WO2001044270A2

WO2001044270A2 - Methods for treating and preventing diabetic complications by use of angiotensin-related peptides

Info

Publication number: WO2001044270A2
Application number: PCT/US2000/032133
Authority: WO
Inventors: Kathleen E. Rodgers; Gere S. Dizerega
Original assignee: University of Southern California USC
Current assignee: University of Southern California USC
Priority date: 1999-12-16
Filing date: 2000-11-27
Publication date: 2001-06-21
Anticipated expiration: 2002-06-16
Also published as: WO2001044270A3; AU1792901A

Abstract

The present invention provides improved methods, kits, and pharmaceutical compositions for treating or preventing diabetic complications by administering an effective amount of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII) analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists to treat or prevent diabetic complications.

Description

METHODS FOR TREATING AND PREVENTING DIABETIC COMPLICATIONS

Field of the Invention

This present invention relates to methods to treat and prevent diabetic complications.

Background of the Invention Diabetes is a major cause of morbidity and mortality in the United States. One of the most common complications of diabetes is nephropathy. Diabetic nephropathy is thought to be caused by the progressive glycosylation of proteins, leading to a progressive loss of renal function. (U.S. Patent No. US 6,031,006) Diabetic nephropathy generally results in a chronic and progressive degradation of kidney function, to the point where the patient must undergo dialysis or transplant to survive. Approximately 40 percent of all diabetic patients developing nephropathy which requires either kidney dialysis or transplantation. (U.S. Patent No. 5,302,609; incorporated by reference herein in its entirety.) Diabetic nephropathy is believed responsible for at least 25% of all renal dialysis patients, with an increase in morbidity and costs of medical care. Diabetes mellitus is the leading cause of end stage renal disease and therefore, any newly diagnosed patient with diabetes mellitus would be considered at risk for the development of diabetic nephropathy. Several years prior to the development of renal insufficiency, diabetic patients also exhibit renal disease manifested by renal hypertrophy and hyperfiltration. Currently the primary treatment for preventing or reversing the renal hypertrophy of diabetic nephropathy is rigorous insulin therapy.

Thus, there is a need in the art for other methods of treating and preventing diabetic nephropathy. Summary of the Invention

The present invention provides methods and kits for treating and preventing diabetic complications by administering to a patient in need thereof an amount effective of angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues thereof, angiotensin II (All) analogues, All fragments or analogues thereof or All AT type 2 receptor agonists, either alone, combined, or in further combination with other compounds, for treating or preventing diabetic complications, such as insulin or angiotensin converting enzyme inhibitors (ACE inhibitors).

Brief Description of the Figures

Figure 1. Effect of angiotensin peptides on diabetic nephropathy.

Detailed Description of the Preferred Embodiments

All cited patents, patent applications and references are hereby incorporated by reference in their entirety.

Unless otherwise indicated, the term "angiotensin converting enzyme inhibitors" or "ACE inhibitors" includes any compound that inhibits the conversion of the decapeptide angiotensin I to angiotensin II, and include but are not limited to alacepril, alatriopril, altiopril calcium, ancovenin, benazepril, benazepril hydrochloride, benazeprilat, benzazepril, benzoylcaptopril, captopril, captopril-cysteine, captopril-glutathione, ceranapril, ceranopril, ceronapril, cilazapril, cilazaprilat, converstatin, delapril, delapril-diacid, enalapril, enalaprilat, enalkiren, enapril, epicaptopril, foroxymithine, fosfenopril, fosenopril, fosenopril sodium, fosinopril, fosinopril sodium, fosinoprilat, fosinoprilic acid, glycopril, hemorphin-4, idapril, imidapril, indolapril, indolaprilat, libenzapril, lisinopril, lyciumin A, lyciumin B, mixanpril, moexipril, moexiprilat, moveltipril, muracein A, muracein B, muracein C, pentopril, perindopril, perindoprilat, pivalopril, pivopril, quinapril, quinapril hydrochloride, quinaprilat, ramipril, ramiprilat, spirapril, spirapril hydrochloride, spiraprilat, spiropril, spiropril hydrochloride, temocapril, temocapril hydrochloride, teprotide, trandolapril, trandolaprilat, utibapril, zabicipril, zabiciprilat, zofenopril and zofenoprilat. (See for example Jackson, et al., Renin and Angiotensin in Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th ed., eds. Hardman, et al. (McGraw Hill, 1996); and U.S. Patent No. 5,977,159.)

Unless otherwise indicated, the term "active agents" as used herein refers to the group of compounds comprising angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues thereof, angiotensin II analogues, All fragments or analogues thereof or All AT₂ type 2 receptor agonists.

As defined herein, the term "diabetic complications" encompass decreases in renal function, including but not limited to proteinuria, renal hypertrophy, and diabetic nephropathy. The method of the present invention finds use in any patient diagnosed as having diabetes mellitus. The active agents are administered to the diabetic patient as soon as possible after being diagnosed with diabetes in order to prevent or ameliorate diabetic complications.

U.S. Patent No. 5,015,629 to DiZerega (the entire disclosure of which is hereby incorporated by reference) describes a method for increasing the rate of healing of wound tissue, comprising the application to such tissue of angiotensin II (All) in an amount which is sufficient for said increase. The application of All to wound tissue significantly increases the rate of wound healing, leading to a more rapid re-epithelialization and tissue repair. The term

All refers to an octapeptide present in humans and other species having the sequence Asp- Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:l]. The biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen (Circulation Research 60:786-790 (1987); Clouston et al., Genomics 2:240-248 (1988); Kageyama et al., Biochemistry 23:3603-3609; Ohkubo et al., Proc. Natl Acad. Sci. 80:2196-2200 (1983)); all references hereby incorporated in their entirety). The substance so formed is a decapeptide called angiotensin I (Al) which is converted to All by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from Al, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe- His-Leu [SEQ ID NO:37]. All is a known pressor agent and is commercially available.

Studies have shown that All increases mitogenesis and chemotaxis in cultured cells that are involved in wound repair, and also increases their release of growth factors and extracellular matrices (diZerega, U.S. Patent No. 5,015,629; Dzau et. al., J. Mol Cell. Cardiol. 21:S7 (Supp III) 1989; Berk et. al., Hypertension 13:305-14 (1989); Kawahara, et al., BBRC 150:52-9 (1988); Naftilan, et al., J. Clin. Invest. 83:1419-23 (1989); Taubman et al., J. Biol Chem. 264:526-530 (1989); Nakahara, et al., BBRC 184:811-8 (1992); Stouffer and Owens, Circ. Res. 70:820 (1992); Wolf, et al., Am. J. Pathol. 140:95-107 (1992); Bell and Madri, Am. J. Pathol. 137:7-12 (1990)). In addition, All was shown to be angiogenic in rabbit corneal eye and chick chorioallantoic membrane models (Fernandez, et al., J. Lab. Clin. Med. 105:141 (1985); LeNoble, et al., Eur. j. Pharmacol. 195:305-6 (1991)).

We have previously demonstrated that angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof, and All AT₂ type 2 receptor agonists are effective in accelerating wound healing and the proliferation of certain cell types. See, for example, co- pending U.S. Patent Application Serial Nos. 09/012,400, filed January 23, 1998; 09/198,806 filed November 24, 1998; 09/264,563, filed March 8, 2000; 09/287,674, filed April 7, 1999; 09/255,136 filed February 19, 1999; 09/245,680, filed February 8, 1999; 09/250,703 filed February 15, 1999; 09/246,525 filed February 8, 1999; 09/266,293 March 11, 1999; 09/332,582 filed June 14, 1999; 09/373,962 filed August 13, 1999; and 09/352,191 filed July 12, 1999; as well as U.S. Patent Serial Nos. 5,015,629; 5,629,292; 5,716,935; 5,834,432; and 5,955,430; 6,096,709; 6,110,895.

The effect of All on a given cell type has been hypothesized to be dependent, in part, upon the All receptor subtypes the cell expresses (Shanugam et al., Am. J. Physiol. 268:F922- F930 (1995); Helin et al., Annals of Medicine 29:23-29 (1997); Bedecs et al., Biochem J. 325:449-454 (1997)). These studies have shown that All receptor subtype expression is a dynamic process that changes during development, at least in some cell types. All activity is typically modulated by either or both the ATI and AT2 All receptors. However, All has recently been shown to stimulate proliferation of primary human keratinocytes via a non- ATI, non-AT2 receptor. (Steckelings et al., Biochem. Biophys. Res. Commun. 229:329-333 (1996)). These results underscore the cell-type (ie: based on receptor expression) specific nature of All activity.

Many studies have focused upon AII(l-7) (All residues 1-7) or other fragments of All to evaluate their activity. AII(l-7) elicits some, but not the full range of effects elicited by AIL (Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther. 265:664-673 (1991); Jaiswal, et al., Hypertension 17:1115-1120 (1991); Portsi, et a., Br. J. Pharmacol. 111:652-654 (1994)). Other data suggests that the All fragment AII(l-7) acts through a receptor(s) that is distinct from the ATI and AT2 receptors which modulate All activity. (Ferrario et al., J. Am. Soc. Nephrol. 9:1716-1722 (1998); Iyer et al., Hypertension 31 :699-705 (1998); Freeman et al., Hypertension 28:104 (1996); Ambuhl et al., Brain Res. Bull. 35:289 (1994)). Thus, AII(l-7) activity on a particular cell type cannot be predicted based solely on the effect of All on the same cell type. In fact, there is some evidence that AII(l-7) often opposes the actions of AIL (See, for example, Ferrario et al., Hypertension 30:535-541 (1997))

The use of ACE inhibitors for the treatment of diabetic nephropathy is well known. (See, for example, Sharma et al., Am. J. Kidney Disease 34:818-823 (1999)) The use of ATI receptor antagonists has also been suggested for the treatment of diabetic nephropathy. (See for example Mogyorosi and Sonkodi, Diabetes Metab. Res. Rev. 15:55-58 (1999) and Timmermans and Smith, Cardiologia 39:397-400 (1994)). All is believed to be involved in the pathogenesis of diabetic nephropathy. (See, for example, Singh et al., Diabetes 48:2066- 73 (1999)) AII(l-7) has been shown to elevate urinary sodium excretion and water and glomerular filtration rates in rats, but has not been suggested for use to treat diabetic patients, nor to treat and prevent diabetic complications such as diabetic nephropathy and proteinuria. (Hilchey et al., Hypertension 25:1238-1244 (1995); Handa et al., Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39) F141-F147 (1996); Vallon et al., J. Card. Pharm. 32:164-167 (1998)) Based on the above, there would be no expectation by one of skill in the art that angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues thereof, angiotensin II (All) analogues, All fragments or analogues thereof, or All AT₂ type 2 receptor agonists could be used to treat and prevent diabetic complications, such as proteinuria and diabetic nephropathy. A peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for

AT2 than ATI) is p-aminophenylalanine6-AII ["(p-NH₂-Phe)6-AII)"], Asp-Arg-Val-Tyr-Ile- Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH₂-Phe (Speth and Kim, BBRC 169:997- 1006 (1990). This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93-97 (1994); Bryson, et al., Eur. J. Pharmacol. 225:119-127 (1992). The effects of All receptor and All receptor antagonists have been examined in two experimental models of vascular injury and repair, which suggest that both All receptor subtypes (ATI and AT2) play a role in wound healing (Janiak et al., Hypertension 20:737-45 (1992); Prescott, et al., Am. J. Pathol. 139:1291-1296 (1991); Kauffman, et al., Life Sci. 49:223-228 (1991); Viswanathan, et al., Peptides 13:783-786 (1992); Kimura, et al., BBRC 187:1083-1090 (1992)).

As hereinafter defined, a preferred class of AT2 agonists for use in accordance with the present invention comprises All analogues or active fragments thereof having p-NH-Phe in a position corresponding to a position 6 of AIL In addition to peptide agents, various nonpeptidic agents (e.g., peptidomimetics) having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.

The active All analogues, fragments of All and analogues thereof of particular interest in accordance with the present invention comprise a sequence of at least three contiguous amino acids of groups R -R in the sequence of general formula I R'-R²-R³-R⁴-R⁵-R⁶-R^7"R⁸

wherein R¹ is suitably selected from H, Asp, Glu, Asn, Acpc (1-

aminocyclopentane carboxylic acid), Ala, Me²Gly, Pro, Bet, Glu(NH ), Gly,

Asp(NH₂) and Sue, or R¹ is absent

R² is suitably selected from Arg, Lys, Ala, Citron, Orn, Ser(Ac), Sar, D-Arg

and D-Lys,

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly,

Lys, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, homoSer,

azaTyr, and Ala; R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly; R is selected fro n the group consisting of His, Arg or 6-NH₂-Phe; R is selected fro n the group consisting of Pro or Ala; and R is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group, and wherein the active agent is not All (SEQ ID NO: 1).

In alternate embodiments, the active agents comprise a sequence of at least four, five, six, or seven contiguous amino acids of groups R -R in the sequence of general formula I. In a further alternative, the active agents consist essentially of a sequence of at least four, five, six, or seven contiguous amino acids of groups R'-R⁸ in the sequence of general formula I.

Compounds falling within the category of AT2 agonists useful in the practice of the invention include the All analogues set forth above subject to the restriction that R⁶ is p-NH₂- Phe.

1

Particularly preferred combinations for R and R are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys. Particularly preferred embodiments of this class include the following: AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3-8), also known as desl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(l-7), Asp-Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:4]; AII(2-7). Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:5]; AII(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8), Ile-His-Pro-Phe [SEQ ID NO:7]; AII(l-6), Asp-Arg- Val-Tyr-Ile- His [SEQ ID NO:8]; AII(l-5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AII(l-4), Asp-Arg-Val- Tyr [SEQ ID NO:10]; and AII(l-3), Asp-Arg-Val [SEQ ID NO:l l]. Other preferred embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO: 12] and Arg-Val-Tyr- norLeu-His-Pro-Phe [SEQ ID NO: 13]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His- Pro-Phe [SEQ ID NO:31]. AII(6-8), His-Pro-Phe [SEQ ID NO: 14] and AH(4-8), Tyr-Ile- His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be effective.

Another class of compounds of particular interest in accordance with the present invention are those comprising an amino acid sequence of the general formula II

R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸

in which R² is selected from the group consisting of H, Arg, Lys, Ala, Orn, Citron, Ser(Ac), Sar, D-Arg and D-Lys;

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr; R⁴ is selected from the group consisting of Tyr, Tyr(PO ) , Thr, Ser, homoSer, azaTyr, and Ala;

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, wherein the active agent is not AIL A particularly preferred subclass of the compounds of general formula II has the

formula

R²-R³-Tyr-R⁵-His-Pro-Phe [SEQ ID NO: 16]

wherein R², R³ and R⁵ are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO: 18]. The fragment AII(4-8) was ineffective in repeated tests; this is believed to be due to the exposed tyrosine on the N-terminus.

Other particularly preferred embodiments include:

1GD Ala4-AII(l-7) DRVAIHP SEQ ID NO:38

2GD Pro3-AII(l-7) DRPYIHP SEQ ID NO:39

5GD Lys3-AII(l-7) DRKYIHP SEQ ID NO:40

9GD NorLeu-AII(l-7) DR(nor)YIHP SEQ ID NO:41

GSD 28 Ile⁸-AII DRVYIHPI SEQ ID NO: 42

Ala3aminoPhe6 AIL RVAIHPF SEQ ID NO:43

Ala3-AIII RVAIHPF SEQ ID NO:44

Gly¹ -All GRVYIHPF SEQ ID NO:45

NorLeu⁴-AIII -RVYnLHPF SEQ ID NO:46

Acpc³-AII DR(Acpc)YIHPF SEQ ID NO:47

GSD 37B Orn²-AII D(Orn)VYIHPF SEQ ID NO:48

GSD38B Citron²-AII D(Citron)VYIHPF SEQ ID NO:49

3GD Pro³Ala⁴-AII(l-7) DRPAIHP SEQ ID NO:50

In the above formulas, the standard three-letter abbreviations for amino acid residues are employed. In the absence of an indication to the contrary, the L-form of the amino acid is intended. Other residues are abbreviated as follows:

It has been suggested that All and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974). In general, it is believed that neutral side chains in position R³, R⁵ and R⁷ may be involved in maintaining the appropriate distance between active groups in positions R⁴, R⁶ and R⁸ primarily responsible for binding to receptors and/or intrinsic activity. Hydrophobic side chains in positions R³, R⁵ and R⁸ may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.

Appropriate side chains on the amino acid in position R may contribute to the affinity of the compounds for target receptors, and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R . Alternatively, R₂ may be H, Ala, Orn, Citron, Ser(Ac), Sar, D-Arg, or D-Lys.

For purposes of the present invention, it is believed that R may be involved in the formation of linear or nonlinear hydrogen bonds with R⁵ (in the gamma turn model) or R⁶ (in the beta turn model). R³ would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R³ may suitably be selected from Lys, Val, Ala, Leu, norLeu,

He, Gly, Pro, Aib, Acpc and Tyr. With respect to R⁴, conformational analyses have suggested that the side chain in this position (as well as in R³ and R⁵) contributes to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus, R is preferably selected from Tyr, Thr,

Tyr (PO₃) , homoSer, Ser and azaTyr. In this position, Tyr is particularly preferred, as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra). It has also been found that R⁴ can be Ala. In position R⁵, an amino acid with a β aliphatic or alicyclic chain is particularly desirable. Therefore, while Gly is suitable in position R⁵, it is preferred that the amino acid in this position be selected from He, Ala, Leu, norLeu, and Val.

In the active agents of particular interest in accordance with the present invention, R⁶ is His, Arg or 6-NH₂-Phe. The unique properties of the imidazole ring of histidine (e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R⁶. For example, conformational models suggest that His may 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 presently considered that R⁷ should be Pro or Ala in order to provide the most desirable orientation of R . In position R , both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr, He, Phe(Br), and especially Phe are preferred for purposes of the present invention.

Analogues of particular interest include the following: TABLE 2 Angiotensin II Analogues

The polypeptides of the instant invention may be synthesized by any conventional method, including, but not limited to, those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, 111. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, 77ze Peptides, Vol. 1 , Academic Press, New York, (1965) for solution synthesis. The disclosures of the foregoing treatises are incorporated by reference herein.

In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616, herein incorporated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.

Preferably, peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif), according to the manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well known to those skilled in the art. Alternatively, the peptides can be produced by standard molecular biological techniques.

In one aspect, the present invention provides a method of treating or preventing diabetic complications, such as proteinuria and nephropathy, by administering to a patient in need thereof an amount effective to treat or prevent diabetic complications of angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All analogues, AH fragments and analogues thereof, AH AT₂ type 2 receptor agonists, ("active agents"), either alone, combined, or in further combination with other compounds effective for treating or preventing diabetic nephropathy, including but not limited to insulin and ACE inhibitors.

For use in treating or preventing diabetic nephropathy, the active agents may be administered by any suitable route, including orally, parentally, by inhalation spray, rectally, transdermally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.

In one embodiment, the active agent is applied subcutaneously in effective amounts for a period of time beginning after diagnosis of diabetes, and continuing for a period of time sufficient for treating or preventing diabetic nephropathy or other diabetic complications. The active agent is preferably administered as soon as possible after the diagnosis of diabetes.

A large variety of alternatives are known in the art as suitable for purposes of sustained release and are contemplated as within the scope of the present invention. Suitable delivery vehicles include, but are not limited to, the following: microcapsules or microspheres; liposomes and other lipid-based release systems; crystalloid and viscous instillates; absorbable and/or biodegradable mechanical barriers; and polymeric delivery materials, such as polyethylene oxide/polypropylene oxide block copolymers (e.g. poloxamers), poly-orthoesters, cross-linked polyvinyl alcohol, polyanhydrides, polymethacrylate and polymethacrylamide hydrogels, anionic carbohydrate polymers, etc. Useful delivery systems are well known in the art and are described in, e.g., U.S. Pat. No. 4,937,254, the entire disclosure of which is hereby incorporated by reference.

The active agents may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions), and may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as stabilizers, wetting agents, emulsifiers, preservatives, cosolvents, suspending agents, viscosity enhancing agents, ionic strength and osmolality adjustors and other excipients in addition to buffering agents. Suitable water soluble preservatives which may be employed in the drug delivery vehicle include sodium bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol, phenylethanol or antioxidants such as Vitamin E and tocopherol and chelators such as EDTA and EGTA. These agents may be present, generally, in amounts of about 0.001% to about 5% by weight and, preferably, in the amount of about 0.01 to about 2% by weight.

For administration, the active agents are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tra acanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art. The dosage regimen for treating or preventing diabetic complications with the active agents is based on a variety of factors, including the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Dosage levels on the order of between 0.1 ng/kg and 10 mg/kg of the active agents per body weight are useful for all methods of use disclosed herein, dosages between about 1 ng/kg and 1 mg/kg are preferred; more preferred

are dosages between about 100 ng/kg and 500 μg/kg, and most preferred is a dosage range of

between about 1 μg/kg and about 100 μg/kg.

In a further aspect, the present invention provides kits for treating or preventing diabetic complications, wherein the kits comprise an effective amount of the active agents of the invention to treat or prevent diabetic complications, and instructions for using the amount effective of active agent to treat or prevent diabetic complications. In a preferred embodiment, the kits also contain an effective amount to treat or prevent diabetic complications of one or more other compounds, such as insulin or ACE inhibitors, to be used in combination with the active agents of the invention.

Effective dosages of the active agents of the invention to treat or prevent diabetic nephropathy are between about 0.1 ng/kg and 10 mg/kg, as discussed above.

In another aspect of the invention, pharmaceutical compositions are provided that comprise an amount effective to treat or prevent diabetic complications of one or more of the active agents of the invention, either alone or in combination with an amount effective to treat or prevent diabetic complications of insulin or an ACE inhibitor, including but not limited to those described above.

The present invention, by providing methods for treat or prevent diabetic complications is broadly useful for all diabetic patients. The present invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed to limit the scope of the invention.

Example 1. Effect of All and analogues on proteinuria and diabetic nephropathy Male diabetic mice (db/db) that are genetically predisposed to develop the disorders associated with diabetes were purchased at 6 weeks of age for Jackson Laboratories. Upon arrival and quarantine (1 week), the mice were given daily subcutaneous injections of

Ringers' Lactate Solution (RLS) (placebo, 100 μl) or peptide (100 μg/kg/day or 10

μg/kg/day). At 9, 9.5, and 10 weeks of age, the amount of glucose (as a measure of disease)

and protein (proteinuria; as a measure of kidney damage or nephropathy) in the urine was evaluated by URISTIX™. (Bayer Corporation, Diagnostics Divisions, Elkhart, IN)

Administration of AH accelerated the development of kidney pathology in these mice

(Figure 1). Administration of AII(l-7) or, at later time points, 9GD (NorLeu-3-AII(l-7)), reduced the protein measured in the urine of diabetic mice. These data are represented as the rank order analysis of the values: Trace, 1+, 2+, 3+, 4+, which have the following meanings as used herein:

Trace (or 0): less than 30 mg/dL l+: 30 mg/dL 2+: 100 mg/dL 3+: 300 mg/dL

4+: over 2000 mg/dL As is known to those of skill in the art, "rank order" is the ranking of the non- parametric scores in order and assigning consecutive values to the rank. Within a score of trace, 1+, 2+, 3+ or 4+, all ranks with the same score are given an average of the values. All tied scores are then given that rank and the mean and standard error of those ranks are given on the y axis. The individual scores were as follows:

Group 9 w 9.5 wk 10 wk score score score

Control 2 1 1

Control 2 2 2

Control 2 2 3

Control 2 2 4

Control 3 4 4

All 100 1 1 1

All 100 3 2 2

All 100 4 2 2

All 100 4 3 3

All 100 4 3 4

A7 100 2 1 0

A7 100 0 0

A7 100 1 0

A7 100 1 1

A7 10 0 0 0

A7 10 0 0

A7 10 1 1

A7 10 2 2 2

9gd 100 1 0

9gd 100 2 1

9gd 100 3 2 2

9gd 10 1 1

9gd 10 2 2 2

9gd 10 3 3 3

It is to be understood that the invention is not to be limited to the exact details of operation, or to the exact compounds, compositions, methods, procedures or embodiments shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the full scope of the appended claims.

Claims

We claim

1. A method for treatirg and preventing diabetic complications comprising administering to a diabetic subject an amount effective to treat or prevent diabetic complications of at least one active agent comprising a sequence of at least three contiguous

1 8 amino acids of groups R -R in the sequence of general formula I

R¹ -R²-R³-R⁴-R⁵-R⁶-R^7"R⁸ wherein R¹ is selected from the group consisting of H, Asp, Glu, Asn, 1- aminocyclopentane carboxylic acid, Ala, N,N-dimethylglycyl, Pro, 1-carboxy- N,N,N-trimethylmethanaminium hydroxide inner salt, Glu(NH ), Gly, Asp(NH₂) and

Succinyl, wherein R 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, Nle, He, Gly, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, diphospho tyrosine, Thr, Ser,

homoserine, and aza-α'-homo-L-tyrosyl;

R⁵ is selected from the group consisting of He, Ala, Leu, Nle, Val and Gly;

R⁶ is selected from the group consisting of His, Arg or 6-NH -Phe; R⁷ is selected from the group consisting of Pro or Ala; and

R⁸ is selected from the group consisting of Phe, p-bromo-L-phenylalanyl, He and Tyr; excluding sequences including R⁴ as a terminal Tyr group, and wherein the active agent is not SEQ ID NO: 1.

2. The method of claim 1, wherein the active agent comprises a sequence of at least four contiguous amino acids of groups R^]-R⁸ in the sequence of general formula I.

3. The method of claim 1, wherein the active agent comprises a sequence of at least five contiguous amino acids of groups R^]-R⁸ in the sequence of general formula I.

4. The method of claim 1, wherein the active agent comprises a sequence of at least six

1 8 contiguous amino acids of groups R -R in the sequence of general formula I.

5. The method of claim 1, wherein the active agent comprises a sequence of at least

1 8 seven contiguous amino acids of groups R -R in the sequence of general formula I.

6. The method of claim 1 wherein the active agent consists essentially of a sequence of

1 8 at least three contiguous amino acids of groups R -R in the sequence of general formula I.

7. The method of claim 1 wherein the active agent consists essentially of a sequence of

1 8 at least four contiguous amino acids of groups R -R in the sequence of general formula I.

8. The method of claim 1 wherein the active agent consists essentially of a sequence of at least five contiguous amino acids of groups R'-R⁸ in the sequence of general formula I.

9. The method of claim 1 wherein the active agent consists essentially of a sequence of

1 8 at least six contiguous amino acids of groups R -R in the sequence of general formula I.

10. The method of claim 1 wherein the active agent consists essentially of a sequence of

1 8 at least seven contiguous amino acids of groups R -R in the sequence of general formula I.

11. The method of claim 1 wherein the active agent comprises an amino acid sequence selected from the group consisting of angiotensinogen, SEQ ID NO:2, SEQ ID NO:3, SEQ

ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17,

SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ

ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO.26, SEQ ID NO:27, SEQ ID

NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,

SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.

12. The method of claim 1 wherein the active agent comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:41.

13. The method of claim 1 wherein the active agent consists essentially of an amino acid sequence selected from the group consisting of angiotensinogen, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.

14. The method of claim 1 wherein the active agent consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:41.

15. The method of claim 1 wherein the dosage of active agent is between about 0.01 ng/kg and about 10.0 mg/kg body weight of the subject.

16. The method of claim 1 wherein the diabetic complication is selected from the group consisting of proteinuria and diabetic nephropathy.

17. A pharmaceutical composition comprising:

(a) the active agent of claim 1;

(b) an amount effective for treating or preventing diabetic complications, of a compound selected from the group consisting of insulin and ACE inhibitors; and (c) a pharmaceutically acceptable carrier.

18. A pharmaceutical composition comprising:

(a) the active agent of claim 11 ;

(b) an amount effective for treating or preventing diabetic complications, of a compound selected from the group consisting of insulin and ACE inhibitors; and

(c) a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising:

(a) the active agent of claim 12;

(c) a pharmaceutically acceptable carrier.