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USOO8716268B2

(12) United States Patent (10) Patent No.: US 8,716,268 B2 Liu et al. (45) Date of Patent: May 6, 2014

(54) NITRATE ESTERS OF CORTICOID OTHER PUBLICATIONS COMPOUNDS USEFUL AS DURETCS Paul-Clarket al. 21-NO- is a novel nitric oxide-releas (76) Inventors: Chao Liu, Shijiazhuang (CN); Kunshen ing derivative of prednisolone with enhanced anti-inflammatory Liu, Shijiazhunag (CN) properties; British Journal of Pharmacology (2000) vol. 131, 1345 1354. (*) Notice: Subject to any disclaimer, the term of this Paul-Clark et al. Receptor Nitration Leads to patent is extended or adjusted under 35 Enhanced Anti-Inflammatory Effects of Novel Ligands; The Journal of Immunology (2003) p. 3245-3252. U.S.C. 154(b) by 243 days. Paul-Clark et al. Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis; PNAS (21) Appl. No.: 13/151,628 Feb. 5, 2002 vol. 99 No. 3 1677-1682. Liu et al. Potent Potentiating Diuretic Effects of in Con (22) Filed: Jun. 2, 2011 gestive Heart Failure; J Cardiovasc Pharmacol vol. 48, No. 4, Oct. 2006. (65) Prior Publication Data Di Fillipo et al. The Distinct Alterations Produced in Cardiovascular Functions by Prednisolone and Nitro-prednisolone (NCX-1015) in US 2011 FO3O1136A1 Dec. 8, 2011 the Rat Highlight a Causal Role for Endothelin-1; The Journal of Pharmacology and Experimental Therapeutics vol. 310, No. 3 2004. Related U.S. Application Data Liu et al. Potent Diuretic Effects of Prednisone in Heart Failure Patients with Refractory Diuretic Resistance; Can J Cardiol vol. 23 (60) Provisional application No. 61/352.254, filed on Jun. 2007. 7, 2010. Sica et al. Diuretic Resistance and Strategies toOvercome Resistance in Patients With Congestive Heart Failure; Pharmacotherapy in CHF Mar/Apr. 2002. (51) Int. Cl. Potter et al. Natriuretic Peptides, Their Receptors, and Cyclic A6 IK3I/56 (2006.01) Guanosine Monophosphate-Dependent Signaling Functions; Endo A6 IK3I/567 (2006.01) crine Reviews 27(1):47-72 2006. A 6LX3/573 (2006.01) A.J. De Bold et al. A Rapid and Potent Natriuretic Response to A 6LX3/575 (2006.01) Intravenous Injection of Atrial Myocardial Extract in Rats; Life Sci (52) U.S. Cl. ences, vol. 28, pp. 89-94 Oct. 21, 1980. Qiong Ye, Endothelin Inhibits NPR-A and Stimulates eNOS Gene CPC ...... A61 K3I/567 (2013.01); A61K3I/573 Expression in Rat IMCD Cells; Journal of the American Hear Asso (2013.01); A61 K3I/575 (2013.01) ciation Mar. 2003. USPC ...... 514/170; 514/180: 514/182 Robert W. Schrier et al. Hormones and Hemodynamics in Heart (58) Field of Classification Search Failure; Mechanisms of Disease Aug. 19, 1999. CPC. A61 K31/567; A61 K31/573; A61 K31/575 Haya Yechieli et al. Regulation of renal glomerular and papillary ANP receptors in rats with experimental heart failure; American USPC ...... 514/170, 180, 182 Physiological Society 1993. See application file for complete search history. Paula M. Bryan et al. Renal hyporesponsiveness to atrial natriuretic peptide in congestive heart failure results from reduced atrial (56) References Cited natriuretic peptide receptor concentrations; Am J Physiol Renal Physiol 292: F1636-F1644, 2007. U.S. PATENT DOCUMENTS Jean-Baptiste Micheletal. Urinary cyclic guanosine monophosphate as an indicator of experimental congestive heart failure in rats; Car 3.494,941 A 2/1970 Ledig et al. diovascular Research, 1990, 24,946952. 5,824,669 A 10/1998 Garvey et al. Liu Chao et al., “Potent Potentiating Diuretic Effects of Prednisone in 5,837,698 A 11/1998 Tjoeng et al. Congestive Heart Failure'. Journal of Cardiovascular Pharmacology, 6, 197,762 B1 3/2001 Garvey et al. Oct. 1, 2006, 173-176, vol. 48 No. 4, Raven Press, New York, U.S. 6,579,863 B1 6/2003 Garvey et al. 6,610,676 B1 8, 2003 Del Soldato 7,056,905 B2 6, 2006 Del Soldato Primary Examiner — San-Ming Hui 7,157,450 B2 1/2007 Del Soldato (74) Attorney, Agent, or Firm — Schmesier, Olsen & Watts, 7, 160,871 B2 1/2007 Del Soldato LLP 7,196,075 B2 3/2007 Del Soldato 7,205.288 B2 4/2007 Del Soldato (57) ABSTRACT 7,368.442 B2 5, 2008 Del Soldato 7,605,151 B2 10/2009 Del Soldato The present invention relates to the diuretic effects of nitrate 2005, 0004089 A1 1/2005 Soldato esters of corticoid compounds. A patient in heart failure may be treated by administering atherapeutically effective dosage FOREIGN PATENT DOCUMENTS of a pharmaceutical composition comprising a nitrate ester of corticoid compound as a diuretic having the general formula EP O929565 7, 1999 FR 2143664 A1 2, 1973 B X—NO. A method of improving kidney function by WO 98.15568 A2 4f1998 administering atherapeutically effective dosage of a pharma WO 0049.993 A2 8, 2000 ceutical composition having a nitrate ester of corticoid com WO WOOO49.993 8, 2000 pound of the same formula is also described. WO O3064443 A2 8, 2003 WO 2010015529 A1 2, 2010 11 Claims, No Drawings US 8,716,268 B2 1. 2 NITRATE ESTERS OF CORTCOD sated heart failure. Moreover, ANP levels in the circulation COMPOUNDS USEFUL AS DURETCS are proportional to the severity of systemic volume overload and mortality. In the kidney, renal tubular epithelial cells, CROSS REFERENCE TO RELATED especially the medullary collecting duct (MCD) cells are the primary site for renal water and sodium excretion, which are APPLICATION 5 also the primary sites for ANP's action. This application claims priority to U.S. Provisional Patent Therefore, there is a need to develop pharmaceutical com Application to Chao Liu, et al, entitled “Nitrate Esters of positions which could increase the density of NPR-A in renal Corticoid Compounds Useful as Diuretics’ Ser. No. 61/352. tubular epithelial cells, especially the medullary collecting 254, filed Jun. 7, 2010, the disclosure of which is hereby 10 duct (MCD) cells, which potentiates ANP's action in the incorporated entirely herein by reference kidney. There is a further need to develop diuretics for use with diseases which result in severe body fluid and sodium BACKGROUND OF THE INVENTION retention. Natriuretic peptide resistance in heart failure is a major problem faced by physicians. There is still a further Technical Field 15 need for physicians treating heart failure to develop drugs that could upregulate NPR-A in renal tubular epithelial cells. This invention relates generally to a method of using phar maceutical compositions having corticoid nitrate derivatives DISCLOSURE OF THE INVENTION as a diuretic. More specifically, the invention relates to a method of treating a patient with a pharmaceutical composi The present invention relates to the diuretic effects of tion having corticoid nitrate esters to improve kidney func nitrate esters of corticoid compounds. The nitrate esters of tion. corticoid compounds of the present invention have the gen Heart failure (HF) is a major and growing public health eral formula: problem in the world. Over 5 million individuals in the United States alone have HF and this condition is one of the leading (1) causes of deaths in the western world. As a complex clinical 25 hemodynamic disorder, HF is characterized by progressive or their esters or salts, where B has the following structure: pump failure and fluid accumulation. One out of three patients with HF is resistant to diuretic therapy, and devel oped fluid accumulation. See Ravnan, S. L., Ravnan, M. C. & Deedwania, P. C. Pharmacotherapy in congestive heart fail 30 ure: diuretic resistance and strategies to overcome resistance in patients with congestive heart failure. Congest Heart Fail 8, 80-85 (2002). Atrial natriuretic peptide (ANP) has potent body fluid-eliminating effects in normal physical setting. See Potter, L. R., Abbey-Hosch, S. & Dickey, D. M. Natriuretic peptides, their receptors, and cyclic guanosine monophos 35 phate-dependent signaling functions. Endocr Rev 27, 47-72 (2006). Since ANP was discovered in 1981, people have been trying to use it in the treatment of HF with fluid accumulation. where, in place of the hydrogens H in the CH groups or two However, all attempts have failed eventually because patients hydrogens H. in the CH group shown in the general formula, with HF become resistant to both endogenously secreted and there may be the following substituents: exogenously administered ANP. See de Bold, A. J., Boren at position 1-2: there may be a double bond; stein, H. B., Veress, A.T. & Sonnenberg, H. A rapid and potent at position 2-3: there may be the following substituent: natriuretic response to intravenous injection of atrial myocar dial extract in rats. Life Sci 28,89-94 (1981); Schrier, R. W. & 45 Abraham, W. T. Hormones and hemodynamics in heart fail ure. NEnglJMed341,577-585 (1999). This resistance is due to reduced renal natriuretic peptide receptor A (NPR-A) den sity in HF. See Bryan, P. M., Xu, X., Dickey, D. M., Chen, Y. & Potter, L. R. Renal hyporesponsiveness to atrial natriuretic 50 peptide in congestive heart failure results from reduced atrial natriuretic peptide receptor concentrations. Am J Physiol Renal Physiol 292, F1636-1644 (2007); Schrier, R. W. & Abraham, W. T. Hormones and hemodynamics in heart fail ure. N Engl J Med 341, 577-585 (1999); Michel, J. B., et al. 55 Urinary cyclic guanosine monophosphate as an indicator of at position 2: there may be Cl, Br; experimental congestive heart failure in rats. Cardiovasc Res at position 3: there may be —O. —O CH, CH, C1, OH: 24, 946-952 (1990); and Yechieli, H., Kahana, L., Haramati, A., Hoffman, A. & Winaver, J. Regulation of renal glomerular at position 4-5: there may be a double bond; and papillary ANP receptors in rats with experimental heart at position 5-6: there may be a double bond; failure. Am J Physiol 265, F119-125 (1993). 60 at position 6: there may be C1, F, CH, —CHO: ANP plays a crucial role in body fluid control. Blood at position 7: there may be Cl; volume expansion acts directly on the heart by stretch of atrial at position 9: there may be C1, F: myocytes to increase the release of ANP, which activates renal natriuretic peptide receptor A (NPR-A) and induces at position 11: there may be OH, CO. Cl; potent diuresis. However, since the discovery of ANP people 65 at position 16: there may be CH, OH, =CH: are frustrated by the fact that its favorable effects are blunted at position 17: In addition to R", the other H can be substituted in the diseases with body fluid overload, such as, decompen with the following: OH, CH, OCO(O)(CH), CH, or US 8,716,268 B2 4

O -CH-O- -(CH-CH-CH-O) - or ( ) 21 ONO. S. where ua is an integer equal to 0 or 1, Va is an integer from 0 Sás-X to 4: COOH CH at positions 16-17: there may be the following groups: 10 where inf is an integer from 1 to 6, preferably from 2 to 4: CH -(CH-CH-O)- Rif I6 O I6 15 CH where R–H, CH, and nfis an integer from 1 to 6, preferably from 2 to 4. The compounds which can be mentioned, and which are those preferred, are the ones listed below where B can be I6 O obtained according to the known processes of the art. For example, the precursors and related processes described for example in The Merck Index, 12th Ed. of 1996, herein incor RandR' are equal or different one from the other and may be porated by reference, can be mentioned as precursors and hydrogen or linear or branched alkyls having from 1 to 4 related processes. The precursors (according to the Merck carbon atoms, preferably R—R’—CH: 25 nomenclature) include the following, where H. H. R. R. R" have the meaning as defined in the compounds listed below: B being a residue: , , , algestone, R" is —(CO-L)-(X)— beclomethasone, , , clobeta where t and t are integers equal or different one from the Sol, , , , , corti other and equal to 0 or 1, provided that they cannot be both 30 costerone, , , , dexam equal to 0 when B contains no —OH groups; ethasone, , , , the bivalent bridging group L is selected from: , flucloronide, flumethasone, , fluocino lone acetonide, , fluocortyn butyl, , (CRRs) (O),(CRRs), (CO), (O), n(CO), n, , acetate, acetate, (CRRs), , flurandrenolide, , , 35 halobetasol propionate, , acetate, where na, n'a and n'a are equal or different one from the other , loteprednoletabonate, , mepred and are integers from 0 to 6, preferably from 1 to 3; nb, n'b, nisone, , furoate, parameta n"band n"b are equal or different one from the other and are Sone, , prednisolone, prednisolone 25-diethy integers equal to 0 or 1; R and Rs are equal or different one laminoacetate, prednisolone sodium phosphate, prednisone, from the other and are chosen from H. linear or branched alkyl 40 prednival, , , , triamci having from 1 to 5 carbon atoms, preferably from 1 to 3: nolone acetonide, 21-acetoxypregnenolone, , amci X is equal to Xo-0, NH, NR, where R is a linear or nonide, proprionate, maZipredone, , tri branched alkyl having from 1 to 10 C atoms; or equal to X amcinolone hexacetonide, as described in U.S. Pat. Nos. where X is equal to OH, CH, Cl, N(—CH2—CH), 7,368,442, 7,205,288, 7,196,075, 7,157,450, 6,610,676, SCHF, SH, 45 7,160.871, and 7,056,905, which are incorporated entirely herein by reference. Accordingly, the present invention relates to a method of treating a patient with heart failure by administering a thera peutically effective dosage of a pharmaceutical composition 50 comprising a nitrate ester of a corticoid compound described by formula (1). In addition the present invention relates to a method of improving kidney function by administeringatherapeutically X is a bivalent connecting bridge chosen from: effective dosage of a pharmaceutical composition comprising YO, where Y is a linear or whenever possible branched 55 a nitrate ester of a corticoid compound described by formula C-C alkylene, preferably having from 2 to 5 carbonatoms, (1). The nitrate esters of corticoid compounds potentiate the or an optionally substituted cycloalkylene having from 5 to 7 natriuretic peptides action in the kidney. carbon atoms; The foregoing and other features and advantages of the Y selected from: present invention will be apparent from the following more 60 detailed description of the particular embodiments of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION -(CH2) 65 The present invention relates to a method of using pharma where n is an integer from 0 to 3: ceutical compositions having nitrate esters of corticoid com US 8,716,268 B2 5 pounds as diuretics. It has now been found that pharmaceu -continued tical compositions having nitrate esters of corticoid CH3 compounds have potent diuretic effects in heart failure with body fluid retention. Generally, diuretic effects are described as the ability of the kidney to excrete water and sodium. The nitrate esters of corticoid compounds of the present I6 O invention have the general formula: RandR' are equal or different one from the other and may be or their esters or salts, where B has the following structure: 10 hydrogen or linear or branched alkyls having from 1 to 4 carbon atoms, preferably R—R'—CH:

B being a corticosteroid residue: R" is —(CO-L)-(X), 15 where t and t are integers equal or different one from the other and equal to 0 or 1, provided that they cannot be both equal to 0 when B contains no —OH groups; the bivalent bridging group L is selected from:

(CRRs). (O),(CRRs), (CO), (O), (CO), n, (CRRs), where, in place of the hydrogens H in the CH groups or two where na, n'a and n'a are equal or different one from the other hydrogens H in the CH group shown in the general formula, and are integers from 0 to 6, preferably from 1 to 3; nb, n' b, there may be the following substituents: 25 n"band n"b are equal or different one from the other and are at position 1-2: there may be a double bond; integers equal to 0 or 1; R and Rs are equal or different one at position 2-3: there may be the following substituent: from the other and are chosen from H. linear or branched alkyl having from 1 to 5 carbon atoms, preferably from 1 to 3:

30 X is equal to Xo-0, NH, NR, where R is a linear or branched alkyl having from 1 to 10 C atoms; or equal to X where X is equal to OH, CH, Cl, N(-CH2-CH-), SCHF, SH,

35

at position 2: there may be Cl, Br; 40 at position 3: there may be —O. —O CH, CH, C1, OH: at position 4-5: there may be a double bond; X is a bivalent connecting bridge chosen from: at position 5-6: there may be a double bond; YO, where Y is a linear or whenever possible branched at position 6: there may be C1, F, CH, —CHO: C-C alkylene, preferably having from 2 to 5 carbonatoms, at position 7: there may be Cl; 45 or an optionally substituted cycloalkylene having from 5 to 7 at position 9: there may be C1, F: carbon atoms; at position 11: there may be OH, CO. Cl; at position 16: there may be CH, OH, =CH: Y selected from: at position 17: In addition to R", the other H can be substituted with the following: OH, CH, OCO(O)(CH), CH, or 50

O -(CH2) or ( ) 55 where n is an integer from 0 to 3: where ua is an integer equal to 0 or 1, Va is an integer from 0 to 4: at positions 16-17: there may be the following groups: -CH-O- 60 21 -(CH-CH-CH-O) - CH3 S. ONO.

COOH S-XCH I6 O 65 where inf is an integer from 1 to 6, preferably from 2 to 4: US 8,716,268 B2

-(CH-CH-O)- Rif where R, H, CH, and nfis an integer from 1 to 6, preferably CHONO from 2 to 4. The compounds which can be mentioned, and which are those preferred, are the ones listed below where B can be 10 obtained according to the known processes of the art. For O example, the precursors and related processes described for example in The Merck Index, 12th Ed. of 1996, herein incor and NCX-1022 (NO-hydrocortisone): hydrocortisone 21-4'- porated by reference, can be mentioned as precursors and (nitro-oxymethyl)benzoate having the chemical structure: related processes. The precursors (according to the Merck 15

nomenclature) include the following, where H. H. R. R', R" have the meaning as defined in the compounds listed below: budesonide, hydrocortisone, alclometasone, algestone, beclomethasone, betamethasone, chloroprednisone, clobeta Sol, clobetaSone, clocortolone, cloprednol, cortisone, corti CHONO costerone, deflazacort, desonide, desoximetaSone, dexam ethasone, diflorasone, diflucortolone, difluprednate, fluazacort, flucloronide, flumethasone, flunisolide, fluocino lone acetonide, fluocinonide, fluocortyn butyl, fluocortolone, fluorometholone, , , O fluprednisolone, flurandrenolide, formocortal, halcinonide, 25 We have found that pharmaceutical compositions having halobetasol propionate, halometaSone, halopredone acetate, nitrate esters of corticoid compounds are sensitizers of natri hydrocortamate, loteprednoletabonate, medrysone, mepred uretic peptide, such as ANP and B-type natriuretic peptide nisone, methylprednisolone, mometasone furoate, parameta (BNP). They can increase the density of NPR-A in renal Sone, prednicarbate, prednisolone, prednisolone 25-diethy tubular epithelial cells, especially renal MCD cells, therefore laminoacetate, prednisolone sodium phosphate, prednisone, 30 potentiating ANP's action in heart failure. Consistent with the physiological consequences of ANP's action, pharmaceutical prednival, prednylidene, rimexolone, triamcinolone, triamci compositions having nitrate ester of corticoid compounds nolone acetonide, 21-acetoxypregnenolone, cortivaZol, amci administration produces potent diuresis and natriuresis. nonide, fluticasone proprionate, maZipredone, tiXocortol, tri Results received point to the possibility that nitrate esters amcinolone hexacetonide, as described in U.S. Pat. Nos. of corticoid compounds according to the invention may pro 7,368,442, 7,205,288, 7,196,075, 7,157,450, 6,610,676, 35 duce potent diuretic effects in patients with heart failure. It is 7,160.871, and 7,056,905, which are incorporated entirely believed that the diuretic effects induced by nitrate esters of herein by reference. corticoid compounds are (GR) medi In addition, U.S. Pat. Nos. 7,605,151, 5,824,669, 5,837, ated and can be abolished by GR antagonist RU486 (i.e. 698, 6,197.762, 6,579,863, and 7,056,905, incorporated ). The corticoid nitrate esters of the present herein by reference, disclose nitrate esters of corticoid com 40 invention induced potent diuretic effects are mediated by pounds. upregulation of the natriuretic peptide receptor A (NPR-A) in The diuretic effect induced by the nitrate esters of corticoid renal tubular epithelial cells, particularly the MCD cells. It compounds according to the invention is both dose and time has now been found that pharmaceutical compositions having dependent. Of note, human response to prednisone, another nitrate esters of corticoid compounds increase the density of glucocorticoid, is rather slow. There is usually a 3- or 4-day 45 NPR-A in renal tubular epithelial cells, especially the MCD (sometime even longer) silent waiting period for the diuretic cells in vitro and promote their cyclic guanosine monophos effects of prednisone therapy to burst out (Liu C, Chen H, phate (cGMP, the second messenger for the natriuretic effects Zhou C, Ji Z. Liu G. Gao Y, et al. Potent potentiating diuretic of atrial natriuretic peptide) generation in both a time- and effects of prednisone in congestive heart failure. J Cardiovasc dose-dependent manner. 50 In vivo, Systemic administration with pharmaceutical com Pharmacol. 2006: 48(4): 173-6.). The lag time of diuretic positions having nitrate esters of corticoid compounds of the effects induced by prednisone and the dosage of prednisone present invention produce potent diuresis in the rats with (1 mg/kg/day) in humans hampers its use in clinical practice. decompensated heart failure given ad libitum access to food We have now found that nitrate esters of corticoid com and water, and there was no diuresis-induced increase in pounds according to the invention can dramatically upregu water drinking, therefore leading to remarkable systemic Vol late the NPR-A expression in renal tubular epithelial cells in 55 ume depletion. Thus, therapy with pharmaceutical composi much shorter time and with much lower dose in vitro study tions having nitrate esters of corticoid compounds according when compared with their parent compounds. (For example, to the method of the present invention represents a promising it takes about half the time and about /10 of the dose of therapeutic strategy for diseases with fluid retention. These NCX-1015 prednisolone nitrate ester derivatives to attain effects are glucocorticoid receptor (GR) mediated and are the same effect of prednisolone on renal NPR-A in vitro. 60 abolished by GR antagonist RU486 (i.e. mifepristone). Nitric oxide (NO)-donating group of nitrate esters of corti The therapeutically effective dosages are those that contain coid compounds may synergize with the glucocorticoid drug an effective dose, or an appropriate fraction thereof, of the moiety to produce a more potent diuretic effect. Suitable active ingredient. The therapeutically effective dosage of the nitrate esters of corticoid compounds include but are not nitrate esters of corticoid compounds useful in the present limited to NCX-1015 (NO-prednisolone): prednisolone 21 65 invention is in an amount in the range of 0.001 to about 1000 (4'-nitro-oxymethyl)benzoate having the chemical struc mg/kg/day. The treatment time is in the range from about 1 to ture: about 360 days. US 8,716,268 B2 10 The pharmaceutical compositions having nitrate esters of Generally, the pharmaceutical compositions of the present corticoid compounds of the present invention release nitric invention are administered by oral administration or intrave oxide (NO) in biological fluids both demonstrated in vitro and nous infusion. Suitable methods of oral administration in vivo. NO-donating group of nitrate esters of corticoid include, but are not limited to oral Solid preparations, such as compounds can clearly synergize with the glucocorticoid capsules or tablets, or oral liquid preparations. Tablets may be moiety to produce a potent cardiorenal protective effect. It is coated by conventional aqueous or nonaqueous techniques. believed that this synergy is the result of glucocorticoid The pharmaceutical compositions of the present invention receptor (GR) nitration by NO-donating group of nitrate may be administered as the raw chemical, or together with esters of corticoid compounds and/or rapid alterations of the one or more pharmaceutically acceptable carriers. Generally, microcirculation within minutes of application of a NO 10 the pharmaceutical compositions of the present invention are donor. prepared by uniformly and intimately admixing the active Pharmaceutical compositions comprising the nitrate esters ingredient with liquid carriers or finely divided solid carriers of corticoid compounds according to the invention increase or both, and then, if necessary shaping the pharmaceutical the density of NPR-A in renal tubular epithelial cells, particu composition into the desired presentation. larly the MCD cells in vitro and vivo. This potentiates ANPs 15 The following examples further illustrate, not limit, the action in the kidney. Therefore, pharmaceutical compositions invention. comprising the nitrate esters of corticoid compounds accord ing to the invention can be used as diuretics in the diseases with severe body fluid and sodium retention, including but not Example 1 limited to heart failure, and cirrhotic ascites. Pharmaceutical compositions comprising the nitrate esters of corticoid compounds according to the invention also The Effect of on Diuretic Effect and inhibit renin-angiotensin-aldosterone system (RAAS) in NPR-A Expression in Renal Medulla. TABLE 1. The effect of (Dex) on diuretic effect and NPR-A expression in renal medulla Urinary NPR-A expression Urinary volume sodium Compound Dose (mg/kg) (100% of vehicle) (ml/24 h) (mmol/24 h) Dex 1 132.29.7 16.42.4 3.SO 0.39* Vehicle 1OO.O. 10.7 8.91.1 1.69 0.24 Dex + RU486 Dex (1 mg/kg), 95.5 - 102 8.6 1.O 1.67 + O.27 RU486 (100 mg/kg) The data is expressed as meant standard deviation, *P<0.01 compared with vehicle. The receptor expressions in renal medulla were assessed by western blotting analysis and expressed as a relative value compared with the average density measured in the vehicle treated rats, heart failure. The kidney contains all elements of the RAAS. To determine the effect of glucocorticoids on diuretic Both angiotensin II (ANG II) and aldosterone has stimulatory 40 effect and NPR-A expression in renal medulla, 15 intact effects on sodium reabsorption in the kidney. ANG II take Wistar rats were randomized to receive Dexamethasone effects by binding to angiotensin II type I (AT) receptors. (Dex), vehicle and glucocorticoid receptor (GR) antagonist Plasma ANG II and aldosterone levels are significantly RU486. As shown in Table 1 above, after 24-hour treatment, elevated in decompensated heart failure, accompanied by an Dex dramatically increased urinary Volume and urinary increased renal AT receptor density. Renal NPR-A activation 45 Sodium compared with vehicle treated rats, which was asso induced by glucocorticoids can inhibit the activated RAAS. ciated with a dramatic NPR-A overexpression. But the Glucocorticoids not only decreases ANG H and aldosterone diuretic effect and NPR-A expression in renal medulla levels in the circulation, but also down-regulates AT receptor induced by Dex was completely abolished by RU486, Sug expression in the kidney. gesting that the diuretic effect and NPR-A overexpression Pharmaceutical compositions comprising the nitrate esters 50 induced by glucocorticoids was mediated by GR. of corticoid compounds according to the invention also inhibit vasopressin axis in heart failure. The actions of argi Example 2 nine vasopressin (AVP) are mediated by plasma membrane Study of NPR-A Expression in Inner Medullary Collecting receptors. Three different subtypes of vasopressin receptors Duct (IMCD) Cells. have been cloned. Only arginine vasopressin receptor 2 (V) is located in the kidney. AVP causes antidiuresis by activating 55 TABLE 2 V receptors on the basolateral surface of the principal cells in Study of the effect of NCX-1015 versus prednisolone on NPR-A the collecting duct. Renal NPR-A activation induced by glu expression in IMCD cells cocorticoids can down-regulate V2 receptor expression in the kidney. Moreover, NPR-A activation in hypothalamus NPR-A NPR-A induced by glucocorticoids can inhibit AVP release into the 60 concentration expression expression circulation. Compound (mol/L) (24 hours) (48 hours) The pharmaceutical compositions of the present invention NCX-101S 10-7 ------may be administered, for example by oral, rectal, parenteral prednisolone 10-6 ------route (including Subcutaneous, intradermal, intramuscular, Vehicle -- -- 65 intravenous and intraarticular) or by local (dermal, topical, Density of membrane NPR-A in IMCD cells were visualized by immunofluorescence transdermal, osculatory, inhalatory, topical (including der expressed with the number of "+” (+ = low, ++ = moderate, +++ = high). mal, buccal, Sublingual, and intraocular) application. US 8,716,268 B2 11 12 IMCD cells from Wistar rats were isolated and cultured Example 3 using the method previously described by Ye Q et al.: Endot helin inhibits NPR-A and stimulates eNOS gene expression Study of Diuretic Effects in Decompensated Heart Failure in rat IMCD cells. Hypertension 41, 675-681 (2003). IMCD 5 cells were cultured with Dulbecco's modified Eagle medium TABLE 4 plus fetal calf serum that was glucocorticoid free for 3-4 days Study of diuretic s of NCX- episolone in rats W CCOIIPCISIC e 8. until the cells attained confluence. The IMCD cells were then treated with NCX-1015, prednisolone, or vehicle for 48 10 compound (iii)D Urirayne UriNE2 di y hours. The Density of membrane NPR-A in IMCD cells were NCX-101S 1 8.8 + 1.5*f 1.43 + 0.15* visualized by immunofluorescence. As shown in Table 2 prednisolone 1 6.1 O.7* 1.08 O.1* Vehicle 3.30.5 O.72 O.O7 above, NCX-1015 dramatically increased NPR-A density in 15 IMCD cells using less time and dose compared with- its0 parent Thes dGo N.R.d S.+ standard deviationdeviation, compound prednisolone. "P<0.05 compared with prednisolone, To determine the effects of on systemic 2O Volume in HF, 15 Wistar rats with HF were randomized to TABLE 3 receive NCX-1015 (n=5), or prednisolone (n=5), or vehicle (n=5). HF model was accomplished by left anterior descend ing artery ligation, and then the Survived rats were raised for The effect of NCX-1022 on NPR-A expression in IMCD cells 12 weeks to have decompensated HF (i.e. HF with fluid accumulation). Parameters on urinary Volume and urinary NPR-A NPR-A 25 sodium in 12 hours were collected. As shown in Table 4 concentration expression expression above, both NCX-1015 and prednisolone dramatically C d (mol/L) (24 hours) (48 hours) increased renal water and sodium excretion as compared with ompoun O OS OS vehicle. Of note, NCX-1015 treated rats excreted much more water and sodium than prednisolone treated rats. Western NCX-1022 10-7 ------30 blotting analysis showed that heart failure rats treated with hydrocortisone 10-6 ------prednisolone or NCX-1015 had higher NPR-A expression in Vehicle -- -- the renal medulla (26.5% higher in prednisolone group, P=0.008; and 42.8% higher in NCX-1015 group, P=0.008: puttianDensity of membrane NSA.M.E.:NPR-A in MCD cell Sirisualized byEunofluorescence i f is respectively)1015 treated thanrats didhad vehicle-treatedhigher NPR-A rats. expression However, in NCXrenal medulla than did prednisolone treated rats (P=0.032). Similarly, as shown in Table 3 above, NCX-1022 dramati Example 4 cally increased NPR-A density in IMCD cells using less time Study of Renin-Angiotensin-Aldosterone System and dose compared with its parent compound hydrocortisone. (RAAS) in Decompensated Heart Failure

TABLE 5

Study of NCX-1015 on RAAS in rats with decompensated heart failure

AT receptor density in the Plasma Plasma kidney (% of angiotensin II aldosterone Compound Dose (mg/kg) vehicle) (pg/ml) (ng/ml)

NCX-101S 1 43.14 + 7.07*" 412.20 + 64.99* 0.99 + 0.09" Prednisolone 1 67.06 12.96* 665.86+ 113.90 1.39 O.26* Vehicle 1OOOO 949 878.S. 117.91 2.16 - 0.41

The data are expressed as meant standard deviation, *P<0.01 compared with vehicle. P<0.05 compared with vehicle, &P < 0.01 compared with prednisolone, "P<0.05 compared with prednisolone, The receptor densities were assessed by western blotting analysis and expressed as a relative value compared with the average density measured in the vehicle treated rats, US 8,716,268 B2 13 14 To determine the effects of corticosteroids on RAAS in HF, 15 Wistar rats with HF were randomized to receive NCX 1015 (n=5), or prednisolone (n=5), or vehicle (n=5). After 24-hour treatment, as shown in Table 5 above, both NCX 1015 and prednisolone dramatically decreased plasma angio HO OH tensin II and aldosterone levels, and AT receptor density in CHONO the kidney as compared with vehicle. Of note, NCX-1015 is superior over prednisolone in inhibiting RAAS in rats with heart failure. 10 O Example 5 and wherein renal natriuretic peptide receptor A expression in Study of Vasopressin Axis in Decompensated Heart Failure the patient kidney is increased with the therapeutically effec 15 tive amount of the pharmaceutical composition. TABLE 6 2. A method of treating a patient with heart failure by administering a therapeutically effective amount of a phar Study of NCX-1015 on vasopressin axis in rats with maceutical composition comprising a nitrate ester of a corti coid compound wherein the compound is prednisolone 21 decompensated heart failure (4'-nitro-oxymethyl)benzoate having the chemical Structure: Dose V2 receptor density in the Plasma vasopressin compound (mg/kg) kidney (% of vehicle) (pg/ml) 25 NCX-101S 1 54.06 it 16.15* 32.14+ 5.47%f prednisolone 1 79.80 +9.44 45.72 + 8.35 Vehicle 100.00 1282 68.26 - 14.09 CHONO 30 The data are expressed as meant standard deviation, *P<0.01 compared with vehicle. P<0.05 compared with vehicle. O P<0.05 compared with prednisolone, 35 The receptor densities were assessed by western blotting analysis and expressed as a relative value compared with the average density measured in the vehicle treated rats, and wherein vasopressin axis is inhibited. 3. The method of claim 2, wherein the therapeutically To determine the effects of corticosteroids on vasopressin effective amount of the nitrate ester of the corticoid com axis in HF 15 Wistar rats with HF were randomized to receive pound is in an amount in the range of from about 0.001 to NCX-1015 (n=5), or prednisolone (n=5), or vehicle (n=5). 40 about 1000 mg/kg/day. After 24-hour treatment, as shown in Table 6 above, both 4. The method of claim 2, wherein treatment time is in the NCX-1015 and prednisolone dramatically decreased plasma range of from about 1 to about 360 days. vasopressin levels, and V receptor density in the kidney as 5. A method of treating a patient with heart failure by compared with vehicle. NCX-1015 was superior over pred administering a therapeutically effective amount of a phar 45 maceutical composition comprising a nitrate ester of a corti nisolone in inhibiting vasopressin axis in rats with heart fail coid compound wherein the compound is hydrocortisone 21 le. 4'-(nitro-oxymethyl)benzoatehaving the chemical structure: The embodiments and examples set forth herein were pre sented in order to best explain the present invention and its practical application and to thereby enable those of ordinary 50 skill in the art to make and use the invention. However, those of ordinary skill in the art will recognize that the foregoing description and examples have been presented for the pur HO OH poses of illustration and example only. The description as set CHONO forth is not intended to be exhaustive or to limit the invention 55 to the precise form disclosed. Many modifications and varia tions are possible in light of the teachings above without departing from the spirit and scope of the forthcoming claims. O 60 The invention claimed is: and wherein renal natriuretic peptide receptor A expression in 1. A method of treating a patient with heart failure by the patient kidney is increased with the therapeutically effec administering a therapeutically effective amount of a phar tive amount of the pharmaceutical composition. maceutical composition comprising a nitrate ester of a corti 6. The method of claim 5, wherein the therapeutically coid compound wherein the compound is prednisolone 21 65 effective amount of the nitrate ester of the corticoid com (4'-nitro-oxymethyl)benzoate having the chemical pound is in an amount in the range of from about 0.001 to Structure: about 1000 mg/kg/day. US 8,716,268 B2 15 16 7. The method of claim 5, wherein treatment time is in the range of from about 1 to about 360 days. 8. The method of claim 1, wherein the therapeutically effective amount of the nitrate ester of the corticoid com pound is in an amount in the range of from about 0.001 to about 1000 mg/kg/day. 9. The method of claim 1, wherein treatment time is in the range of from about 1 to about 360 days. 10. The method of claim 1, wherein the therapeutically effective amount of the pharmaceutical composition is a 10 diuretic. 11. The method of claim 1, wherein renin-angiotensin aldosterone system is inhibited. k k k k k