agonist. Figure 22 illustrates the percentage of control response in wound closure in relation to a control treated with vehicle; Figure 23 illustrates how the granulation tissue decreases with time as a result of tracing with All and There peptide analogs in relation to vehicle control. Example 7 describes the methods used to demonstrate that the additional analogues of All and There were useful for accelerating tissue healing in the in vivo model described above.

EXAMPLE 7 THE ANALOGS OF AH AND THERE ACCELERATE THE SCREENING OF DERMAL WOUNDS OF TOTAL THICKNESS The analogues of All and There that have the structures described in Table 4 were prepared using an automated peptide synthesizer and methods familiar to those of skill in the art. The ability of each of the analogs to accelerate wound healing was tested essentially according to the method of Example 6. The results of the procedures used to determine the degree of wound healing at 4 and 9 days measured as a percentage of a control wound treated by vehicle are also presented in Table 4. Because all analogs were not tested during the same experimental procedure, All was included as a positive coniol for each group of tested peptides. As indicated below, the peptide analogs are identified as All or There analogs that have amino acid substitutions at the positions identified by superscripts. By tante, for example, Gly4-AIII is the designation for an analogue of There that has a Gly residue substituted at position 4 of There. The results of the analogous procedures that were tested as a group are grouped together in the following table.

TABLE 4 Compilation of Peptide Efficacies TABLE 4 (CONTINUED) The numerical results presented in Table 4 show that almost all the analogues tested in the procedure effectively accelerated the closure of full-thickness wounds. The following example generally describes how compositions consisting of All, an AT2 agonist, an All analogue, an All fragment or analogue thereof., angiotensinogen and analogs thereof, fragments of angiotensinogen and analogs thereof, angiotensin I and analogs thereof, and angiotensin I fragments and analogs thereof can be used to promote the healing of skin grafts. Those of skill in the art will appreciate that skin grafts represent structures that differ from the open wounds described in the previous examples.More specifically, previous studies have shown that both angiotensin II and platelet-derived growth factor (PDGF) can accelerate the healing of wounded tissue and can increase the formation of granulation tissue in animal models (diZeraga, US Patent 5,015,629, Grotendorst et al., J. Clin. Invest. 76: 2323 (1985), Pierce et al., J. Exp. Med. 167: 974 (1988)). Recent studies have shown that PDGF, while increasing granulation tissue formation in rabbit ear ulcers does not effectively increase skin graft survival (Brown et al., Am. J. Surg. 171: 247 (1996)). These data, together with those described by Eberhard and others (Annals of Plastic Surgery 32: 361 (1994)), Hom et al., (Ann Otol Rhinol Laryngol 105: 109 (1996)), Stepnick and others, (Arch Otolaryngol Head Neck Surg 121: 667 (1995)) and Nail et al. (Arch Otolaryngol Head Neck Surg 122: 171 (1996)), indicate that the agents useful for accelerating wound healing can not be reasonably predicted as being useful for increasing the survival of the wound. graft or "grab". As used herein, a skin graft is a piece of graft material that can be transplanted to replace a lost portion of the skin surface of the body; it can be a graft of a total thickness, split thickness, or split skin. The graft material used in connection with the invention may be a graft material taken from the same organism that receives the graft, but may also be a allograft material taken or prepared from a non-identical member of the same species. Also contemplated to be used with the invention are graft materials that are xenoinjeríos taken from an organism that is a member of a different species with respect to the organism that receives the graft. In all cases, the graft material can be processed further after being isolated from the donor organism. In some cases, the graft material may be artificial skin of the type described by Hansbrough et al., In J. Burn Care & Rehab 15: 346 (1994). Additionally, the nugget materials contemplated for use in connection with the present invention may be live, dead or inanimate. The inanimate material contemplated will be composed of acellular material. Particular examples of graft materials useful in connection with the invention include: INTEGRA, a dermal acellular xenograft of bovine collagen and condrofine sulfa (Integra Life Sciences); DERMAGRAFT, an allogenic dermal graft of human fibroblasts on a Vicrylo mesh base structure (Advanced Tissue Sciences, San Diego, CA); DERMAGRAFT TC a temporary allograft of human fibroblasts on a nylon grid with a silicone sheet; and ALLODERM, a cadaveric dermis allograft that has been processed to remove cells (Life Cell Inc.). Example 8 illustrates how therapeutic peptides of the above-described type may advantageously promote the incorporation of a skin graft into the underlying tissue. More particularly, this example describes how therapeutic peptides can be used in combination with a live cultured skin replacement to aid in the closure of full-thickness wound defects in a mammal.

EXAMPLE 8 METHODS TO IMPROVE THE INCORPORATION OF A SKIN GRAFT INSIDE THE UNDERLYING TISSUE Eighteen female shaved rats were first obtained from a commercial vendor. On the day of surgery, the rats received intramuscular ketamine / anesthesia rompum before preparation for surgery. After carving the rats with betadine, a skin wound of 1 x 1 cm was created on the dorsal surface of each rat. Nine of the rats received grafts consisting of 1 x 1 cm sections of a live replacement skin graft sutured in place at the corners of the grafts. Before contacting the niche and the wound bed, a 100 μl sample of a pharmaceutically acceptable carrier consisting of 10% carboxymethylcellulose was applied to the wound bed. After the suture was complete, an additional 100 μl sample of vehicle was applied to the outer surface of the graft. This first group of six rats, which did not receive any therapeutic peptide, represents a negative control group. The second group of nine rats represents the rat test group. The rats of the rest of the group similarly received 1 x 1 cm grafts of artificial skin, but additionally received an application of a 100 μl sample of a drug to the wound bed before the graft was sutured into place. The drug consists of the peptide All dispersed in 10% carboxymethylcellulose of low viscosity. After the suture was complete a sample of additional 100 μl of the drug is applied to the outer surface of the graft material. Rats in the negative control group and in the test group were bandaged and allowed to recover from anesthesia. The two groups of rats received daily applications of either vehicle or medication, as appropriate. The results of the grafting procedure were evaluated macroscopically and microscopically. Three rats in each of the groups were sacrificed at 6 and 15 days after surgery, and sections of tissue from the graft area were prepared for histological analysis. The results of the microscopic analyzes indicate that the dermis of the grafts administered with the drug that confers the therapeutic peptide are more highly vascularized than the grafts of the negative control both in 6 and 15 days post-surgery. The number and size of the vessels increases during the time interval between 6 and 15 days after surgery. The macroscopic examination at 21 days after surgery of the remaining pairs of rats indicates that both grafts receiving the therapeutic peptide are firmly attached to the underlying tissue. In contrast, one of the grafts with vehicle only is non-adherent. This vascularity and increased adherence of the graft with the drug indicates that the application of the ipaeuic peptide promotes venfajosamenie the cicarization of skin grafts in mammals. Example 9 describes the methods used to demonstrate that a medicament including All was useful for promoting "grabbing" or incorporation of an autologous skin graft into underlying tissue. In these procedures, the medication that All was applied was applied to the wound bed underlying a skin flap. The flaps were created on the back of experimental animals making incisions of total thickness until the panniculus carnosus, raising and then replacing the flap. Although phosphate pH regulated saline and 10% carboxymethylcellulose were used as carriers in the following procedures, numerous other vehicles useful for administering All, active All analogues, All fragments or analogs thereof, angiotensin and analogs thereof. , angiotensin fragments and analogs thereof, angiotensin I and analogs thereof, and angiotensin I fragments and analogs thereof will be familiar to those skilled in the art.

EXAMPLE 9 ANGIOTENSIN II IMPROVES GRAFTING IN A COLLAPSE MODEL RANDOM Rajas Sprague Dawley were anesthetized with intramuscular ketamine / xylazine anesthesia. The animals were shaved using animal staples and then carved with povidone-iodine and isopropyl alcohol. Artificial tears were placed in the eyes of anesthetized animals. From the cauda to the encephalic, two incisions of 7 cm long and 1.2 cm apart were made on the dorsum starting immediately below the scapula. At the caudal end of the incisions, the 1.2 cm length was cut crosswise so that the incisions could be connected to define three edges of a skin flap. The flap was lifted from the fascia and any connective tissue was cut. A volume of 0.3 ml of medication was then placed on the wound bed. The skin flap was replaced and maintained in contact with contact skin using 8 baseline 5-0 ETHILON suture obtained from Ethicon (Ranian, NJ). The drug formulations tested in this procedure were: (1) a phosphate buffered saline (PBS) negative control solution, (2) 1 mg / ml All in PBS, (3) 10 mg / ml All in PBS, (4) 10% low viscosity carboxymethylcellulose as a second negative control, and (5) 1 mg / ml All in 10% low viscosity carboxymethylcellulose. The sutured flaps were bandaged with TEGADERM which was obtained from 3M Corp. (Minneapolis, MN), and sealed on the edges with benzoin. The animals were allowed to recover post-operatively and were then periodically observed to evaluate the level of nondiatation incorporation. The incorporation of graft was measured by: (1) the number or percentage of grafts that were viable, as determined by a healthy iniacious appearance (table 5), and (2)% graft encostramienio as indicated by discoloration, dryness and non-viable necrotic tissue (Figures 24 and 25). The results presented in Figures 24 and 25 and Table 5 indicated that the drugs that included All substantially improved the efficiency of graft incorporation and reduced the proportion of necrotic tissue that was present in viable grafts. More particularly, the results in Table 5 indicated that administration of a single dose of either 10 mg / ml All in PBS or 1 mg / ml All in 10% carboxymethylcellulose at the time of flap formation substantially improved the graft grip efficiency within 12 days. In fact, only the grafts that received one of the medications that are All were successfully incorporated into the underlying bed of the wound. The All of the conírol grafts with only one of the two vehicles had graft necrosis on days 12 and 16. In the groups of rats administered with 10 mg / ml of All in PBS or 1 mg / ml of All in carbocimethylcellulose At 10%, 67% of the grafts were viable and the incisions were sickened. Some graft necrosis was observed in the resfaníes animals. The resulting graphs presented in figures 24 and 25 show that almost 50% of the areas of the graft showed crusting or necrosis in the negative control groups that have been treated with one of the two vehicles. The percentage of graft encrustation was reduced for grafts that were brought with one of the drugs that included All. Irritable grafts with either 10 mg / ml All in PBS or 1 mg / ml All in 10% carboxymethylcellulose showed particularly good results, although tests conducted using 1 mg / ml All in PBS also produced decreased tissue formation necrotic TABLE 5 All Improve the Incorporation of Autologous Skin Grafts The results presented in the previous example proved that the dramatic differences between success and failure of autologous graft incorporation within the underlying bed of a wound were attributable to the presence or absence of All in the drug that was applied between the wound bed and the superimposed graft. The results of the time course presented in figures 24 and 25 showed that the degree of graft necrosis in the experimental system was essentially stable between 8-12 days post-surgery. Agree with this, the time frame was used in subsequent procedures that were used to establish the dose response relationship between the administration of All and the degree of incorporation of allograft graft. Example 10 describes the methods that were used to determine the optimum dosages to promote the incorporation of a denier skin graft of an underlying tissue. In these procedures, an allograft skin graft was applied to the underlying wound bed. Although All was used in the following procedure, those skilled in the art will appreciate that similar procedures can be used to establish optimal dosages of active All analogs, All fragments or analogs thereof, angiogenesis and analogues thereof, fragments of angiogenesis. and analogs thereof, angioinensin I and analogs thereof, and fragments of angioinensin I and analogs thereof according to the present invention.

EXAMPLE 10 ANGIOTENSIN II IMPROVES GRAFTING IN A COLLAPSE MODEL RANDOM Skin flaps were created according to the method of the previous example and treated with one of six experimental formulations. After suturing the flaps brought in their position, the animals were bandaged, allowed to recover and evaluated for tissue incorporation 8 and 12 days after surgery. The medical formulations tested in this procedure were: (1) a negative control consisting of 10% low viscosity carboxymethylcellulose, and (2) 0.01-1 mg / ml All in 10% low viscosity carboxymethylcellulose. The concentrations of All tested in this procedure were 1 .0, 0.3, 0.1, 0.03 and 0.01 mg / ml. The results shown in figures 26 and 27 confirmed that All improved the graft in a dose-responsive manner. More particularly, Fig. 26 shows that administering a single dose of 0.01 mg / ml All in 10% carboxymethyl cellulose at the time of flap formation increased the efficiency of graft incorporation into underlying tissue, as judged for the percentage of flaps determined as completely viable in the 8 and 12 days after the surgery. Additionally, Figure 27 shows that the percentage of the flap that was viable was increased in the tests administered with the drug containing All. All the experimental animals administered with 10% carboxymethylcellulose as a negative control exhibited graft necrosis at 8 weeks. and 12 days after the surgery In confrasfe, even the lowest dosage of All tested in the procedure substantially increased the viability of fractional flap when compared to vehicle-borne control. These results proved that the positive effects observed in the incorporation of graft were attributable to All's activity. Moreover, these results demonstrate methods to optimize the dosage of therapeutic compounds that promote the incorporation of skin grafts. An optimum dosage will be the amount of medicament that provides substantially the highest level of% complete complex flap at the lowest corresponding dosage of medication. For example, if maximum flap survival is met when the drug contains 0.03 mg / ml of a particular compound related to All, so that higher concentrations do not provide an added benefit, then the optimal dosage would be 0.03. mg / ml. The above procedures used a model of allograft graft to demonstrate how the compositions described herein could be used to promote graft incorporation. Given this demonstration, it was of interest to determine if the described compositions were also useful to promote the incorporation of non-arogenic grafts. In the example that follows, an artificial skin or "living skin equivalent" formed a viable niche on a wound bed that had been created by excision of tofal thickness only after irradiation with All. An example below describes a similar result obtained in a debrided burn model. Living skin equivalents of the type used in the methods described herein can be obtained from commercial sources such as Organogenesis, Inc. (Canton, Mass.) And Ortec Inc. (New York, NY). Skin replacements used in the methods described herein were composed of human fibroblasts that condense a bovine collagen grid that is then seeded with human culíivated keratinocytes. The collagen grid with fibroblasts serves as a dermal payroll, and the overlying human keratinocytes form the epidermal component of the composite skin replacement. A description of the composition of this skin replacement has been presented by Hansbrough et al., In J. Burn Care &; Rehabil. 15: 346 (1994), the full description of this article being incorporated herein by reference. Example 1 1 describes the methods that were used to demonstrate that All could be used to improve the graft using a living skin equivalent in an ionic thickness excision model. Similar procedures can be used to improve the grafting of different living skin equivalents by using All-active analogues, All fragments or analogs thereof, Angioinensinogen and analogues thereof, Angiotensinogen fragments and analogs thereof, Angioinens I and the like thereof, fragments of angiotensin I and its analogues.

EXAMPLE 11 ARTIFICIAL LEATHER GRAFTING ON THE SITE OF A THICK EXCISION TOTAL Six Swiss shaved male mice (22-24 grams) were purchased from Taconic laboratories (Germanfown, NY) and quarantined at least 2 days before surgery. After anesthesia by intramuscular injection of KETASET / ROMPUM which had been obtained from Western Medical Supply, (Arcadia, CA), excisions of total thickness of 1 x 1 cm were made on the dorsum of each mouse. The living skin equivalents produced essentially as described by Hansbrough et al., (Supra) were obtained from a commercial source. This material was placed on the wound defect and cut with a microfiber so that no gap was observed between the edges of the mouse skin and the nugget material. The mice were divided into three groups of 2 mice per group based on the treatment administered to the graft material before placement in the wound bed. In group 1, the living skin equivalent was removed from its culture dish and placed directly on the wound bed. Mice in group 2 were grafted with the living skin equivalent that had been soaked in the Ringer's solution with 5% dextrose for 10 minutes prior to placement in the wound bed. In group 3, the living skin equivalent was soaked for 10 minutes in the lactated Ringer's solution with 5% dextrose containing 1 mg / ml All before being placed in the wound bed. After the graft material was placed, the dorsal surface of the mouse was covered with a gauze soaked in peir followed by two adhesive bandages. After recovering from anesthesia, the mice were returned to their individual cages and observed daily until euthanasia. All mice received intramuscular analgesics during the first three days after surgery. No mouse lost the bandage before the necropsy on day 7 (1 mouse of each group) or on day 9 (1 mouse of each group). At necropsy, the degree of incorporation of the graft and the appearance of the graft was noted before placing the biopsy in 10% pH regulated formalin in preparation for paraffin crushing, sectioning, and hemaxilillin and eosin anointing. The results of these procedures indicated that All had a positive effect on the incorporation of denim skin equivalent of full-thickness wounds. All the grafts appeared healthy and the inosculation was nofó for 80-100% of the graft edges on days 7 and 9. Thus, soaking the living skin equivalent in lactated Ringer solution did not adversely affect the graft material. Microscopic analyzes indicated that one of the All-framed grafts had numerous vessels adhered to the inferior side confraining the fascia on day 7 of post-surgery. This was not noted for any of the two grafts that were not frayed, neither for the grafts that had been irritated with Ringer's solution, nor in one of the grafts that had been irradiated with the All solution. Histological analyzes were also used to evaluate the number of endothelial cells and the number of vascular channels that contain red blood cells as a measure of tissue integration in 20x microscopic fields at the interface between the equivalent of living skin and adjacent mouse tissue. Between 9 and 20 fields were counted for each graft to make this analysis. Synthetic data were obtained by using grafts that had been soaked in laxative Ringer's solution with 5% dexfrosa for approximately 10 minutes before being placed in the wound bed. The results presented in Figures 28 and 29 show that soaking the living skin equivalent in an All solution for 10 minutes before placement increases the number of endothelial cells and vascular channels that contain red blood cells on days 7 and 9 after of surgery, suggesting that integration with murine tissue occurs more rapidly. At both time points the resins obtained for the grafts that were brought with All were similar. However, from day 7 to 9 there was an increase in the number of endoihelial cells and vascular channels at the interface between the living skin equivalent and mouse tissue for the vehicle-treated grafts. Because the All-containing preparations improved the incorporation of allogenic grafts and living skin equivalents of underlying tissue, it was of interest to further explore the scale of graft applications that would be improved by the administration of All, analogues of active All, fragments of All and analogs thereof, angiogenesis and analogues thereof, fragments of angiogenesis and analogs thereof, angiogenesis I and analogs thereof, fragments of angiofensin I and analogs thereof. The following examples illustrate how All can be used to promote the incorporation of a living skin equivalent into a debrided burn injury. Similar results are expected when analogs of All acfivos, All fragments or analogs thereof are subsumed by All, angiotensinogen and analogs thereof, fragments of angiotensinogen and analogues thereof, angiotensin I and its analogues and fragments of angiotensin I and analogs thereof. same. Example 12 describes the methods used to demonstrate that compositions of the type described herein were useful in promoting the incorporation of living skin equivalents within the site of a debrided burn injury.

EXAMPLE 12 ARTIFICIAL LEATHER GRAFTING TO A DEBRIDUAL SITE 48 AFTER INJURY OF TOTAL GROSS BURN IN RASURED MICE Shaved / shaved Swiss male mice (26 grams) were purchased from Taconic laboratories (Germantown, NY) and quarantined for a period of 5 days before the induction of a full-thickness burn lesion. The burn injury occurred by contacting the skin on the back for a period of 10 seconds with a brass rod that had been heated to 100 ° C. Two days after the induction of the burn, an incision was made on the site and the area was grafted with an equivalent of living skin that had been pretreated either by soaking for 10 minutes in: (saline, or (2) saline solution of 1 mg / ml of All. The results of these procedures indicated that All improved the incorporation of a living skin equivalent at the site of the burn injury, only one of the mice grafted with a living skin equivalent that had been soaked in saline remained alive on day 21. The graft on this surviving mouse contracted and was not viable.A second mouse that had received a similar graft died with necrotic necrosis 6 days after surgery. 21 days after surgery a mouse that had been grafted with an equivalent of living skin soaked in the All solution before the placement had a graft It was completely viable, and two other mice had grafts that were partially convalescent and were not viable. Therefore, the only graft that was successfully incorporated into the underlying tissue of a debrided burn injury was the graft that had been treated with All prior to placement. This confirmed that the compounds described herein were useful for improving graft grip at the site of debrided burn injuries. The following procedures were carried out to study, by quantitative histology, the effect on neovascularization of pre-rinsing an equivalent of living skin in a Rinse solution laced with Dexrose and containing All before grafting.

Example 13 describes the methods used to demonstrate that soaking a living skin equivalent in a solution including All before placing the graft advantageously displayed increased numbers of endothelial cells and numbers of vascular channels in the graft interface.

EXAMPLE 13 PACKING A LIVING LEVEL EQUIVALENT IN A SOLUTION THAT CONSISTS OF ALL IMPROVES INCORPORATION WITHIN UNDERLYING TISSUE Twelve shaved male Swiss mice (22-24 grams) were purchased from Taconic laboratories (Germantown, NY) and quarantined at least 2 days before surgery. The mice were anesthetized with an intramuscular injection of Ketaset / Rompum and excisions were made total thickness of 1 x 1 cm on the dorsal surface of each mouse. An equivalent w of living skin essentially of the type described by Hansbrough and others, (supra) was placed on the wound defect and cut with a micro-sieve so that no gap was observed between the edges of the mouse skin and the equivalent of living skin. The mice were divided into 4 groups (three per group) based on the All concentration in the solution that was used to soak the living skin equivalents before placement. The All concentrations used in the procedures were: 0, 0.01, 0.1 and 1.0 mg / ml. After the graft was placed, the dorsal surface of the mouse was covered with a gauze soaked in petrolatum followed by two adhesive bandages (Baxter). After recovering from anesthesia, the mice were returned to their individual cages and observed daily until euthanasia. The mice received intramuscular anesthetics during the first three days after anesthesia. No mice lost their bandages before the necropsy on day 7. At necropsy, the degree of graft grip and the appearance of the grafted tissue were noted before the replacement of the regulated formalin biopsy at 10% pH in preparation for paraffin incrustation and haematoxylin section and eosin staining. The results of these procedures indicated that all grafts had a healthy appearance (except one control animal that lost its graft) and inosculation was noted for 80-100% of the graft edges. One of the frayed grafts with All was numerous vessels adhered to the inferior side of the graft coníra the fascia of the shaved mouse after excision of total thickness. This was not noticed in any of the mice. The number of endothelial cells and vascular channels containing red blood cells per 20 × microscope field (between 9 and 20 fields per mouse) present at the interface between the living skin equivalent and the shaved mouse tissue were determined. The control data were obtained from grafts that had been soaked in laxative Ringer's saline solution with dextrose for 10 minutes as a vehicle control. These data are presented in figures 30 and 31. Soaking the living skin equivalent in a solution of 0.01-1 mg / ml All for 10 minutes before the placement showed that it increases the number of endothelial cells and vascular channels that contain globules red Therefore, this dose response experiment showed that: (1) the living skin equivalent could be soaked in lactated Ringer's saline solution for 10 minutes before graft placement without negative effects; and (2) an equivalent of living skin pretreated with All showed accelerated neovascularization, as determined by the number of endothelial cells and vascular channels, in the graft site. Additionally, these data showed that the effect was concentration dependent. In general, the aggregated results presented above indicated that the compounds shown above useful for accelerating wound healing are also useful in promoting the incorporation of skin grafts into underlying tissue. From the aforementioned description, one skilled in the art can easily evaluate the essential characteristics of the invention and, without departing from the spirit and scope thereof, can adapt the invention to various uses and conditions. Changes in form and substitution of equivalents are contemplated as circumstances may suggest and become adequate, and although specific terms have been used in the present, they are designed in a descriptive sense and not for limitation purposes.

LIST OF SEQUENCES (1) General information: (i) Applicant: The University of Southern California (ii) Title of the Invention: Method to promote the Incorporation of Healing in Skin Grafts (iii) Number of Sequences: 46 (v ) Address for Correspondence: (A) Recipient: Knobbe, Martens, Olson & Bear (B) Street: 620 Newport Center Drive 16th Floor (C) City: Newporí Beach (D) State: California (E) Country: E.U.A. (F) Zip Code: 92660 (v) Compliant form in Computing: (A) Type of Media: Diskette (B) Computing: IBM Compafible (C) Operating System DOS (D) Software: FastSEQ Version 1.5 (vi) Data of the Current Application (A) Application Number: (B) Application Date: (C) Classification: (vii) Previous Application Data: (A) Application Number: 60 / 028,310 (B) Application Date: 16-Dec- 1996 (viii) Attorney / Agent Information: (A) Name: Alíman, Daniel E. (B) Record Number: 34,115 (C) Reference Number / Case: USC012.001A (ix) Telecommunications Information: (A) Telephone: 714-760-0404 (B) Téiefax 714-760-9502 (C) Telex: (2) Information for Sec. From ID No .: 1: (i) Sequence Characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Seq. ID No.: 1: Asp Arg Val Tyr He His Pro Phe 1 5 (2) Information for Sec. Id. No .: 2: (i) Sequence characteristics: (A) Longifud: 8 amino acids (B) Type: amino acid (C) Type of chain: Simple (D) Topology: Linear (ii) Type of Molecule: Pépíido (ix) Feature: (A) Name / Password: Other (B) Location: 6 ... 6 (D) Other Information: position 6 is p-NH2-Phe (xi) Sequence Description: Seq. Id. No.:2: Asp Arg Val Tyr He Xaa Pro Phe 1 5 (2) Information for Seq. Id. No .: 3: (i) Characteristics of sequence: (A) Longifud: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Id. From Sec. No.:3: Arg Val Tyr lie His Pro Phe 1 5 (2) Information for Sec. Id. No .: 4: (i) Sequence characferics: (A) Longifud: 6 amino acids (B) Type : amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) De Sequence description: Seq. Id. No.:4: Val Tyr He His Pro Phe 1 5 (2) Information for Seq. Id. No .: 5: (i) Sequence characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Single (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Seq. Id. No.:5: Asp Arg Val Tyr lie His Pro 1 5 (2) Information for Sec. From ID No .: 6: (i) Sequence characteristics: (A) Length: 6 amino acids (B) Type: amino acid (C) Chain type: Single (D) ) Topology: Linear Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:6 Arg Val Tyr lie His Pro 1 5 (2) Information for Seq. Id. No .: 7: (i ) Sequence characteristics: (A) Length: 5 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Id. Sec. No.:7 Val Tyr lie His Pro 1 5 (2) Information for Sec. From ID No .: 8: (i) Sequence characteristics: (A) Length: 4 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:8 He His Pro Phe 1 (2) Information for Seq. Id. No .: 9: (i) Sequence characteristics: (A) Length: 6 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Id From Sec. No.:9 Asp Arg Val Tyr lie His 1 5 (2) Information for Sec. From ID No .: 10: (i) Sequence characteristics: (A) Length: 5 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. ID No .: 10 Asp Arg Val Tyr lie 1 5 (2) Information for Sect. Id. No .: 11: (i) Sequence characteristics: (A) Longiud: 4 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (¡i) Molecule type: Pépíido (xi) Sequence description: id. From Sec. No.:1 1 Asp Arg Val Tyr 1 (2) Information for Sec. From ID No .: 12: (i) Sequence Characteristics: (A) Length: 3 amino acids (B) Type: amino acid (C) ) Chain type: Simple (D) Topology: Linear (¡i) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:12 Asp Arg Val 1 (3) Information for Sec. No .: 13: (i) Sequence characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (ix) ) Feature: (A) Name / Password: Other (B) Location: 2 ... 2 (D) Oira Information: Position 2 is norLeu (xi) Sequence Description: Seq. ID No .: 13 Arg Xaa Tyr lie His Pro Phe 1 5 (2) Information for Sec. From ID No .: 14: (i) Sequence characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Type of Molecule: Peptide (ix) Caraciérisíica: (A) Name / Cl bird: Ofro (B) Location: 4 ... 4 (D) Oíra Information: Position 4 is norLeu (xi) Sequence description: Se. ID No.:14 Arg Val Tyr Xaa His Pro Phe 1 5 (2) Information for Sec. From ID No .: 15: (i) Sequence Characteristics: (A) Length: 3 amino acids (B) Type: amino acid (C) Type of chain : Simple (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. ID No .: 15 His Pro Phe 1 (2) Information for Sec. From ID No .: 16: (i) Sequence Characteristics: (A) Length: 5 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. ID No .: 16 Tyr He His Pro Phe 1 5 (2) Information for Sec. From ID No .: 17: (i) Sequence Characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule Type: Pepido (xi) Sequence Description: Sec. Id. No.:17 Xaa Xaa Tyr Xaa His Pro Phe 1 5 (2) Information for Sec. .: 18: (i) Sequence features: (A) Longiid: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear Molecule type: Peptide (xi) Sequence description: Seq ID No.:18 Arg Val Tyr Gly His Pro Phe 1 5 (2) Information for Seq. Id. No.:19 (i) Sequence Characteristics: (A) Length: 7 amino acids (B) Type : amino acid (C) Chain type: Single (D) Topology: Linear (¡i) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:19 Arg Val Tyr Ala His Pro Phe 1 5 (2) Information for Sec. Id. No .: 20: (i) Characteristic Sequences: (A) Longifud: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Sec. No.:20 Asp Arg Val Tyr Hal His Pro Phe 1 5 (2) Information for Sec. From ID No .: 21: (i) Sequence Characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:21 Asn Arg Val Tyr Val His Pro Phe 1 5 (2 ) Information for Sec. Of ID No .: 22: (i) Sequence characteristics: (A) Length: 1 1 amino acid (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) ) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:22 Wing Pro Gly Asp Arg He Tyr Val His Pro Phe 1 5 10 (2) Information for Sect. Id. No .: 23 : (i) Sequence characteristics: (A) Length: 8 amino acids (B) Type: a minoacid (C) Chain type: Single (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:23 Glu Arg Val Tyr He His Pro Phe 1 5 ( 2) Information for Sec. Id. No .: 24: (i) Sequence characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) ) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:24 Asp Lys Val Tyr He His Pro Phe 1 5 (2) Information for Seq. Id. No .: 25: (i) Sequence characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Sec. No.:25 Asp Arg Ala Tyr He His Pro Phe 1 5 (2) Information for Sec. From ID No .: 26: (i) Sequence Characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Type of chain: Simple (D) Topology: Linear (ii) Type of Mole Cyp: Pepido (xi) Sequence Description: Seq. Id. No.:26 Asp Arg Val Thr He His Pro Phe 1 5 (2) Information for Seq. Id. No .: 27: (i) Sequence Characteristics : (A) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Seq. ID No. : 27 Asp Arg Val Tyr Leu His Pro Phe 1 5 (2) Information for Sec. From ID No .: 28: (i) Sequence Characteristics: (A) Longifud: 8 amino acids (B) Type: amino acid (C) Chain type: Single (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:28 Asp Arg Val Tyr lie Arg Pro Phe 1 5 ( 2) Information for Sec. From ID No .: 29: (i) Sequence characteristics:. (A) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Single (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Seq. ID No .: 29 Asp Arg Val Tyr He His Wing Phe 1 (2) Information for Sec. Id. No .: 30: (i) Sequence Characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Type of chain: Simple (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. Id. No.:30 Asp Arg Val Tyr He His Pro Tyr 1 5 (2) Information for Sec. From Id. No .: 31: (i) Sequence characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence Description: Seq. Id. No.:31 Pro Arg Val Tyr He His Pro Phe 1 5 (2) Information for Seq. Id. No .: 32: (i) Sequence Characteristics: (A ) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule Type: Peptide (xi) Sequence Description: Seq. ID No.:32 Asp Arg Pro Tyr He His Pro Phe 1 5 (2) Information for Seq. No .: 33: (i) Sequence features: (A) Longilud: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (ix) Caracíerísíica: (A) Name / Key: Oíro (B) Location: 4 ... 4 (D) Oíra Information: The position 4 is Tyr (PO3) 2 (xi) Description of Sequence: Id. Of Sec. No .: 33 Asp Arg Val Xaa He His Pro Phe 1 5 (2) Information for Sec. From ID No: 34: (i) Sequence Characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Type Chain: Simple (D) Topology: Linear (ii) Type of Molecule: Peptide (ix) Characteristic (A) Name / Key: Ofro (B) Location: 3 ... 3 (D) Oíra Information: Position 3 is norLeu: (xi) Sequence description: Sec. Id. No.:34 Asp Arg Xaa Tyr lie His Pro Phe 1 5 (2) Information for Sec. From ID No .: 35: (i) Sequence characteristics: (A) Length: 8 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule Type: Peptide (ix) Feature: (A) Name / Password: Other (B) Location: 5 ... 5 (D) Other Information: Position 5 is norLeu (x) Description of Sequence: Seq ID No.:35 Asp Arg Val Tyr He Xaa His Pro Phe 1 5 (2) Information for Seq. Id. No .: 36: (i) Sequence Characteristics: (A) Length: 9 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (ix) Characteristic: (A) Name / Password: Other (B) Location: 4 .. .4 (D) Other Information: Position 4 is homoSer (x) Sequence Description: Sec. Id. No.:36 Asp Arg Val Xaa Tyr He His Pro Phe 1 5 (2) Information for Sec. No .: 37: (i) Sequence characteristics: (A) Longiud: 8 amino acids (B) Type: am inoacid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Seq. ID No.:37 Ser Arg Val Tyr He His Pro Phe 1 5 ( 2) Information for Sec. Id. No .: 38: (i) Sequence characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) ) Molecule Type: Peptide (xii) Sequence Description: Seq. ID No.:38 Arg Val Tyr lie His Pro He 1 5 (2) Information for Seq. Id. No .: 39: (i) Characteristics sequence: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide (xi) Sequence description: Seq. No.:39 Arg Val Tyr Val His Pro Phe 1 5 (2) Information for Sec. From ID No .: 40: (i) Features sequence: (A) Length: 7 amino acids (B) Type: amino acid (B) Chain type: Single (C) Topology: Linear (ii) Molecule type: Peptide (xii) Sequence description: Seq. No.:40 Lys Val Tyr He His Pro Phe 1 5 (2) Information for Sec. From ID No .: 41: (1) Sequence Characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) ) Chain type: Simple (D) Topology: Linear (iii) Molecule Type: Peplido (xi) Sequence description: Seq. Id. No.:41 Arg Allah Tyr He His Pro Phe 1 5 (2) Information for Sec. From ID No .: 42: (i) Sequence features: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type : Peptide (xi) Sequence Description: Seq ID No.:42 Arg Val Thr He His Pro Phe 1 5 (2) Information for Seq. Id. No .: 43: (i) Sequence Characteristics: ( A) Length: 7 amino acids (B) Type: amino acid (C) Type of chain : Simple (D) Topology: Linear (¡i) Molecule Type: Peptide (xi) Sequence Description: Seq. ID No.:43 Arg Val Tyr Leu His Pro Phe 1 5 (2) Information for Sec. Id. No .: 44: (i) Sequence characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (ii) Molecule type: Peptide ( xi) Sequence Description: Seq. Id. No.:44 Arg Val Tyr He Arg Pro Phe 1 5 (2) Information for Sec. Id. No .: 45: (i) Sequence Characteristics: (A) Length : 7 amino acids (B) Type: amino acid (C) Chain type: Simple (D) Topology: Linear (i) Molecule type: Peptide (xi) Sequence description: Seq. ID No.:45 Arg Val Tyr He His Wing Phe 1 5 (2) Information for Sec. From ID No .: 46: (i) Sequence characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) Chain type: Simple ( D) Topology: Linear (i) Type of Molecule: Peptide (xi) Description of S Ecuen: Sec. Id. No.:46 Arg Val Tyr lie His Pro Tyr 1 5

Claims (15)

NOVELTY OF THE INVENTION CLAIMS

1. The use of angioinensin II, an angiofensin II analogue, an angiotensin II fragment, or an analogue of an angiotensin II fragment in the preparation of a medicament for promoting the incorporation of a denier skin graft of underlying tissue of a mammal , wherein said angiotensin II, an angiotensin II analog, an angiotensin II fragment, or an analogue of an angiotensin II fragment consists of a peptide consisting of at least three contiguous amino acids of groups R1-R8 in the general formula; R1-R2-R3-R4-R5-R6-R7-R8; wherein R1 is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, BeI, Glu (NH2), Gly, Asp (NH2) and Suc; R2 is selected from the group consisting of Arg, Lys Ala, Orn, 5 Sec (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc, and Tyr; R4 is selected from the group consisting of Tyr, Tyr (PO3) 2, Thr, Ser, homoSer and azaTyr; R5 is selected from the group consisting of He, 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), He and Tyr, or excluding sequences that include R4 as a terminal Tyr group.

2. The use according to claim 1, wherein said medicament is for soaking the skin skin and consists of a saline solution regulated by pH and / or as a pharmaceutically acceptable vehicle.

3. - The use according to claim 2, wherein the medicament consists of a pharmaceutically acceptable carrier which consists of carboxymethylcellulose and wherein the medicament is for application to tissue underlying said skin graft.

4. The use according to claim 1, wherein the angiotensin II, an angiotensin II analog, an angiotensin II fragment or an angiotensin II fragment analog consists of a peptide having the general formula; R2-R3-R4-R5-R6-R7-R8: wherein R2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc, and Tyr; R4 is selected from the group consisting of Tyr, Tyr (P03) 2, Thr, Ser, homoSer, and azaTyr; R5 is selected from the group consisting of He, 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 He, Phe, Phe (Br) and Tyr.

5. The use according to claim 1, wherein the skin graft is an autograft.

6. The use according to claim 1, wherein the skin graft is an allograft.

7. The use according to claim 1, wherein the skin graft is a xenograft.

8. The use of a composition consisting of a pharmaceutically acceptable carrier and a peptide in the manufacture of a medicament for promoting the incorporation of a skin graft on underlying tissue of a mammal, wherein said peptide consists of at least three amino acids contiguous of groups R1-R8 in the general formula; R1 - R2 - R3 - R4 - R5 - R6 - R7 - R8: in which R1 is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bei, Glu (NH2), Gly, Asp (NH2) and Suc; R2 is selected from the group consisting of Arg, Lys Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc, and Tyr; R4 is selected from the group consisting of Tyr, Tyr (PO3) 2, Thr, Ser, homoSer and azaTyr; R5 is selected from the group consisting of He, 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), He and Tyr, excluding sequences that include R4 as a terminal Tyr group.

9. The use according to claim 8, wherein said peptide has the general formula; R2-R3-R4-R5-R6-R7-R8: wherein R2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys; R3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc, and Tyr; R4 is selected from the group consisting of Tyr, Tyr (PO3) 2, Thr, Ser, homoSer, and azaTyr; R5 is selected from the group consisting of He, 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 He, Phe, Phe (Br) and Tyr; 10.

The use according to claim 8, wherein said skin graft is an equivalent of living skin. 1.

The use according to claim 8, wherein said skin graft is an allograft.

12. The use according to claim 8, wherein said skin graft is an allograft.

13. - The use according to claim 8, wherein said skin graft is a xenograft.

14. The use according to claim 10, wherein the living skin equivalent is an allograft selected from the group consisting of allogeneic dermal skin or human fibroblasts on a Vicryl grid structure, a temporary allograft of human fibroblasts on a grid of nylon with a silicone sheet, and an allograft from corpus dermis that has been processed to remove the cells.

15. The use according to claim 10, wherein the living skin equivalent is an acellular dermal xenograft of bovine collagen and chondroitin sulfate.