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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2013/028501 Al 28 February 2013 (28.02.2013) P O P C T

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 31/201 (2006.01) A61K 31/401 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/20 (2006.01) A61P 13/12 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, A61K 31/41 (2006.01) A61P 43/00 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, A61K 31/4178 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/US20 12/05 1304 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (22) International Filing Date: NO, NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, 17 August 2012 (17.08.2012) SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (25) Filing Language: English zw. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 61/525,543 19 August 201 1 (19.08.201 1) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (71) Applicant (for all designated States except US): THE TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, UNIVERSITY OF UTAH RESEARCH FOUNDATION EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, LV, [US/US]; Suite 310, 615 Arapeen Drive, Salt Lake City, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, UT 84108 (US). TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (72) Inventor; and (75) Inventor/Applicant (for US only): YANG, Tianxin Published: [US/US]; 4343 S. 1350 E., Salt Lake City, UT 84124 (US). — with international search report (Art. 21(3)) (74) Agents: MEARA, Joseph, P. et al; Foley & Lardner LLP, Verex Plaza, 150 E. Oilman Street, Madison, WI 53703 (US).

(54) Title: COMBINATION THERAPY WITH NITRATED LIPIDS AND INHIBITORS OF THE -- AL DOSTERONE SYSTEM

5 1 ¾

© (57) Abstract: The present technology provides compositions and methods for treating chronic kidney disease, end-stage renal dis - o ease, or diabetic nephropathy. The compositions comprise a nitrated lipid and an inhibitor of the renin-angiotensin-aldosterone sys - tem. The methods comprise administering a nitrated lipid in combination with an inhibitor of the renin-angiotensin-aldosterone sys tem to a subject in need thereof, in an amount effective to treat diabetic nephropathy, chronic kidney disease, and/or end-stage renal o disease. The use of a nitrated lipid with an inhibitor of the renin-angiotensin-aldosterone system exhibits a synergistic effect in treat ing chronic kidney disease and diabetic nephropathy. COMBINATION THERAPY WITH NITRATED LIPIDS AND INHIBITORS OF THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional

Application No. 61/525,543, filed August 19, 201 1, the entire disclosure of which is incorporated herein by reference.

STATEMENT OF GOVERNMENT RIGHTS

[0002] This invention was made with government support under 079162 by the National Institutes of Health. The government has certain rights to this invention.

FIELD OF THE INVENTION

[0003] The present technology relates to compositions and methods for treating chronic kidney disease (CKD). More specifically, a combination of nitrated lipid and an inhibitor of the renin-angiotensin-aldosterone system may be used for treatment of CKD and end-stage renal disease, including, but not limited to, that associated with diabetes.

BACKGROUND OF THE INVENTION

[0004] Diabetes and diabetic complications represent a major public health problem, affecting 25 million Americans. In particular, diabetes and diabetic nephropathy constitute a major cause of CKD which progressively develops to end-stage renal disease (ESRD); patients with ESRD typically require dialysis or a kidney transplant. Currently, PPARy agonists, thiazolidinediones (TZDs), are effective antidiabetic agents but are associated with severe edema, body weight gain and cardiovascular events. Inhibitors of the renin- angiotensin-aldosterone system (RAAS), which are widely used as anti-hypertensive agents, can help alleviate high blood pressure accompanying CKD but fail to stop the progression of CKD to ERSD. Indeed, in some instances, combination treatment with both an ACE inhibitor and an angiotensin receptor inhibitor has been shown to worsen major renal outcomes such as increasing serum creatinine and causing a greater decline in estimated glomerular filtration rate. Yusuf, S., et al., New England J. Med. (2008) 358 (15): 1547-59. SUMMARY

[0005] The present technology provides methods, compositions and medicaments useful in the treatment of chronic kidney disease and/or diabetic nephropathy. The methods involve administration of a nitrated lipid in combination with an inhibitor of RAAS to a subject in need thereof in amounts effective to treat the chronic kidney disease and/or diabetic nephropathy. This combination appears to be synergistic for reducing albuminuria, urinary and renal TBARS, and COX-2 mRNA expression in the kidney, all of which are diagnostic for diabetic nephropathy and chronic kidney disease (see FIGS. 1-4). Conversely, the combination of nitrated lipid and RAAS inhibitor synergistically increase the expression of renoprotective heme oxygenase 1 (HO-1) (FIG. 5). Hence, in one aspect there are provided compositions including a nitrated lipid and an inhibitor of RAAS for separate, simultaneous or sequential administration. Further, the use of a nitrated lipid and an inhibitor of RAAS in the preparation of a medicament for treatment of chronic kidney disease and/or diabetic nephropathy are provided.

[0006] The present methods include of treatment include administering an effective amount of a nitrated lipid and an inhibitor of the RAAS to a subject suffering from chronic kidney disease, diabetic nephropathy or hypertensive nephropathy. A variety of nitrated lipids may be used, including, but not limited to, nitro-fatty acids or esters thereof. Similarly, a variety of fatty acids are compatible with the disclosed methods, including, but not limited to, monounsaturated and polyunsaturated fatty acids. In some embodiments, the nitrated lipid is 9-nitrooleic acid, 10-nitrooleic acid or combinations thereof. In certain embodiments the RAAS inhibitor is an ACE inhibitor, a or an angiotensin receptor inhibitor, e.g., .

BRIEF DESCRIPTION OF THE FIGURES

[0007] FIG. 1 is a bar graph comparing the amounts of urinary albumin in db/db mice before and after a 12 day (12-d) administration of vehicle, losartan, nitrooleic acid (OA-N02), or losartan + OA-N02. Lean mice (non-diabetic) with vehicle treatment were used as controls losartan was administered via diet, while OA-N02 and vehicle were each infused via osmotic mini-pump. Lean: n = 5; vehicle: n = 10; losartan: n = 8; OA-N02: n = 9; losartan + OA-N02: n = 10. Data are mean ± SE. [0008] FIG. 2 is a bar graph comparing the amounts of urinary thiobarbituric acid reactive substances (TBARS) in db/db mice before and after 12-d infusion with vehicle, losartan, nitrooleic acid (OA-N02), or losartan + OA-N02. Lean mice (non-diabetic) with vehicle treatment were used as controls. Losartan was administered via diet, while vehicle and OA-N02 were each infused via osmotic mini-pump. Lean: n = 5; vehicle: n = 10; losartan: n = 8; OA-N02: n = 9; losartan + OA-N02: n = 10. Data are mean ± SE.

[0009] FIG. 3 is a bar graph showing the amounts of renal TBARS in db/db mice after a 12-d administration of vehicle, losartan, nitrooleic acid (OA-N02), or losartan + OA- N02. Lean mice (non-diabetic) with vehicle treatment were used as controls. Losartan was administered via diet, while vehicle and OA-N02 were infused via osmotic mini-pump. Lean: n = 5; vehicle: n = 10; losartan: n = 8; OA-N02: n = 9; losartan + OA-N02: n = 10. Data are mean ± SE.

[0010] FIG. 4 shows the amounts of renal COX-2 protein in db/db mice after a 12-d administration of vehicle, losartan, nitrooleic acid (OA-N02), or losartan + OA-N02. Lean mice (non-diabetic) with vehicle treatment were used as controls. Renal COX-2 protein was analyzed by using immunoblotting. Shown are representative results from 2 animals per group.

[0011] FIG. 5 shows the amounts of renal heme oxygenase- l(HO-l) protein in db/db mice after a 12-d administration of vehicle, losartan, nitrooleic acid (OA-N02), or losartan + OA-N02. Lean mice (non-diabetic) with vehicle treatment were used as controls. Renal HO-1 protein was analyzed by using immunoblotting. Shown are representative results from 2 animals per group.

DETAILED DESCRIPTION

[0012] The following terms are used throughout as defined below.

[0013] "ACE inhibitor" is an inhibitor of the angiotensin I converting enzyme (ACE). ACE is a zinc proteinase that converts the peptide hormone angiotensin I to angiotensin II. Inhibitors of ACE include Zn chelating functionality such as carboxyl or sulfhydryl groups.

[0014] Alkyl groups include straight chain, branched chain, or cyclic alkyl groups having 1 to 24 carbons or the number of carbons indicated herein. In some embodiments, an alkyl group has from 1 to 16 carbon atoms, from 1 to 12 carbons, from 1 to 8 carbons or, in some embodiments, from 1 to 6, or 1, 2, 3, 4 or 5 carbon atoms. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups.

[0015] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 24 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2, 3 or 4 carbon atoms. Examples include, but are not limited to vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), and -C(CH2CH )=CH2, among others.

[0016] "Diabetic nephropathy" refers to progressive kidney disease or damage due to diabetes. While the exact cause is not clear, it appears that high blood sugar leads to damage of the glomeruli and associated nephrons of the kidney. The resulting glomerulosclerosis, scarring and obstruction of the glomeruli, causes the kidney structures to begin to leak and protein begins to pass into the urine. The main sign of diabetic nephropathy is thus persistent protein in the urine (urinary albumin, also known as albuminuria).

[0017] Diabetic nephropathy is a leading cause of chronic kidney disease (CKD), where kidney function is lost over time until end-stage renal disease (ESRD) develops. ESRD is the complete or near-complete failure of the kidneys to function at a level needed for day-to-day life. ESRD usually occurs when diabetic nephropathy induced CKD worsens to the point where kidney function is less than 10% of normal. Subjects with ESRD typically require dialysis or a kidney transplant.

[0018] Oxidative stress is known to play an essential role in the pathogenesis of diabetic nephropathy. NADPH oxidase 4 (NOX4) is a major oxidant generating enzyme that is implicated to play a major pathogenic role in the development of diabetic nephropathy. A product from the oxidation of lipids by NOX4 or other oxidant generating systems is thiobarbituric acid reactive substances (TBARS). Thus, urinary and renal TBARS can be measured as an index of oxidative stress in diabetic nephropathy. [0019] Cyclooxygnase-2 (COX-2) has been implicated to play a major role in the pathogenesis of diabetic nephropathy. In particular, increased renal COX-2 expression has been demonstrated in animal models of diabetic nephropathy and may serve as a marker of such a condition.

[0020] Heme oxygenase- 1 (HO-1) is an enzyme that catalyzes the degradation of heme, resulting in the production of biliverdin, iron, and carbon monoxide. Abundant evidence supports a renoprotective role for HO-1 in various types of kidney injury. Raised levels of HO-1 may therefore indicate an enhanced renoprotective effect against chronic kidney disease and diabetic nephropathy.

[0021] The renin-angiotensin-aldosterone system (RAAS) is a hormonal system that is traditionally considered to play a major role in regulation of blood pressure and blood volume. Emerging evidence suggests that this system also plays a pro-inflammatory and pro-oxidative role in the pathogensis of CKD. In the RAAS, renin converts angiotensinogen (secreted by the liver) to angiotensin I. Angiotensin I is converted to angiotensin II by angiotensin converting enzyme (ACE). Angiotensin II binds to AT receptors to cause, among other effects, blood vessels to constrict and aldosterone to be released by the adrenal cortex, and also to elicit pro-inflammatory and pro-oxidant responses. Inhibitors of this system (e.g., ACE inhibitors, renin inhibitors and angtiotensin receptor inhibitors) are used therapeutically to lower blood pressure in hypertensive subjects and also to treat CKD.

[0022] "Renin inhibitor" is an inhibitor of the aspartic proteinase, renin. The latter enzyme, as part of the RAAS, converts the peptide angiotensinogen to angiotensin I.

[0023] "Treating" means an alleviation, in whole or in part, of symptoms associated with a condition or disorder (e.g., disease), or halt of further progression or worsening of those symptoms. Similarly, as used herein, an "effective amount" of a compound disclosed herein refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with a condition or disorder, or halts further progression or worsening of those symptoms. For example, in treating diabetic nephropathy or chronic kidney disease, a slowed or halted increase in albuminuria or a decrease in albuminuria are examples of desirable treatment results. In another example, treating diabetic or chronic kidney disease may include slowing or halting the increase in urinary or renal TBARS or a decrease in COX-2 expression. Further, treating does not necessarily occur by administration of one dose of the compound, but often occurs upon administration of a series of doses. Thus, an effective amount may be administered in one or more doses.

[0024] The compositions and methods disclosed herein make use of nitrated lipids.

Nitrated lipids are lipids comprising at least one nitro (N0 2) group covalently bonded to the lipid. The methods disclosed herein encompass administration of a single type of nitrated lipid or a mixture of two or more different types of nitrated lipids. By way of example, 9- nitro-9-czs-octadecenoic acid is one type of nitrated lipid. A single type of nitrated lipids is distinguished from other types by the identity of the lipid and number and position of N0 2 groups.

[0025] A variety of lipids may be used to form the nitrated lipids. In general, useful lipids include, but are not limited to, fats and fat-derived materials. In some embodiments, the lipid is a fatty alcohol, sterol, or complex lipid. Examples of complex lipids include, but are not limited to, glycerolipids (e.g., compounds having a glycerol backbone including, but not limited to, phospholipids, glycolipids, monoglycerides, diglycerides, triglycerides) or cholesterol (e.g., cholesterols having fatty acids attached to it such as cholesterol linoleate). Other examples of nitrated lipids include, but are not limited to, those disclosed in U.S. Patent Publication No. 2007/0232579.

[0026] Alternatively, the lipid is a fatty acid or ester thereof such as a C -C2 4 fatty C10-C22, C12-C20, acid or ester. In some embodiments the fatty acid or ester is a C14-C 1 or C14-C22 fatty acid or ester. A fatty acid is alkyl or alkenyl in which the terminal carbon is a

COOH group. In some embodiments, the alkyl or alkenyl is a C -C2 4 alkyl or alkenyl. In other embodiments, the alkyl or alkenyl has 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 carbons or falls in a range between and including any two values thereof. In some embodiments, the alkyl or alkenyl is branched or unbranched. A wide variety of fatty acids may be used, including, but not limited to monounsaturated fatty acids and polyunsaturated fatty acids. In some embodiments, the monounsaturated fatty acid is oleic acid or linoleic acid. In some embodiments, the oleic acid is 9-nitrooleic acid, 10-nitrooleic acid, or combinations thereof.

[0027] Nitrated lipids may be synthesized according to known procedures. For example, U.S. Patent Publication No. 2007/0232579 discloses a procedure comprising the steps of reacting a lipid with a mercuric salt, a selenium compound, and a nitrating compound to produce a first intermediate and reacting the first intermediate with an oxidant. Useful mercuric salts, selenium compounds, nitrating compounds, oxidants, relative amounts of reactants, and reaction conditions are also disclosed in U.S. Patent Publication No. 2007/0232579. Such synthetic procedures may provide mixtures of two or more types of nitrated lipids which may be separated or purified by techniques known in the art, if desired.

[0028] The lipids described above may be obtained from a variety of sources. For example, lipids may be commercially available or may be obtained from natural sources. Plant oils, including, but not limited to olive oil, linseed oil, flaxseed oil, rapeseed oil, and perilla oil are possible natural sources of fatty acid lipids. Fish oils or other marine oils are other possible sources of fatty acids. Nitrated lipids present in any of these or other natural sources may be extracted and/or purified for use in the methods disclosed herein.

[0029] In one aspect, the present technology provides compositions including a nitrated lipid and an inhibitor of the renin-angiotensin-aldosterone system. In some embodiments of the present compositions, the nitrated lipid is a nitrated monounsaturated fatty acid or a nitrated polyunsaturated fatty acid. In some embodiments, the nitrated lipid is a nitrooleic acid or a nitrolinoleic acid such as, e.g., 9-nitrooleic acid, 10-nitrooleic acid, or a combination thereof.

[0030] The inhibitor of the RAAS may be any such inhibitor known in the art such as an ACE inhibitor, a renin inhibitor, or an angiotensin receptor antagonist. In some embodiments, the inhibitor of the RAAS is an ACE inhibitor such as , , , , , , , , , or . In certain embodiments, the inhibitor of the RAAS is an angiotensin receptor inhibitor such as losartan, , , , , , , or . For example, in some embodiments, the inhibitor of the RAAS is losartan. In certain embodiments, the inhibitor of the RAAS is a renin inhibitor such as , remikiren, or renin siRNA. In some embodiments, the present compositions may include two or more inhibitors of the renin-angiotensin-adosterone system. For example, compositions of the present technology may include an ACE inhibitor and losartan.

[0031] In some embodiments, the composition of the present technology is a pharmaceutical composition that includes any of the compositions disclosed herein and a pharmaceutically acceptable additive, such as, e.g., pharmaceutically acceptable carriers and excipients. The pharmaceutical composition may be any number of pharmaceutical formulations capable of various administration routes e.g., oral administration, topical administration, transdermal administration, by nasal administration, rectal administration, subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection. The formulations can take the form of granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. These formulations may further include a variety of well-known pharmaceutically acceptable additives, carriers ,and/or excipients as necessary. Any of the formulations, delivery methods, and pharmaceutically acceptable additives, carriers, and excipients disclosed in U.S. Patent Publication No. 2007/0232579 may also be used in the pharmaceutical composition described herein.

[0032] Combinations of the present technology may be administered separately, simultaneously, or sequentially. Thus, the present technology provides for the administering of each of the components separately but as part of the same therapeutic treatment program or regimen, and it is contemplated that separate administration of each compound, at different times and by different routes, will sometimes be recommended. Thus the two components need not necessarily be administered at essentially the same time. In the one embodiment the nitrated lipid will be given one or more days prior to or after the administration of the inhibitor of the RAAS either daily or "on demand". In another embodiment, administration is timed so that the peak pharmacokinetic effect of the nitrated lipid precedes or coincides with the peak pharmacokinetic effect of RAAS inhibitor. In some embodiments, both components are administred in an oral dosage form.

[0033] In some embodiments, the present technology provides a kit that includes a separate or combined composition(s) that include the nitrated lipid and the inhibitor of the renin-antiotensin-aldosterone system. The kit may include a container for containing the separate compositions such as a divided bottle or a divided foil packet, wherein each compartment contains a plurality of dosage forms (e.g., tablets) comprising either the nitrated lipid or the RAAS inhibitor. Alternatively, rather than separating the active ingredient-containing dosage forms, the kit may contain separate compartments each of which contains a whole dosage which comprises separate compositions. An example of this type of kit is a blister pack wherein each individual blister contains two tablets, one tablet comprising the nitrated lipid, the other comprising the RAAS inhibitor. The nitrated lipid and the inhibitor of the renin-antiotensin-aldosterone system may also be formulated as a single composition for simultaneous administration, e.g., a single tablet or capsule.

[0034] Typically the kit comprises directions for the administration of the separate components. Such instructions would cover situations such as: i) the dosage form in which the components are administered (e.g. oral and parenteral), ii) when the component parts of the product are administered at different dosage intervals, or iii) when titration of the individual components of the combination is desired by the prescribing physician. The container may have deposited thereon a label that describes the contents therein and any appropriate warnings.

[0035] In another aspect the present technology provides method of treatment for chronic kidney disease, diabetic nephropathy and/or hypertensive nephropathy. In some embodiments of the methods, the subject has chronic kidney disease or end-stage renal disease. The methods include administering a nitrated lipid in combination with an inhibitor of the RAAS to a subject in need thereof, in an amount effective to treat chronic kidney disease, end-stage renal disease, diabetic nephropathy and/or hypertensive nephropathy. In some embodiments of the methods, the nitrated lipid is a nitrated monounsaturated fatty acid or a polyunsaturated fatty acid. In some embodiments, the nitrated lipid is a nitrooleic acid or a nitrolinoleic acid. For example, the nitrated lipid may be selected from 9-nitrooleic acid, 10-nitrooleic acid, or combinations thereof.

[0036] In methods of the present technology, the inhibitor of the RAAS can be an ACE inhibitor, a renin inhibitor, or a angiotensin receptor antagonist. Thus, the inhibitor of the RAAS maybe, e.g., enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, zofenopril, fosinopril, or captopril. In some embodiments, the inhibitor of the RAAS can be losartan, valsartan, telmisartan, irbesartan, azilsartan, olmesartan, candesartan, or eprosartan. In some embodiments, the inhibitor of the RAAS is losartan. In certain embodiments, the inhibitor of the RAAS can be aliskiren, remikiren, or renin siRNA. In the present methods , two or more inhibitor of the RAAS may be employed. In some embodiments of the methods, the inhibitor of the RAAS is an ACE inhibitor and losartan.

[0037] In another aspect of the present technology, the combinations and compositions disclosed herein may be used in a prophylactic manner to prevent diabetic nephropathy or hypertensive nephropathy. In particular, there are provided methods including administering a nitrated lipid in combination with an inhibitor of the RAAS to a subject in need thereof, in an amount effective to prevent chronic kidney disease or end- stage renal disease. Any of the nitrated lipids, the inhibitors of the renin-angitotensin- aldosterone system, or compositions comprising such compounds as disclosed herein may be used in such methods. For example, in some embodiments, the nitrated lipid is selected from 9-nitrooleic acid, 10-nitrooleic acid, or combinations thereof. In some embodiments, the inhibitor of the RAAS is losartan.

[0038] Specific effective amounts of the nitrated lipids to be administered will vary depending upon a variety of factors, e.g., the condition to be treated, the age, body weight, general health, sex, and diet of the subject, the dose intervals, and the administration route. In some embodiments, the effective amount of the nitrated lipid ranges from about 1 µg per day to about 100 mg per day, from about 1 mg per day to about 50 mg per day, from about 1 mg per day to about 25 mg per day, or from about 2 mg per day to about 10 mg per day.

[0039] Any of the nitrated lipids disclosed herein may be administered to the subject alone or in combination with one or more other therapeutic agents. By "administered in combination," it is meant that the nitrated lipids and the therapeutic agents may be administered as a single composition, simultaneously as separate doses, or sequentially. Sequential administration refers to administering the nitrated lipids and at least one therapeutic agent either before or after one another.

[0040] The nitrated lipids may be administered to a subject via any number of pharmaceutical formulations and administration routes. The formulations can take the form of granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. These formulations may further include a variety of well-known pharmaceutically acceptable additives, carriers, and/or excipients as necessary. The formulations may be delivered to the subject by various routes of administration, e.g., by topical administration, transdermal administration, oral administration, by nasal administration, rectal administration, subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection. Any of the formulations, delivery methods, and pharmaceutically acceptable additives, carriers, and excipients disclosed in U.S. Patent Publication No. 2007/0232579 may also be used with the methods described herein. Another possible route of administration includes incorporating the nitrated lipid into various food products. Food products, include, but are not limited to butter, margarine, vegetable oils, and the like.

[0041] The subjects of the disclosed methods include any animal that can benefit from the administration of a nitrated lipid. In some embodiments, the subject is a mammal, e.g., a human, a primate, a dog, a cat, a horse, a cow, a pig, or a rodent, e.g., a rat or mouse. Typically, the mammal is a human.

[0042] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0043] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure. [0044] For the purposes of this disclosure and unless otherwise specified, "a" or "an" means "one or more."

[0045] The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES

Materials and Methods:

[0046] Animals. Male 3-4-month-old db/db mice were from Jackson Laboratories (Bar Harbor, Me). All animals were housed in an air-conditioned room with a 12-hour light/dark cycle. All procedures and protocols were in accordance with guidelines set by the Laboratory Animal Care Committee at the University of Utah.

[0047] Materials. 9-Nitrooleic acid and 10-nitrooleic acid are two regioisomers of nitrooleic acid (OA-N0 2), which are formed by nitration of oleic acid in approximately equal proportions (Hayama et al. Chemistry Letters pp.1 109-1 112,1982). The two compounds were purchased from Cayman Chemicals (Ann Arbor, MI) (9-nitrooleic acid:

Cat#10008042; 10-nitrooleic acid: Cat#10008043) and used as an 1:1 mixture of the isomers.

Protocols for animal experiments.

[0048] db/db mice were implanted with a 3-week osmotic mini-pump which delivered a vehicle or OA-N0 2 at 10 mg/kg/d. Animals were fed with losartan, which is an ATI blocker. 24-h urine samples were collected before and after the treatment.

Example 1: Measurement of urinary albumin in diabetic mice treated with nitrated oleic acid, losartan or both

[0049] As compared with lean mice, untreated db/db mice developed severe albuminuria (FIG. 1) over the course of the 12-day study. Neither losartan or OA-N02 alone exhibited a statistically significant effect on urinary albumin excretion. In contrast, the combination of the two drugs induced a synergistic reduction of urinary albumin excretion (P<0.05). The changes of urinary albumin relative to the vehicle group by Losartan alone, OA-N02 alone, and Losartan + OAN02 were +7%, -13%, and -42.6%. The plus symbol (+) means an increase and the minus symbol (-) indicates a decrease in albuminuria.

Example 2 : Measurement of urinary and renal TBARS in diabetic mice treated with nitrated oleic acid, losartan or both

[0050] Untreated diabetic mice exhibited significant urinary and renal TBARS

(FIGS. 2-3). The combination of OA-N0 2 and losartan induced a striking reduction of urinary TBARS whereas treatment with only one of OA-N0 2 or losartan was without an effect (FIG. 2). The changes of urinary TBARS relative to the vehicle group after 12 days for losartan alone, OA-N02 alone, and losartan + OAN02 were +33.4%, +16.5%, and - 47.6%, %, respectively. Renal TBARS content in each of losartan alone and OA-OA2 alone group showed a trend of reduction but none of these changes reached statistical significance. A statistically significant reduction was only found in the losartan + OA-N02 group (FIG. 3).

Example 3 : Measurement renal COX-2 protein expression in diabetic mice treated with nitrated oleic acid, losartan, or both

[0051] Renal COX-2 expression in db/db mice treated as above was examined by immunoblotting. As compared with lean controls, a marked induction of COX-2 protein expression was found in the db/db vehicle group. Losartan or OA-N02 alone only produced a modest effect on renal COX-2 expression (FIG. 4). In contrast, the COX-2 expression was significantly suppressed in the losartan + OA-N02 group (FIG. 4). The changes of renal COX-2 protein expression relative to the vehicle group by losartan alone, OA-N02 alone, and losartan + OAN02 were -5.3%, -14.8%, and -38.8%, respectively.

Example 4 : Measurement renal heme oxygenase-l protein expression in diabetic mice treated with nitro-oleic acid, losartan, or both

[0052] Renal HO-1 expression was examined using immunoblotting. Either losartan or OA-N02 alone elevated renal HO-1 expression and the combination of the two agents produced a much greater (synergistic) effect (FIG. 5). The changes of renal HO-1 protein expression relative to the vehicle group by losartan alone, OA-N02 alone, and losartan + OA-N02 were +97.6%, +48.8%, and +21 1.4%, respectively. [0053] Overall, consistent results from analysis of the above-mentioned 5 key parameters relevant to the diabetic kidney injury demonstrated that renoprotective action of the combination of Losarton and OA-N02 is greater than the additive effects of the single treatments. These results provide compelling evidence supporting a strong synergy between nitrated fatty acids and an inhibitor of renin-angiotensin system in management of diabetic nephropathy. CLAIMS

WHAT IS CLAIMED IS:

1. A composition comprising a nitrated lipid and an inhibitor of the renin-angiotensin- aldosterone system.

2. The composition of claim 1, wherein the nitrated lipid comprises a monounsaturated fatty acid or a polyunsaturated fatty acid.

3. The composition of claim 1, wherein the nitrated lipid is a nitrooleic acid or a nitrolinoleic acid.

4. The composition of claim 1, wherein the nitrated lipid is selected from 9-nitrooleic acid, 10-nitrooleic acid, or combinations thereof.

5. The composition of claim 1, wherein the inhibitor of the renin-angiotensin- aldosterone system is an ACE inhibitor, a renin inhibitor, or a angiotensin receptor antagonist.

6. The composition of claim 1, wherein the inhibitor of the renin-angiotensin- aldosterone system is enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, zofenopril, fosinopril, or captopril.

7. The composition of claim 1, wherein the inhibitor of the renin-angiotensin- aldosterone system is losartan, valsartan, telmisartan, irbesartan, azilsartan, olmesartan, candesartan, or eprosartan.

8. The composition of claim 1, wherein the inhibitor of the renin-angiotensin- aldosterone system is losartan.

9. The composition of claim 1, wherein the inhibitor of the renin-angiotensin- aldosterone system is aliskiren, remikiren, or siR A.

10. The composition of claim 1, comprising an additional inhibitor of the renin- angiotensin-adosterone system.

11. The composition of claim 10, comprising an ACE inhibitor and losartan. 12. A pharmaceutical composition comprising the composition of any one of claims 1 - 11 and a pharmaceutically acceptable additive.

13. A kit comprising a separate or combined composition comprising the nitrated lipid or the inhibitor of the renin-antiotensin-aldosterone system.

14. A method comprising administering a nitrated lipid in combination with an inhibitor of the renin-angiotensin-aldosterone system to a subject in need thereof, in an amount effective to treat chronic kidney disease or diabetic nephropathy.

15. The method of claim 14, wherein the nitrated lipid is a nitrated monounsaturated fatty acid or a nitrated polyunsaturated fatty acid.

16. The method of claim 14 wherein the nitrated lipid is a nitrooleic acid or a nitrolinoleic acid.

17. The method of claim 14, wherein the nitrated lipid is selected from 9-nitrooleic acid, 10-nitrooleic acid, or combinations thereof.

18. The method of claim 14, wherein the inhibitor of the renin-angiotensin-aldosterone system is an ACE inhibitor, a renin inhibitor, or a angiotensin receptor antagonist.

19. The method of claim 14, wherein the inhibitor of the renin-angiotensin-aldosterone system is enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, zofenopril, fosinopril, or captopril.

20. The method of claim 14, wherein the inhibitor of the renin-angiotensin-aldosterone system is losartan, valsartan, telmisartan, irbesartan, azilsartan, olmesartan, candesartan, or eprosartan.

2 1. The method of claim 14, wherein the inhibitor of the renin-angiotensin-aldosterone system is losartan.

22. The method of claim 14, wherein the inhibitor of the renin-angiotensin-aldosterone system is aliskiren, remikiren, or renin siR A.

23. The method of claim 14, comprising two or more inhibitors of the renin-angiotensin- adosterone system. 24. The method of claim 22, wherein the inhibitor of the renin-angiotensin-aldosterone system is an ACE inhibitor and losartan.

25. The method of any one of claims 14 - 23, where the subject has chronic kidney disease or end-stage renal disease.

26. A method comprising administering a nitrated lipid in combination with an inhibitor of the renin-angiotensin-aldosterone system to a subject in need thereof, in an amount effective to prevent chronic kidney disease or end-stage renal disease.

27. The method of 25, wherein the nitrated lipid is a monounsaturated fatty acid selected from 9-nitrooleic acid, 10-nitrooleic acid, or combinations thereof.

28. The method of 25, wherein the inhibitor of the renin-angiotensin-aldosterone system is losartan.

A. CLASSIFICATION OF SUBJECT MATTER A61K 31/201 (2006.01) A61K 31/20 (2006.01) A61K 31/41 (2006.01) A61K 31/4178 (2006.01) A61K 31/401 (2006.01) A61P 13/12 (2006.01) A61P 43/00 (2006.01)

According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) EPOQUE: EPODOC, WPI, MEDLINE; STN: CHEM ABS; PATENTSCOPE; Keywords: nitrate, nitro, lipid, fatty acid, oleic, linoleic, ace, rennin, angiotensin converting, inhibitor, ras, raas, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, zofenopril, fosinopril, captopril, losartan, valsartan, telmisartan, irbesartan, azilsartan, olmesartan, candesartan, eprosartan, aliskiren, remikiren, diabetes, kidney, renal, nephropathy, ckd, esrd, albuminuria, proteinuria

C . DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

Documents are listed in the continuation of Box C

X Further documents are listed in the continuation of Box C X See patent family annex

* Special categories of cited documents: "A" document defining the general state of the art which is not "T" later document published after the international filing date or priority date and not in considered to be of particular relevance conflict with the application but cited to understand the principle or theory underlying the invention "E" earlier application or patent but published on or after the "X" document of particular relevance; the claimed invention cannot be considered novel international filing date or cannot be considered to involve an inventive step when the document is taken alone "L" document which may throw doubts on priority claim(s) or "Y" document of particular relevance; the claimed invention cannot be considered to which is cited to establish the publication date of another involve an inventive step when the document is combined with one or more other citation or other special reason (as specified) such documents, such combination being obvious to a person skilled in the art "O" document referring to an oral disclosure, use, exhibition or other means "&" document member of the same patent family "P" document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 1 November 2012 0 1 November 2012 Name and mailing address of the ISA/AU Authorised officer

AUSTRALIAN PATENT OFFICE Ross Heisey PO BOX 200, WODEN ACT 2606, AUSTRALIA AUSTRALIAN PATENT OFFICE Email address: [email protected] (ISO 9001 Quality Certified Service) Facsimile No.: +61 2 6283 7999 Telephone No. 0262833 185

Form PCT/ISA/210 (fifth sheet) (July 2009) ont nuat on . PCT/US2012/051304

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

WO 2009/149496 A l (CENTRAL NORTHERN ADELAIDE HEALTH SERVICE) 17 December 2009 X page 8, lines 20-26; page 11, lines 5-14; page 18, lines 21-25; page 24, line 27-page 25, 1-2, 5-6, 10, 12-15, 18-19, line 1; page 26, lines 10-25; page 31, line 20-page 32, line 31; claims 33-35 23, 25-26 Y page 18, lines 6-25; page 26, lines 10-25 3-4, 16-17, 27

WO 2009/129495 A l (THE UNIVERSITY OF UTAH RESEARCH FOUNDATION) 22 October 2009 Y para. [0020], [0025], [0035], [0043] 3-4, 16-17, 27

WO 2005/1 10396 A2 (UAB RESEARCH FOUNDATION) 24 November 2005 Y page 18, line 26-page 19, line 15; page 29, lines 3-16; page 38, lines 22-28; Fig. 10 3-4, 16-17, 27

WO 2009/155439 A2 (THE UNIVERSITY OF UTAH RESEARCH FOUNDATION) 23 December 2009 A para. [0004]-[0006], [0008], [0025], [0028] 1-28

WO 2001/015673 A2 (AVENTIS PHARMA DEUTSCHLAND GMBH) 08 March 2001 A page 2, lines 18-20; page 5, line 1 - page 8, line 2 1-28

WO 2009/017802 A l (UNIVERSITY OF PITTSBURGH - OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION) 05 February 2009 A para. [0008], [0028] 1-28

Form PCT/ISA/210 (fifth sheet) (July 2009) Information on patent family members PCT/US2012/051304 This Annex lists known patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.

Patent Document/s Cited in Search Report Patent Family Member/s

Publication Number Publication Date Publication Number Publication Date

WO 2009/149496 A l 17 Dec 2009 WO 2009149496 A l 17 Dec 2009

WO 2009/129495 A l 22 Oct 2009 CN 102066924 A 18 May 201 1

EP 2271929 A l 12 Jan 201 1

U S 201 1092594 A l 2 1 Apr 201 1 WO 2009129495 A l 22 Oct 2009

WO 2005/1 10396 A2 24 Nov 2005 U S 2007232579 A l 04 Oct 2007

U S 7776916 B2 17 Aug 2010 U S 201033 1268 A l 30 Dec 2010 WO 2005 110396 A2 24 Nov 2005

WO 2009/155439 A2 23 Dec 2009 CN 102099024 A 15 Jun 201 1 EP 2299997 A2 30 Mar 201 1 U S 201 1196037 A l 11 Aug 201 1 WO 2009155439 A2 23 Dec 2009

WO 2001/015673 A2 08 Mar 2001 AU 7648400 A 26 Mar 2001 AU 2005203694 A l 08 Sep 2005 AU 200920055 1 A l 05 Mar 2009 AU 200920055 1 B2 30 Sep 2010

BG 1063 19 A 29 Dec 2002

BG 661 15 B l 31 May 201 1 BR 0013540 A 30 Apr 2002 CA 2382387 A l 08 Mar 2001 CN 1384756 A 11 Dec 2002 CN 1015371 81 A 23 Sep 2009

EE 200200085 A 15 Apr 2003

EE 05130 B l 16 Feb 2009

EP 121208 1 A2 12 Jun 2002 EP 121208 1 B l 26 Oct 2005

EP 143713 1 A l 14 Jul 2004 EP 143713 1 B l 25 Apr 2012 EP 1925303 A2 28 May 2008

EP 22775 19 A2 26 Jan 20 11 HK 1050327 A l 03 Jul 2009 HR P20020169 A2 31 Oct 2005 HU 0202461 A2 28 Dec 2002

Due to data integration issues this family listing may not include 10 digit Australian applications filed since May 2001 . Form PCT/ISA/210 Famil Annex Jul 2009 Information on patent family members PCT/US2012/051304 This Annex lists known patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.

Patent Document/s Cited in Search Report Patent Family Member/s

Publication Number Publication Date Publication Number Publication Date

JP 2003508426 A 04 Mar 2003

JP 4843 172 B2 2 1 Dec 201 1 KR 20080035703 A 23 Apr 2008

ME P28208 A 10 Oct 2010 MX PA02001633 A 06 Aug 2002

NO 20020850 A 2 1 Feb 2002 NO 329245 B l 20 Sep 2010 NO 20100569 A 2 1 Feb 2002 NZ 571901 A 28 May 2010 PL 353066 A l 06 Oct 2003

RU 2002107673 A 10 Jan 2004

RU 2005 128 124 A 20 Mar 2007 U S 2006194868 A l 31 Aug 2006 U S 2008 125472 A l 29 May 2008

U S 2009258919 A l 15 Oct 2009

U S 2010267798 A l 2 1 Oct 2010 U S 201 1263619 A l 27 Oct 201 1

WO 0 115673 A2 08 Mar 2001 ZA 200201471 A 28 May 2003

WO 2009/01 7802 A l 05 Feb 2009 EP 2 180787 A l 05 May 2010 U S 2010216884 A l 26 Aug 2010 WO 2009017802 A l 05 Feb 2009

End of Annex

Due to data integration issues this family listing may not include 10 digit Australian applications filed since May 2001 . Form PCT/ISA/210 Famil Annex Jul 2009