WO2013005216A1

WO2013005216A1 - Compositions and methods for treatment of renal ischemia-reperfusion injury

Info

Publication number: WO2013005216A1
Application number: PCT/IL2012/050238
Authority: WO
Inventors: Andrew Lurie Salzman; Garry Southan
Original assignee: Radikal Therapeutics Inc.
Priority date: 2011-07-05
Filing date: 2012-07-04
Publication date: 2013-01-10

Abstract

The invention provides compositions and methods for prevention or treatment of renal ischemia-reperfusion injury using piperidine, pyrrolidine, or azepane derivatives comprising one to four nitric oxide donor groups and a reactive oxygen species (ROS) degradation catalyst.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF RENAL

ISCHEMIA-REPERFUSION INJURY

TECHNICAL FIELD

[0001] The present invention relates to use of compounds comprising a nitric oxide donor and a reactive oxygen species (ROS) degradation catalyst in pharmaceutical compositions and methods for prevention and treatment of renal ischemia-reperfusion injury (RIRI).

BACKGROUND ART

[0002] RIRI is a frequent consequence of cadaveric renal transplantation, circulatory shock, and major vascular procedures of the abdominal and thoracic aorta (Rectenwald et al, 2002). In the post-operative period after thoracoabdominal aortic aneurysm repair, for example, renal complications occurred in 28% of the patients, and this was a major independent risk factor for early mortality (Rectenwald et al, 2002). Similarly, in the early post-operative period after cadaveric renal transplantation, creatinine clearance and tubular sodium reabsorption were profoundly reduced in kidneys, coinciding with significantly increased urinary concentrations of tubular injury markers, in particular, neutrophil gelatinase- associated lipocalin (NGAL), N-acetyl-p-glucosaminidase, and cystatin C; and an 18-fold increase in renal production of cytokeratin-18, indicating extensive necrotic cell death (Snoeijs et al, 2011).

[0003] RIRI is a multifactorial process, presumably requiring a complex therapeutic response. Aligned with this need, therapeutic resolution of free radical imbalance induces a multi-faceted cellular and tissue response. Exogenous provision of nitric oxide, for example, reduces infarct volume and edema, and improves functional recovery in numerous rodent models of RIRI (Garcia-Criado et al, 1998; Ozturk et al, 2001; Dobashi et al, 2002; Martinez-Mier et al, 2002; Mehta et al, 2002; Rhoden et al, 2002; Mitterbauer et al, 2003; Sekhon et al, 2003; Jeong et al, 2004; Kurata et al, 2004; Chander and Chopra, 2005; Kurata et al, 2005; Lozano et al, 2005; Kucuk et al, 2006) via a number of mechanisms, including the inhibition of polymorphonuclear neutrophil (PMN) infiltration (Garcia- Criado et al, 1998; Martinez-Mier et al, 2002), thwarted expression of pro-inflammatory cytokines (Martinez-Mier et al, 2002; Sekhon et al, 2003; Lozano et al, 2005) and chemokines (Martinez-Mier et al, 2002), inhibition of mitogen- activated protein (MAP) kinase (Martinez-Mier et al. , 2002) and activating protein- 1 (AP-1) (Mehta et al, 2002), suppression of endothelin-1 expression (Kurata et al, 2004; Kurata et al, 2005), inhibition of Nuclear Factor- KappaB (NF- KB) activation (Lozano et al, 2005), scavenging of superoxide anion (O₂ ^~) (Garcia- Criado et al, 1998), and activation of endogenous antioxidant enzymes (Dobashi et al, 2002; Chander and Chopra, 2005). Despite the convincing benefit demonstrated by therapeutic nitric oxide in experimental models of RIRI (Garcia-Criado et al, 1998; Ozturk et al, 2001; Dobashi et al , 2002; Martinez-Mier et al , 2002; Mehta et al, 2002; Rhoden et al, 2002; Mitterbauer et al, 2003; Sekhon et al, 2003; Jeong et al, 2004; Kurata et al, 2004; Chander and Chopra, 2005; Kurata et al, 2005; Lozano et al, 2005; Kucuk et al, 2006), however, there are also reports of nitric oxide-related induction of the toxic species peroxynitrite (ONOO ) during RIRI (Nakajima et al, 2006) and persuasive evidence that ONOO -mediated induction of DNA single strand breakage and poly(ADP-ribose)polymerase ("PARP") activation is a prime instigator of renal dysfunction (O'Valle et al, 2009; Devalaraja-Narashimha et al, 2005; Zheng et al, 2005; Chatterjee et al, 2004; Martin et al, 2000). Thus, the benefit of nitric oxide therapy of RIRI is potentially compromised by its induction of ONOO^" formation.

[0004] Given the benefit in RIRI afforded by either provision of nitric oxide or removal of O₂ , and the demonstration that it is ultimately the imbalance of these two free radical species that dictates outcome in RIRI (Nakajima et al, 2006), it was hypothesized that optimal resuscitation of RIRI by nitric oxide may be achieved by: (i) simultaneous replenishment of nitric oxide and removal of O₂ , such that ONOO^" cannot form; and (ii) co-localization of these two activities (nitric oxide donation, O₂ removal) within a single agent, such that they necessarily act congruently in space and time.

[0005] US Patent Nos. 6,448,267, 6,455,542 and 6,759,430, herewith incorporated by reference in their entirety as if fully described herein, disclose, inter alia, piperidine, pyrrolidine and azepane derivatives comprising a nitric oxide donor and a O₂ scavenger, capable of acting as sources of nitric oxide and as ROS degradation catalysts, their preparation, and their use in the treatment of various conditions associated with oxidative stress or endothelial dysfunction such as diabetes mellitus and cardiovascular diseases.

[0006] International Patent Application No. PCT/IL2011/00879 provides methods and compositions for treatment of sepsis and conditions associated therewith using the piperidine, pyrrolidine, or azepane derivatives disclosed in US Patent Nos. 6,448,267, 6,455,542 and 6,759,430; and International Publication No. WO 2011/092690 discloses methods and compositions for prevention, treatment, or management of pulmonary arterial hypertension (PAH) using those compounds.

SUMMARY OF INVENTION

[0007] It has been found, in accordance with the present invention, that administration of certain 1-pyrrolidinyloxy derivatives, more particularly 3-nitrato methyl-2,2,5,5-tetramethylpyrrolidinyloxy, in a rat RIRI model, with the initial dose given during the final 5 minutes of a 30 minute period of ischemia prior to reperfusion, significantly inhibited renal dysfunction, PMN infiltration into the renal parenchyma, and histological damage, i.e., tubular necrosis, strongly indicating that 3-nitratomethyl-2,2,5,5-tetramethylpyrrolidinyloxy is protective in resuscitating RIRI.

[0008] In one aspect, the present invention thus relates to a method for prevention or treatment of renal ischemia-reperfusion injury (RIRI) in an individual in need thereof, comprising administering to said individual a prophylactically or therapeutically effective amount of a compound of the general formula I:

or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof,

wherein

Ri each independently is selected from H, -OH, -COR₃, -COOR₃, - OCOOR₃, -OCON(R₃)₂, -(C C₁₆)alkylene-COOR₃, -CN, -NO₂, -SH, -SR₃, -(C Ci₆)alkyl, -O-(C Ci₆)alkyl, -N(R₃)₂, -CON(R₃)₂, -SO₂R₃, -S(=O)R₃, or a nitric oxide donor group of the formula -X X₂-X₃, wherein Xi is absent or selected from -O-, -S- or -NH-; X₂ is absent or is (CrC₂₀)alkylene optionally substituted by one or more -ONO₂ groups and optionally further substituted by a moiety of the general formula D:

and X₃ is -NO or -ONO₂, provided that at least one Ri group is a nitric oxide donor group;

R₂ each independently is selected from (Ci-C₁₆)alkyl, (C₂-C₁₆)alkenyl, or (C₂-C₁₆)alkynyl;

R₃ each independently is selected from H, (C₁-C₈)alkyl, (C₃-C₁₀)cycloalkyl, 4-12-membered heterocyclyl, or (C₆-C₁₄)aryl, each of which other than H may optionally be substituted with -OH, -COR₄, -COOR₄, -OCOOR₄, -OCON(R₄)₂, - (C C₈)alkylene-COOR₄, -CN, -NO₂, -SH, -SR₄, -(C C₈)alkyl, -O-(C C₈)alkyl, - N(R₄)₂, -CON(R₄)₂, -SO₂R4, or -S(=O)R₄;

R4 each independently is selected from H, (CrC₈)alkyl, (C₃-C₁₀)cycloalkyl, 4-12-membered heterocyclyl, or (C₆-C₁₄)aryl; and

n and m each independently is an integer of 1 to 3.

[0009] While reducing the present invention into practice, it has further been found that aqueous solutions of 3-nitratomethyl-2,2,5,5-tetramethylpyrrolidinyloxy, having concentrations several times greater than those with commonly used co- solvents can be obtained by stirring said compound in water with 2-hydroxypropyl- β-cyclodextrin (HPCD) in ratios typically between 1: 15 and 1 :20 w/w, depending on the degree of substitution of the cyclodextrin with the hydroxypropyl side chain. Moreover, aqueous solutions containing substantially higher concentrations of said compound with HPCD can be achieved by stirring HPCD in distilled water with said compound; filtering and freeze drying the filtrate; and re-dissolving the resulting freeze dried solid, i.e., the lyophilizate, in a volume of water that is less than that originally used to prepare the solution prior to lyophilization. In certain embodiments, the compound administered according to the method of the present invention is thus formulated as an inclusion complex with an hydroxyalkyl- cyclodextrin.

[0010] In another aspect, the present invention provides a pharmaceutical composition for prevention or treatment of RIRI comprising a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. In certain embodiments, the compound comprised within said pharmaceutical composition is formulated as an inclusion complex with an hydroxyalkyl-cyclodextrin.

[0011] In a further aspect, the present invention provides a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, for use in prevention or treatment of RIRI. [0012] In yet another aspect, the present invention relates to use of a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, for the preparation of a pharmaceutical composition for prevention or treatment of RIRI.

[0013] In another aspect, the present invention provides an inclusion complex of a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and an hydroxyalkyl-cyclodextrin.

[0014] In a further aspect, the present invention provides a pharmaceutical composition comprising an inclusion complex as defined above, and a pharmaceutically acceptable carrier.

[0015] In still a further aspect, the present invention relates to a process for the preparation of a solution of an inclusion complex as defined above in distilled water or a physiological solution, comprising the steps of:

(i) dissolving said hydroxyalkyl-cyclodextrin in distilled water or a physiological solution, with said compound, or enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, under stirring conditions, thus forming a solution of said inclusion complex;

(ii) filtering the solution obtained in step (i) to remove said compound that is not dissolved;

(iii) lyophilizing the solution obtained in step (ii) to obtain a lyophilizate of said inclusion complex; and

(iv) dissolving said lyophilizate in distilled water or physiological solution, wherein the concentration of the inclusion complex, thus the nominal concentration of said compound, or enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, in the solution obtained in step (iv) is higher than that in the solution obtained in step (i). BRIEF DESCRIPTION OF DRAWINGS

[0016] Figs. 1A-1C show representative photomicrographs from the kidney of rats subjected to RIRI. Sections were stained with hematoxylin and eosin. While no histological alterations were observed in the kidney section from sham-operated rats (1A), animals that underwent renal ischemia/reperfusion demonstrated the recognized features of severe acute injury, including brush border loss, nuclear condensation, cytoplasmic swelling, and consistent loss of significant numbers of nuclei from tubular profiles (IB). Compound la (R-100; 80 mg/kg/day, TID IP) significantly reduced the I/R-induced histological alteration (1C).

[0017] Fig. 2 shows the plasma concentrations of urea in the different groups of rats (Sham, I/R+ vehicle, and I/R+R-100) according to the study in Example 3.

[0018] Fig. 3 shows the plasma concentration of creatinine in the different groups of rats (Sham, I/R+ vehicle, and I/R+R-100) according to the study in Example 3.

[0019] Fig. 4 shows the creatinine clearance in the R-100 treated group vs. the vehicle group, according to the study in Example 3.

[0020] Figs. 5A-5B show the FE_Na in the R-100 treated group vs. the vehicle group, calculated using plasma Na⁺ concentrations (5A), urine production (urine flow, ml/min) and urinary concentrations of Na⁺ (5B), according to the study in Example 3.

[0021] Fig. 6 shows the plasma NGAL levels in the different groups of rats according to the study in Example 3.

[0022] Fig. 7 shows the urine NGAL levels in the different groups of rats according to the study in Example 3.

[0023] Figs. 8A-8B show the kidney MPO activity (8A) and MDA levels (8B) in the R-100 treated group vs. the vehicle group, according to the study in Example 3.

[0024] Figs. 9A-9C show representative photomicrographs demonstrating hematoxylin/eo sin- stained kidney sections taken from sham-operated rats (9A), animals that underwent renal I/R (9B), and rats that underwent renal I/R and were treated with R-100 (80 mg/kg, IV) (9C), according to the study in Example 3. [0025] Fig. 10 shows the histological score of the kidney of the rats in the different groups of rats (Sham, I/R+ vehicle, and I/R+R-100) according to the study in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

[0026] In one aspect, the present invention provides a method for prevention or treatment of RIRI, by administration of piperidine, pyrrolidine, or azepane derivatives of the general formula I as defined above, comprising one to four nitric oxide donor groups and a ROS degradation catalyst, i.e., a O₂ scavenger.

[0027] The term "alkyl" as used herein typically means a straight or branched saturated hydrocarbon radical having 1-16 carbon atoms and includes, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2- dimethylpropyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, and the like. Preferred are (C C₆)alkyl groups, more preferably (CrC₄)alkyl groups, most preferably methyl and ethyl. The terms "alkenyl" and "alkynyl" typically mean straight and branched hydrocarbon radicals having 2-16 carbon atoms and 1 double or triple bond, respectively, and include ethenyl, propenyl, 3-buten-l-yl, 2-ethenylbutyl, 3-octen-l- yl, 3-nonenyl, 3-decenyl, and the like, and propynyl, 2-butyn-l-yl, 3-pentyn-l-yl, 3- hexynyl, 3-octynyl, 4-decynyl, and the like. C₂-C₆ alkenyl and alkynyl radicals are preferred, more preferably C₂-C₄ alkenyl and alkynyl.

[0028] The term "alkylene" typically means a divalent straight or branched hydrocarbon radical having 1-20 carbon atoms and includes, e.g., methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene, 2-methylbutylene, hexylene, 2-methylpentylene, 3-methylpentylene, 2,3-dimethylbutylene, heptylene, octylene and the like. Preferred are (C₁-C₈)alkylene, more preferably (C C )alkylene, most preferably (CrC₂)alkylene.

[0029] The term "cycloalkyl" as used herein means a cyclic or bicyclic hydrocarbyl group having 3-12 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like. Preferred are (C₅-C₁₀)cycloalkyls, more preferably (C₅-C₇)cycloalkyls.

[0030] The term "aryl" denotes an aromatic carbocyclic group having 6-14 carbon atoms consisting of a single ring or multiple rings either condensed or linked by a covalent bond such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl.

[0031] The term "heterocyclic ring" denotes a mono- or poly-cyclic non-aromatic ring of 4-12 atoms containing at least one carbon atom and one to three heteroatoms selected from sulfur, oxygen or nitrogen, which may be saturated or unsaturated, i.e., containing at least one unsaturated bond. Preferred are 5- or 6-membered heterocyclic rings. The term "heterocyclyl" as used herein refers to any univalent radical derived from a heterocyclic ring as defined herein by removal of hydrogen from any ring atom. Examples of such radicals include, without limitation, piperidino, 4-morpholinyl, or pyrrolidinyl.

[0032] The term "nitric oxide donor group" as defined herein refers to any group of the formula -XrX₂-X₃, wherein X_x may be absent or is selected from -O-, -S- or -NH-; X₂ may be absent or is (CrC₂₀)alkylene optionally substituted by one or more -ONO₂ groups and optionally further substituted by a moiety of the general formula D as defined above; and X₃ is -NO or -ONO₂. Preferred nitric oxide donor groups are those in which X₁ is absent or is -O-; X₂ is absent or is -(C₁-C₆)alkylene, preferably -(CrC₄)alkylene, more preferably methylene; and X₃ is -NO or -ONO₂, preferably -ONO₂, and said alkylene is optionally substituted as defined hereinabove. According to the method of the present invention, the compound of the general formula I may comprise one nitric oxide donor group or more than one identical or different nitric oxide donor groups.

[0033] In certain embodiments, the compound used according to the method of the present invention is a compound of the general formula I, wherein each independently is selected from H, -COOR₃, -CON(R₃)₂, or a nitric oxide donor group; and R₃ is H. [0034] In certain embodiments, the compound used according to the method of the present invention is a compound of the general formula I, wherein R₂ each independently is (CrC₈)alkyl, preferably (CrC₄)alkyl, more preferably (Q- C₂)alkyl, most preferably methyl. Preferred embodiments are those in which all the R₂ groups in the formula I are identical.

[0035] In certain embodiments, the compound used according to the method of the present invention is a compound of the general formula I, wherein in said nitric oxide donor group, Xi is absent or -O-; X₂ is absent or (C₁-C₂₀)alkylene, preferably -(C C₆)alkylene, more preferably -(CrC )alkylene, most preferably methylene; X₃ is -NO or -ONO₂, preferably -ONO₂; and said alkylene is optionally substituted by one or more -ONO₂ groups and optionally further substituted by a moiety of the general formula D as defined above.

[0036] In certain embodiments, the compound used according to the method of the present invention is a compound of the general formula I, wherein n is 1, 2 or 3, preferably 1 or 2.

[0037] In certain embodiments, the compound used according to the method of the present invention has the general formula I, wherein n is 1, i.e., a 1 -pyrrolidinyloxy derivative of the formula la (see Table 1). In particular embodiments, the compound used according to this method has the general formula la, wherein either the carbon atom at position 3 of the pyrrolidine ring or the carbon atom at position 4 of the pyrrolidine ring, or both, are each linked to a nitric oxide donor group.

[0038] In other certain embodiments, the compound used according to the method of the present invention has the general formula I, wherein n is 2, i.e., a 1- piperidinyloxy derivative of the formula lb (see Table 1). In particular embodiments, the compound used according to this method has the general formula lb, wherein one, two or three of the carbon atoms at positions 3 to 5 of the piperidine ring are each linked to a nitric oxide donor group. In more particular embodiments, (i) the carbon atom at position 3 of the piperidine ring and optionally one or more of the carbon atoms at positions 4 or 5 of the piperidine ring are each linked to a nitric oxide donor group; (ii) the carbon atom at position 4 of the piperidine ring and optionally one or more of the carbon atoms at positions 3 or 5 of the piperidine ring are each linked to a nitric oxide donor group; or (iii) the carbon atom at position 5 of the piperidine ring and optionally one or more of the carbon atoms at positions 3 or 4 of the piperidine ring are each linked to a nitric oxide donor group.

[0039] In further certain embodiments, the compound used according to the method of the present invention has the general formula I, wherein n is 3, i.e., a 1- azepanyloxy derivative of the formula Ic (see Table 1). In particular embodiments, the compound used according to this method has the general formula Ic, wherein one, two, three or four of the carbon atoms at positions 3 to 6 of the azepane ring are each linked to a nitric oxide donor group. In more particular embodiments, (i) the carbon atom at position 3 of the azepane ring and optionally one or more of the carbon atoms at positions 4 to 6 of the azepane ring are each linked to a nitric oxide donor group; (ii) the carbon atom at position 4 of the azepane ring and optionally one or more of the carbon atoms at positions 3, 5 or 6 of the azepane ring are each linked to a nitric oxide donor group; (iii) the carbon atom at position 5 of the azepane ring and optionally one or more of the carbon atoms at positions 3, 4 or 6 of the azepane ring are each linked to a nitric oxide donor group; or (iv) the carbon atom at position 6 of the azepane ring and optionally one or more of the carbon atoms at positions 3 to 5 of the azepane ring are each linked to a nitric oxide donor group.

Table 1: Structures la, lb, and Ic, indicating 1-pyrrolidinyloxy, 1-piperidinyloxy and 1-azepanyloxy derivatives, respectively

[0040] In particular embodiments, the compound used according to the method of the invention is a 1-pyrrolidinyloxy derivative of the formula la, 1-piperidinyloxy derivative of the formula lb, or 1-azepanyloxy derivative of the formula Ic, and each one of the nitric oxide donor groups in said compound independently is of the formula -(CrC₆)alkylene-ONO₂, preferably -(CrC₄)alkylene-ONO₂, more preferably -CH₂-ONO₂, or -O-(C₁-C₆)alkylene-ONO₂, wherein said alkylene is optionally substituted by one or more -ONO₂ groups, or is -ONO₂.

[0041] Specific compounds of the general formulas la, lb and Ic described herein, in which each one of the R groups independently is either H or the nitric oxide donor group -CH₂-ONO₂ or -ONO₂, are herein identified compounds la/b-15a/b in bold (compound la is also identified R-100), and their full chemical structures are depicted in Table 2. Other specific compounds of the general formulas la and lb described herein, in which one group is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂, and another group is not H, are herein identified compounds 16a/b- 17a/b in bold, and their full chemical structures are depicted in Table 3. A further specific compound of the general formula lb described herein, in which one R group is the nitric oxide donor group -O-CH₂-CH(ONO₂)CH₂-ONO₂, and the other Ri groups are H, is herein identified compound 18 in bold, and its full chemical structure is depicted in Table 3.

[0042] In specific embodiments, the compound used according to the method of the invention is the compound of formula la, i.e., a compound of the general formula I in which n is 1, wherein R₂ each is methyl; and (i) the Ri group linked to the carbon atom at position 3 of the pyrrolidine ring is the nitric oxide donor group - CH₂-ONO₂ or ONO₂; and the Ri group linked to the carbon atom at position 4 of the pyrrolidine ring is H, i.e., 3-nitratomethyl-2,2,5,5-tetramethylpyrrolidinyloxy (compound la; R-100) or 3-nitrato-2,2,5,5-tetramethylpyrrolidinyloxy (compound lb), respectively; or (ii) each one of the Ri groups linked to the carbon atoms at positions 3 and 4 of the pyrrolidine ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂, i.e., 3,4-dinitratomethyl-2,2,5,5-tetramethylpyrrolidinyloxy (compound 2a) or 3,4-dinitrato-2,2,5,5-tetramethylpyrrolidinyloxy (compound 2b), respectively.

[0043] In other specific embodiments, the compound used according to the method of the invention is the compound of formula lb, i.e., a compound of the general formula I wherein n is 2, wherein R₂ each is methyl; and (i) the Ri group linked to the carbon atom at position 3 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and each one of the Ri groups linked to the carbon atoms at positions 4 and 5 of the piperidine ring is H, i.e., 3-nitratomethyl- 2,2,6,6-tetramethylpiperidinyloxy (3-nitratomethyl-TEMPO; compound 3a) or 3- nitrato-2,2,6,6-tetramethylpiperidinyloxy (3-nitrato-TEMPO; compound 3b), respectively; (ii) the Ri group linked to the carbon atom at position 4 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and each one of the Ri groups linked to the carbon atoms at positions 3 and 5 of the piperidine ring is H, i.e., 4-nitratomethyl-2,2,6,6-tetramethylpiperidinyloxy (4-nitratomethyl- TEMPO; compound 4a) or 4-nitrato-2,2,6,6-tetramethylpiperidinyloxy (3-nitrato- TEMPO; compound 4b), respectively; (iii) each one of the Ri groups linked to the carbon atoms at positions 3 and 4 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and the Ri group linked to the carbon atom at position 5 of the piperidine ring is H, i.e., 3,4-dinitratomethyl-2,2,6,6-tetramethyl piperidinyloxy (3,4-dinitratomethyl-TEMPO; compound 5a) or 3,4-dinitrato- 2,2,6,6-tetramethylpiperidinyloxy (3,4-dinitrato-TEMPO; compound 5b), respectively; (iv) each one of the Ri groups linked to the carbon atoms at positions 3 and 5 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and the Ri group linked to the carbon atom at position 4 of the piperidine ring is H, i.e., 3,5-dinitratomethyl-2,2,6,6-tetramethylpiperidinyloxy (3,5-dinitratomethyl- TEMPO; compound 6a) or 3,5-dinitrato-2,2,6,6-tetramethylpiperidinyloxy (3,5- dinitrato-TEMPO; compound 6b), respectively; or (v) each one of the Ri groups linked to the carbon atoms at positions 3 to 5 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂, i.e., 3,4,5-trinitratomethyl-2,2,6,6- tetramethylpiperidinyloxy (3,4,5-trinitratomethyl-TEMPO; compound 7a) or 3,4,5- trinitrato-2,2,6,6-tetramethylpiperidinyloxy (3,4,5-trinitrato-TEMPO; compound 7b), respectively.

[0044] In further specific embodiments, the compound used according to the method of the invention is the compound of formula Ic, i.e., a compound of the general formula I wherein n is 3, wherein R₂ each is methyl; and (i) the Ri group linked to the carbon atom at position 3 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and each one of the Ri groups linked to the carbon atoms at positions 4 to 6 of the azepane ring is H, i.e., 3-nitratomethyl-2,2,7,7- tetramethylazepanyloxy (compound 8a) or 3-nitrato-2,2,7,7-tetramethylazepanyloxy (compound 8b), respectively; (ii) the Ri group linked to the carbon atom at position 4 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and each one of the Ri groups linked to the carbon atoms at position 3, 5 and 6 of the azepane ring is H, i.e., 4-nitratomethyl-2,2,7,7-tetramethylazepanyloxy (compound 9a) or 4- nitrato-2,2,7,7-tetramethylazepanyloxy (compound 9b), respectively; (iii) each one of the Ri groups linked to the carbon atoms at positions 3 and 4 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and each one of the Ri groups linked to the carbon atoms at positions 5 and 6 of the azepane ring is H, i.e., 3,4- dinitratomethyl-2,2,7,7-tetramethylazepanyloxy (compound 10a) or 3,4-dinitrato- 2,2,7,7-tetramethylazepanyloxy (compound 10b), respectively; (iv) each one of the Ri groups linked to the carbon atoms at positions 3 and 5 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and each one of the Ri groups linked to the carbon atoms at positions 4 and 6 of the azepane ring is H, i.e., 3,5- dinitratomethyl-2,2,7,7-tetramethylazepanyloxy (compound 11a) or 3,5-dinitrato- 2,2,7,7-tetramethylazepanyloxy (compound lib), respectively; (v) each one of the Ri groups linked to the carbon atoms at positions 3 and 6 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and each one of the Ri groups linked to the carbon atoms at positions 4 and 5 of the azepane ring is H, i.e., 3,6- dinitratomethyl-2,2,7,7-tetramethylazepanyloxy (compound 12a) or 3,6-dinitrato- 2,2,7,7-tetramethylazepanyloxy (compound 12b), respectively; (vi) each one of the Ri groups linked to the carbon atoms at positions 3 to 5 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and the R group linked to the carbon atom at position 6 of the azepane ring is H, i.e., 3,4,5-trinitratomethyl- 2,2,7,7-tetramethylazepanyloxy (compound 13a) or 3,4,5-trinitrato-2,2,7,7- tetramethylazepanyloxy (compound 13b), respectively; (vii) each of the R groups linked to the carbon atoms at positions 3, 4 and 6 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂; and the R group linked to the carbon atom at position 5 of the azepane ring is H, i.e., 3,4,6-trinitratomethyl-2,2,7,7- tetramethylazepanyloxy (compound 14a) or 3,4,6-trinitrato-2,2,7,7-tetramethyl azepanyloxy (compound 14b), respectively); or (viii) each of the R groups linked to the carbon atoms at positions 3 to 6 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or ONO₂, i.e., 3,4,5,6-tetranitratomethyl-2,2,7,7-tetramethyl azepanyloxy (compound 15a) or 3,4,5,6-tetranitrato-2,2,7,7-tetramethyl azepanyloxy (compound 15b), respectively.

[0045] In still other specific embodiments, the compound used according to the method of the invention is the compound of formula la, wherein R₂ each is methyl; the Ri group linked to the carbon atom at position 3 of the pyrrolidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and the R group linked to the carbon atom at position 4 of the pyrrolidine ring is -CONH₂, i.e., 3-nitratomethyl-4- carbamoyl-2,2,5,5-tetramethylpyrrolidinyloxy (compound 16a) or 3-nitrato-4- carbamoyl-2,2,5,5-tetramethylpyrrolidinyloxy (compound 16b), respectively.

[0046] In yet other specific embodiments, the compound used according to the method of the invention is the compound of formula lb, wherein R₂ each is methyl; the Ri group linked to the carbon atom at position 3 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; the Ri group linked to the carbon atom at position 4 of the piperidine ring is -COOH; and the Ri group linked to the carbon atoms at position 5 of the piperidine ring is H, i.e., 3-nitratomethyl-4- carboxy-2,2,6,6-tetramethylpiperidinyloxy (3-nitratomethyl-4-carboxy-TEMPO; compound 17a) or 3-nitrato-4-carboxy-2,2,6,6-tetramethylpiperidinyloxy (3-nitrato- 4-carboxy-TEMPO; compound 17b), respectively. Table 2: Compounds of the general formulas la, lb, and Ic, identified la- 15a

The compounds corresponding to la-15a, in which each one of the -CH₂-ONO₂ groups is replaced by the -ONO2 group, are identified compounds lb-15b

[0047] In still a further specific embodiment, the compound used according to the method of the invention is the compound of formula lb, wherein R₂ each is methyl; the Ri group linked to the carbon atom at position 4 of the piperidine ring is the nitric oxide donor group -O-CH₂-CH(ONO₂)CH₂-ONO₂; and each one of the R groups linked to the carbon atom at position 3 and 5 of the piperidine ring is H, i.e., 4-(2,3-dinitratopropyloxy)-2,2,6,6-tetramethylpiperidinyloxy (4-(2,3-dinitrato propyloxy)-TEMPO; compound 18).

Table 3: Compounds of the general formulas la and lb, identified 16a- 17a^* and 18

* The compounds corresponding to 16a and 17a, in which each one of the -CH₂-ON0₂ groups is replaced by the -ON0₂ group, are identified compounds 16b and 17b

[0048] In other particular embodiments, the compound used according to the method of the present invention is a 1-pyrrolidinyloxy derivative of the formula la, 1-piperidinyloxy derivative of the formula lb, or 1-azepanyloxy derivative of the formula Ic; wherein at least one of the nitric oxide donor groups in said compound is of the formula -O-(CrC₆)alkylene-ONO₂; and said alkylene is substituted by a moiety of the general formula D as defined above, and is optionally further substituted by one or more -ONO₂ groups. The general formula D, in which oxygen atom is linked to the carbon atom at position 3 or 4 of the ring, represents a 3- hydroxy-pyrrolidinoxy, 3- or 4-hydroxy-piperidinyloxy, or 3- or 4-hydroxy- azepanyloxy derivative. Conceptually, the compound used in this case is thus a dimer- or higher multimer-like compound, in which two or more identical or different entities, each independently being selected from 1-pyrrolidinyloxy, 1- piperidinyloxy or 1-azepanyloxy derivatives, are linked via alkylene bridges substituted by one or more -ONO₂ groups, wherein each alkylene bridge links two entities only. [0049] Preferred dimer- or higher multimer-like compounds to be used according to the method of the invention are those in which (i) a 1-pyrrolidinyloxy derivative of the general formula la is linked via one or two nitric oxide donor groups thereof to one or two identical or different moieties of a 3-hydroxy-pyrrolidinoxy, i.e., one or two moieties of the general formula D in which m is 1; (ii) a 1-piperidinyloxy derivative of the general formula lb is linked via one, two or three nitric oxide donor groups thereof to one, two or three identical or different moieties of a 3-, or 4-hydroxy-piperidinyloxy, i.e., one to three moieties of the general formula D in which m is 2; or (iii) a 1-azepanyloxy derivative of the general formula Ic is linked via one, two, three or four nitric oxide donor groups thereof to one, two, three, or four identical or different moieties of a 3-, or 4-hydroxy-azepanyloxy, i.e., one to four moieties of the general formula D in which m is 3.

[0050] Specific compounds of the general formula lb described herein, having a dimer-like structure, are herein identified compounds 19-20 in bold, and their full chemical structures are depicted in Table 4.

[0051] In specific embodiments, the compound used according to the method of the invention is the dimer-like compound of formula lb, wherein each one of R linked to the carbon atoms at positions 3 and 5 of the piperidine ring is H; and (i) linked to the carbon atom at position 4 of the piperidine ring is the nitric oxide donor group -O-CH₂-CH₂-CH(CH₃)-ONO₂, wherein the 1,3 butane diyl is substituted at position 2 with -ONO₂ group and at position 4 with a moiety of the general formula D, wherein m is 2, and the oxygen atom is linked to the carbon atom at position 4 of the piperidine ring in the formula D; and R₂ each is methyl, i.e., l,4-di-(4-oxo-TEMPO)-2,3-dinitratobutane (compound 19); or (ii) linked to the carbon atom at position 4 of the piperidine ring is the nitric oxide donor group - O-CH₂-CH(CH₃)-ONO₂, wherein the 1,2 propane diyl is substituted at position 3 with a moiety of the general formula D, wherein m is 2, and the oxygen atom is linked to the carbon atom at position 4 of the piperidine ring in the formula D; and R₂ each is methyl, i.e., l,3-di-(4-oxo-TEMPO)-2-nitratopropane (compound 20). Table 4: Compounds of the general formula lb, identified 19-20

[0052] The compounds used according to the method of the present invention may be synthesized according to any technology or procedure known in the art, e.g., as described in detail in US 6,448,267, 6,455,542 and 6,759,430.

[0053] The compounds of the general formula I may have one or more asymmetric centers, and may accordingly exist both as enantiomers, i.e., optical isomers (R, S, or racemate, wherein a certain enantiomer may have an optical purity of 90%, 95%, 99% or more) and as diastereoisomers. Specifically, those chiral centers may be, e.g., in each one of the carbon atoms of the 1-pyrrolidinyloxy derivative, 1- piperidinyloxy derivative; and 1-azepanyloxy derivative of the general formulas la, lb and Ic, respectively. It should be understood that according to the method of the present invention, treatment of renal ischemia-reperfusion injury can be carried out by administration of all such enantiomers, isomers and mixtures thereof, as well as pharmaceutically acceptable salts and solvates thereof.

[0054] Optically active forms of the compounds of the general formula I may be prepared using any method known in the art, e.g., by resolution of the racemic form by recrystallization techniques; by chiral synthesis; by extraction with chiral solvents; or by chromatographic separation using a chiral stationary phase. A non- limiting example of a method for obtaining optically active materials is transport across chiral membranes, i.e., a technique whereby a racemate is placed in contact with a thin membrane barrier, the concentration or pressure differential causes preferential transport across the membrane barrier, and separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through. Chiral chromatography, including simulated moving bed chromatography, can also be used. A wide variety of chiral stationary phases are commercially available.

[0055] The compounds of the general formula I, e.g., compound la, have a very limited solubility in aqueous solutions. In order to increase their solubility, organic modifiers or co-solvents can be added; however, the amount of organic solvent required to achieve concentrations of several mg/ml may not be pharmaceutically desirable, especially at high therapeutic doses of said compounds. Furthermore, upon dilution of such solutions in aqueous solution, the compound may precipitate out. This might be critical when delivering such compound to an animal or human, e.g., when such solutions are intravenously injected or infused, as precipitation of the compound upon contact with circulating blood may cause local irritation at the injection site or serious systemic complications and/or organ damage. Similar complications may be anticipated for other delivery routes such as oral or subcutaneous routes.

[0056] While reducing the present invention into practice, it has been found that aqueous solutions of compound la, having concentrations several times greater than those with commonly used co-solvents such as polyethylene glycol 400 (PEG400), propyleneglycol, polyvinylpyrrolidone (PVP), N-methy-2-pyrrolidone (NMP), and dimethylacetamide, can be obtained by stirring said compound in water with 2- hydroxypropyl-P-cyclodextrin (HPCD) in ratios typically between 1 : 15 and 1 :20 w/w, depending on the degree of substitution of the cyclodextrin with the hydroxypropyl side chain, or greater. In particular, HPCDs with degrees of substitution between 3.5 and 7 were tested, and aqueous solutions of compound la having concentrations of 15 mg/ml or higher were achieved. As further found, while compound la precipitates from solutions in dimethyl sulfoxide (DMSO), PEG400, NMP or other organic solvents when diluted in aqueous solutions as the proportion of organic solvent falls below a certain value, upon dilution of aqueous solutions of said compound and HPCD in water, saline or dextrose 5% in water (D5W), the compound remains in the solution. [0057] In certain embodiments, the compound used according to the method of the present invention is thus a piperidine, pyrrolidine, or azepane derivative of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, wherein said compound is formulated as an inclusion complex with a hydroxyalkyl-cyclodextrin.

[0058] The term "inclusion complex" or "inclusion compound", as used herein interchangeably, refers to a complex in which one chemical compound ("the host"), in this particular case an hydroxyalkyl-cyclodextrin, forms a cavity in which molecules of a second chemical compound ("the guest"), in this particular case a compound of the general formula I as defined above or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, are located and actually trapped. There is no covalent bonding between guest and host, and the attractions are generally due to van der Waals forces.

[0059] Cyclodextrins (CDs) are a family of cyclic oligosaccharides composed of 5 or more a-D-glucopyranoside units linked 1→4, in the C_\ chair conformation. The most common cyclodextrins have six, seven or eight glucopyranose units and are referred to as α-, β- and γ-CD, respectively. As a consequence of their peculiar structure, these molecules feature a conical cavity that is essentially hydrophobic in nature and limited by hydroxyl groups of different chemical characters. The hydroxyl groups located at the narrower side are primary, i.e., come from position 6 of the glucopyranose ring, while those located at the wider entrance are secondary and therefore are less prone to chemical transformation. The reactivity of the hydroxyl groups strongly depends on the reaction conditions. The non-reducing character of cyclodextrins makes them behave as polyols. On the other hand, the large number of hydroxyl groups available implies that careful selection of the reaction conditions is required in order to avoid the substitution of more groups than those needed for a particular purpose.

[0060] The inner diameter of the conical cavity in unmodified cyclodextrins varies from 5 to 10 A and its depth is about 8 A. For β-CDs, the internal and external diameters are about 7.8 A and 15.3 A, respectively, and the calculated surface area is approximately 185 A². These dimensions allow the inclusion of several types of guest molecules of appropriate size to form inclusion complexes. As a consequence of the inclusion, some properties of the guest molecules change, and this phenomenon, in fact, constitutes the basis of practically all cyclodextrin applications, including artificial enzymes, sensors, drug formulations, cosmetics, food technology and textiles. Cyclodextrin inclusion complexes can be thermodynamically more or less stable depending on the shape and size of the guest molecule, and the association constants can be measured by a range of physicochemical methods. Absorption and emission spectroscopy along with nuclear magnetic resonance and calorimetry are the most popular techniques used to study these systems and have provided an understanding of the structure and energetics of the inclusion process. Recently, the use of scanning probe techniques such as atomic force microscopy has allowed the measurement of the force involved in these interactions at a single-molecule level, opening new and exciting prospects in supramolecular chemistry.

[0061] In particular embodiments, the hydroxyalkyl-cyclodextrin constituting the inclusion complex used according to the method of the present invention is hydroxyalkyl-α-, hydroxyalkyl- β- or hydroxyalkyl-y-cyclodextrin, preferably hydroxyalkyl-p-cyclodextrin. The term "hydroxyalkyl" as used herein refers to any hydroxyl derivative of a C C₄ alkyl as defined above. In more particular embodiments, said hydroxyalkyl-p-cyclodextrin is hydroxyethyl- -cyclodextrin, hydroxypropyl- -cyclodextrin, dihydroxypropyl- -cyclodextrin, or hydroxybutyl-β- cyclodextrin, preferably hydroxypropyl- -cyclodextrin, more preferably 2- hydroxypropyl- -cyclodextrin.

[0062] The hydroxyalkyl groups are randomly substituted onto the hydroxyl groups of the cyclodextrin and the amount of substitution is called the average degree of substitution or number of hydroxyalkyl groups per cyclodextrin, and it is the preferred manner of describing the substitution. The molecular weight of the hydroxyalkyl cyclodextrin is calculated based upon the degree of substitution, wherein said substitution is, in fact, a distribution around the average degree of substitution of the number of hydroxyalkyl groups per cyclodextrin molecule with some molecules having either more or less than the average degree of substitution. The result is a mixture of many molecular species with respect to the number and location of substitutions around the ring of the cyclodextrin.

[0063] The degree of substitution may have an effect on the binding of guests to the hydroxyalkyl cyclodextrin molecule, wherein at low degrees of substitution, binding is very similar to that of the unmodified cyclodextrin, while increasing substitution can lead to weakened binding due to steric hindreance. The effect on the binding of guests to the host molecule is dependent upon the particular guest and it is also possible to obtain increased binding due to an increase in surface area to which the guest can bind. Still, with most guests, these differences in binding with degree of substitution are small if detectable.

[0064] The hydroxyalkyl cyclodextrins constituting the inclusion complex used according to the method of the present invention may have any degree of substitution, i.e., may be either fully or partially modified with hydroxyalkyl groups, wherein each a-D-glucopyranoside units has three hydroxyl groups which can be substituted. In certain embodiments, the hydroxyalkyl cyclodextrins constituting said inclusion complex is an hydroxyalkyl- β-cyclodextrin, preferably 2- hydroxypropyl- -cyclodextrin, having a degree of substitution in a range of 3 to 8, preferably 3.5 to 7.

[0065] In specific embodiments, the method of the present invention comprises administration of an inclusion complex of a compound selected from the compounds of Tables 2-4 above, preferably compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and an hydroxyalkyl-cyclodextrin as defined above, more particularly, of compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and 2-hydroxypropyl- -cyclodextrin. In more specific such embodiments, the ratio of the guest to the host in the inclusion complex of the invention, i.e., of compound la to 2-hydroxypropyl- -cyclodextrin, is between 1: 15 (w/w) to 1:20 (w/w), respectively. [0066] The term "renal ischemia", as used herein, refers to a deficiency of blood flow in one or both kidneys, or nephrons, usually due to functional constriction or actual obstruction of a blood vessel or surgical removal of the kidney. More particularly, renal ischemia may result from various medical conditions including, without being limited to, hemorrhagic shock, septic shock, asphyxia also known as asphyxiation, cardiac arrest also known as cardiopulmonary arrest or circulatory arrest, respiratory arrest, respiratory failure, cardiogenic shock, aortic aneurysm, aortic aneurysm surgery, hypotension, dehydration, spinal shock, trauma, cadaveric renal transplantation, living related donor renal transplantation, liver transplantation, a liver disease, drug-induced renal ischemia, hydronephrosis, urethral obstruction, cardiopulmonary bypass surgery, radiocontrast administration, endovascular renal artery catheterization, renovascular stenosis, renal artery thrombosis, ureteral obstruction, hypoxia, and hypoxemia.

[0067] The term "renal ischemia-reperfusion injury" (RIRI), as used herein, refers to the damage caused to the kidney(s) when blood supply returns to the tissue after a period of renal ischemia. RIRI is characterized by renal dysfunction and tubular damages, and considered as a major cause of acute renal failure that may also be involved in the development and progression of some forms of chronic kidney disease. In general, the absence of oxygen and nutrients from blood during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function.

[0068] As stated above, RIRI is a frequent consequence of cadaveric renal transplantation, circulatory shock, and major vascular procedures of the abdominal and thoracic aorta. For example, in the post-operative period after thoracoabdominal aortic aneurysm repair, renal complications occurred in 28% of the patients, and this was a major independent risk factor for early mortality (Rectenwald et at, 2002). Similarly, in the early post-operative period after cadaveric renal transplantation, creatinine clearance and tubular sodium reab sorption were profoundly reduced in kidneys, coinciding with significantly increased urinary concentrations of tubular injury markers, in particular, NGAL, N-acetyl- -glucosaminidase, and cystatin C; and an 18-fold increase in renal production of cytokeratin-18, indicating extensive necrotic cell death (Snoeijs et at, 2011).

[0069] The term "treatment" as used herein with respect to RIRI refers to administration of a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, after the onset of symptoms of RIRI, i.e., after blood supply to the ischemic tissue has been renewed, regardless of the cause for the renal ischemia. The term "prevention" as used herein with respect to RIRI refers to administration of said compound, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, prior to the onset of symptoms, i.e., either prior to the onset of renal ischemia or following the onset of renal ischemia but prior to reperfusion, and it is mainly relevant in cases wherein the renal ischemia and/or reperfusion is/are associated with a surgical intervention, e.g., with aortic aneurysm surgery, cadaveric renal transplantation, living related donor renal transplantation, liver transplantation, cardiopulmonary bypass surgery, or endovascular renal artery catheterization. According to the present invention, administration of said compound either for treatment or prevention of RIRI is aimed at inhibiting, i.e., limiting or reducing, renal dysfunction, PMN infiltration into the renal parenchyma, and histological damage, i.e., tubular necrosis. The terms "prophylactically effective amount" and "therapeutically effective amount" as used herein refer to the quantity of the compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, that is useful to prevent or treat RIRI, respectively.

[0070] As shown in the Examples section hereinafter, administration of R-100 (80 mg/kg/day, formulated with HPCD) three times a day via an intraperitoneal (IP) route in a rat RIRI model, with the initial dose given during the final 5 minutes of a 30 minute period of ischemia prior to reperfusion, inhibited renal dysfunction, determined based on serum creatinine and NGAL levels; PMN infiltration into the renal parenchyma, determined based on myeloperoxidase (MPO) activity; and histological damage, i.e., tubular necrosis, determined semiquantitatively as described hereinafter, by 75%-90%. Despite its administration at the end of the ischemia period, these results constitute dramatic affirmation that R-100 is protective in resuscitating RIRI.

[0071] As shown in a different study described herein, administration of R-100 (80 mg/kg, formulated with HPCD) as a 10 minute IV infusion beginning 20 minutes after onset of ischemia in a rat RIRI model, partially or entirely inhibited the various manifestations associated with renal injury at the levels of morphology, function, and biochemical damage. In particular, R-100 therapy entirely inhibited the RIRI- induced elevation in serum urea and partially inhibited the RIRI-induced elevation in serum creatinine, strongly indicating that R-100 therapy profoundly blocks RIRI- induced renal dysfunction; significantly reduced the elevation in FE_Na resulting from the impaired ability of the kidney to reabsorb filtered Na⁺ as a consequence of RIRI, and partially inhibited elevations of both serum and urinary NGAL, indicating that R-100 therapy blocks RIRI-induced renal tubular damage; partially reversed the increase in PMN infiltration into renal tissue; and blocked the increase in malondialdehyde (MDA) formation in the renal tissue.

[0072] In certain embodiments, the compound used according to the method of the present invention is thus administered prior to the onset of renal ischemia or following the onset of renal ischemia but prior to reperfusion, so as to prevent, i.e., limit or reduce, RIRI.

[0073] In other embodiments, the compound used according to the method of the invention is administered after reperfusion, so as to treat, i.e., limit or reduce, RIRI.

[0074] In another aspect, the present invention provides a pharmaceutical composition for prevention or treatment of RIRI comprising a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. Particular such pharmaceutical compositions comprise a compound selected from the compounds of Tables 2-4 above, preferably compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof.

[0075] In certain embodiments, the compound comprised within the pharmaceutical composition of the present invention is a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, formulated as an inclusion complex with a hydroxyalkyl-cyclodextrin.

[0076] In particular embodiments, the pharmaceutical composition of the present invention comprises an inclusion complex of a compound selected from the compounds of Tables 2-4 above, preferably compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and an hydroxyalkyl-cyclodextrin as defined above, more particularly, of compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and 2-hydroxypropyl- -cyclodextrin. Specific such pharmaceutical compositions comprise an inclusion complex of compound la and 2- hydroxypropyl- -cyclodextrin, wherein the ratio of compound la to 2- hydroxypropyl- -cyclodextrin is between 1: 15 (w/w) to 1:20 (w/w), respectively.

[0077] The pharmaceutical compositions of the present invention can be provided in a variety of formulations, e.g., in a pharmaceutically acceptable form and/or in a salt form, as well as in a variety of dosages.

[0078] In one embodiment, the pharmaceutical composition of the present invention comprises a non-toxic pharmaceutically acceptable salt of a compound of the general formula I. Suitable pharmaceutically acceptable salts include acid addition salts such as, without being limited to, the mesylate salt, the maleate salt, the fumarate salt, the tartrate salt, the hydrochloride salt, the hydrobromide salt, the esylate salt, the p-toluenesulfonate salt, the benzoate salt, the acetate salt, the phosphate salt, the sulfate salt, the citrate salt, the carbonate salt, and the succinate salt. Additional pharmaceutically acceptable salts include salts of ammonium (NH₄ ⁺) or an organic cation derived from an amine of the formula R N⁺, wherein each one of the Rs independently is selected from H, Q-C₂₂, preferably C C₆ alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n- pentyl, 2,2-dimethylpropyl, n-hexyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3-7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine. Furthermore, where the compounds of the general formula I carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include metal salts such as alkali metal salts, e.g., lithium, sodium or potassium salts, and alkaline earth metal salts, e.g., calcium or magnesium salts.

[0079] Further pharmaceutically acceptable salts include salts of a cationic lipid or a mixture of cationic lipids. Cationic lipids are often mixed with neutral lipids prior to use as delivery agents. Neutral lipids include, but are not limited to, lecithins; phosphatidylethanolamine; diacyl phosphatidylethanolamines such as dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine; phosphatidylcholine; diacyl phosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoyl phosphatidylcholine; phosphatidylglycerol; diacyl phosphatidylglycerols such as dioleoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol and distearoyl phosphatidylglycerol; phosphatidylserine; diacyl phosphatidylserines such as dioleoyl- or dipalmitoyl phosphatidylserine; and diphosphatidylglycerols; fatty acid esters; glycerol esters; sphingolipids; cardiolipin; cerebrosides; ceramides; and mixtures thereof. Neutral lipids also include cholesterol and other 3β hydroxy- sterols.

[0080] Examples of cationic lipid compounds include, without being limited to, Lipofectin^® (Life Technologies, Burlington, Ontario) (1 : 1 (w/w) formulation of the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidyl-ethanolamine); Lipofectamine™ (Life Technologies, Burlington, Ontario) (3: 1 (w/w) formulation of polycationic lipid 2,3-dioleyloxy-N- [2(spermine-carboxamido)ethyl] -Ν,Ν-dimethyl- 1 -propanamin-iumtrifluoroacetate and dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (Life Technologies, Burlington, Ontario) (Lipofectamine and Plus reagent), Lipofectamine 2000 (Life Technologies, Burlington, Ontario) (Cationic lipid), Effectene (Qiagen, Mississauga, Ontario) (Non liposomal lipid formulation), Metafectene (Biontex, Munich, Germany) (Polycationic lipid), Eu-fectins (Promega Biosciences, San Luis Obispo, Calif.) (ethanolic cationic lipids numbers 1 through 12: C₅₂H₁₀₆N₆O₄-4CF₃CO₂H, C₈₈H₁₇₈N₈O₄S₂4CF₃CO₂H, C₄₀H₈₄NO₃P CF₃CO₂H, C₅₀H₁₀₃N₇O₃-4CF₃CO₂H, C₅₅H₁₁₆N₈O₂6CF₃CO₂H, C₄₉H₁₀₂N₆O₃4CF₃CO₂H, C₄₄H₈₉N₅O₃-2CF₃CO₂H, C₁₀oH₂o₆N₁₂O₄S₂-8CF₃CO₂H, C₁₆₂H₃₃₀N₂₂O₉ 13CF₃CO₂H, C₄₃H₈₈N₄O₂2CF₃CO₂H, C₄₃H₈₈N₄O₃-2CF₃CO₂H, C₄iH₇₈NO₈P); Cytofectene (Bio- Rad, Hercules, Calif.) (mixture of a cationic lipid and a neutral lipid), GenePORTER^® (Gene Therapy Systems, San Diego, Calif.) (formulation of a neutral lipid (Dope) and a cationic lipid) and FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, Ind.) (Multi-component lipid based non-liposomal reagent).

[0081] The pharmaceutically acceptable salts of the present invention may be formed by conventional means, e.g., by reacting a free base form of the active agent or ingredient, i.e., the compound of the general formula I, with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is removed in vacuo or by freeze drying, or by exchanging the anion/cation of an existing salt for another anion/cation on a suitable ion exchange resin.

[0082] The present invention encompasses solvates of the compounds of the general formula I as well as salts thereof, e.g., hydrates.

[0083] The pharmaceutical compositions provided by the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19^th Ed., 1995. The compositions can be prepared, e.g., by uniformly and intimately bringing the active ingredient, i.e., the compound of the general formula I, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation. The compositions may be in liquid, solid or semisolid form and may further include pharmaceutically acceptable fillers, carriers, diluents or adjuvants, and other inert ingredients and excipients. In one embodiment, the pharmaceutical composition of the present invention is formulated as nanoparticles.

[0084] The compositions can be formulated for any suitable route of administration, but they are preferably formulated for parenteral, e.g., intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal or subcutaneous, administration, as well as for inhalation. The dosage will depend on the state of the patient, and will be determined as deemed appropriate by the practitioner.

[0085] The pharmaceutical composition of the invention may be in the form of a sterile injectable aqueous or oleagenous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include, without limiting, water, Ringer's solution and isotonic sodium chloride solution.

[0086] Pharmaceutical compositions according to the present invention, when formulated for inhalation, may be administered utilizing any suitable device known in the art, such as metered dose inhalers, liquid nebulizers, dry powder inhalers, sprayers, thermal vaporizers, electrohydrodynamic aerosolizers, and the like.

[0087] Pharmaceutical compositions according to the present invention, when formulated for administration route other than parenteral administration, may be in a form suitable for oral use, e.g., as tablets, troches, lozenges, aqueous, or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets. These excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., corn starch or alginic acid; binding agents, e.g., starch, gelatin or acacia; and lubricating agents, e.g., magnesium stearate, stearic acid, or talc. The tablets may be either uncoated or coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated using the techniques described in the US Patent Nos. 4,256,108, 4,166,452 and 4,265,874 to form osmotic therapeutic tablets for control release. The pharmaceutical composition of the invention may also be in the form of oil-in-water emulsion.

[0088] The pharmaceutical compositions of the invention may be formulated for controlled release of the active agent. Such compositions may be formulated as controlled-release matrix, e.g., as controlled-release matrix tablets in which the release of a soluble active agent is controlled by having the active diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastro-intestinal fluid (in vivo). Many polymers have been described as capable of forming such gel, e.g., derivatives of cellulose, in particular the cellulose ethers such as hydroxypropyl cellulose, hydroxymethyl cellulose, methylcellulose or methyl hydroxypropyl cellulose, and among the different commercial grades of these ethers are those showing fairly high viscosity. In other configurations, the compositions comprise the active agent formulated for controlled release in microencapsulated dosage form, in which small droplets of the active agent are surrounded by a coating or a membrane to form particles in the range of a few micrometers to a few millimeters.

[0089] Another contemplated formulation is depot systems, based on biodegradable polymers, wherein as the polymer degrades, the active ingredient is slowly released. The most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules. Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLG).

[0090] In a further aspect, the present invention provides a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, for use in prevention or treatment of RIRI.

[0091] In yet another aspect, the present invention relates to use of a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, for the preparation of a pharmaceutical composition for prevention or treatment of RIRI.

[0092] In another aspect, the present invention provides an inclusion complex of a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and an hydroxyalkyl-cyclodextrin as defined above.

[0093] The inclusion complex of the invention may be prepared according to any procedure known in the art utilizing any technology available. In particular embodiments, the inclusion complex of the invention is prepared as shown in the Examples section hereinafter, i.e., by (i) stirring the hydroxyalkyl-cyclodextrin, preferably hydroxypropyl- -cyclodextrin, more preferably 2-hydroxypropyl- - cyclodextrin, in distilled water or a physiological solution, with a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof; and optionally (ii) lyophilizing the solution obtained in (i).

[0094] Thus, in a particular aspect, the present invention provides an inclusion complex as defined above, more particularly, an inclusion complex of hydroxypropyl- -cyclodextrin, preferably 2-hydroxypropyl- -cyclodextrin, with a compound selected from the compounds of Tables 2-4 above, preferably compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, in a lyophilized form. [0095] In a further aspect, the present invention provides a pharmaceutical composition comprising an inclusion complex as defined above, and a pharmaceutically acceptable carrier. Particular such pharmaceutical compositions comprise an inclusion complex of a compound selected from the compounds of Tables 2-4 above, preferably compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and hydroxypropyl- -cyclodextrin, preferably 2-hydroxypropyl- -cyclodextrin, in a lyophilized form.

[0096] As surprisingly found, aqueous solutions containing substantially higher concentrations of compound la with HPCD can be achieved by preparing solutions as described above; filtering and freeze drying the filtrate, either as is or diluted further with water optionally containing other additives or excipients; and re- dissolving the resulting freeze dried cake, i.e., the lyophilizate, in a volume of water that is smaller than that originally used to prepare the solution prior to lyophilization. As shown in the Examples section hereinafter, the re-dissolution of the lyophilizate enables obtaining aqueous solutions of compound la having concentrations significantly higher than those which can be theoretically obtained when just mixing said compound with HPCD.

[0097] In still a further aspect, the present invention relates to a process for the preparation of a solution of an inclusion complex of a compound of the general formula I as defined above, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and an hydroxyalkyl- cyclodextrin, as defined above, in distilled water or a physiological solution, comprising the steps of:

(iv) dissolving said lyophilizate in distilled water or physiological solution, wherein the concentration of the inclusion complex, thus the nominal concentration of said compound, or enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, in the solution obtained in step (iv) is higher than that in the solution obtained in step (i).

[0098] The term "lyophilization", also known as lyophilisation, freeze-drying or cryodesiccation, refers to a dehydration process comprising the steps of freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublime directly from the solid phase to the gas phase.

[0099] The term "nominal concentration" as used herein with respect to the compound of the general formula I, or enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, refers to the amount of said compound that is expected to be present in a typical sample of a solution produced according to the process of the present invention at the time said solution is produced, expressed as a percentage by weight.

[00100] In particular embodiments, step (i) of the process described above is conducted by dissolving compound la and 2-hydroxypropyl-P-cyclodextrin, wherein the ratio of compound la to 2-hydroxypropyl-P-cyclodextrin is between 1: 15 (w/w) to 1:20 (w/w).

[00101] The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Example 1. Aqueous solutions of R-100 and hydroxypropyl-p-cyclodextrin

[00102] In this study, various aqueous solutions of compound la (R-100) and hydroxypropyl-P-cyclodextrin (HPCD) were prepared by stirring R-100 in water with HPCD in ratios typically between 1: 15 and 1:20 w/w, depending on the degree of substitution of the cyclodextrin with the hydroxypropyl side chain. In particular, HPCDs with degrees of substitution between 3.5 and 7 were tested, and similar results, more particularly, aqueous solutions having R-100 concentrations of 15 mg/ml or higher, were achieved. As further found, lower ratios could also be used; however, when R-100 was not all dissolved and had to be filtered off, the actual R- 100:HPCD ratio in the resulting solution was in the above range.

[00103] As found in this study, HPCDs can be used to solubilize R-100 and achieve concentrations several times greater than those with commonly used co-solvents such as polyethylene glycol 400 (PEG400), propyleneglycol, polyvinylpyrrolidone, N-methy-2-pyrrolidone (NMP), and dimethylacetamide. Furthermore, while R-100 precipitated from solutions of R-100 in DMSO, PEG400, NMP or other organic solvents when diluted in aqueous solutions as the proportion of organic solvent fell below a certain value, upon dilution of aqueous solutions of R-100 and HPCD in water, saline or dextrose 5% in water (D5W), R-100 remained in the solution.

[00104] A further series of experiments have shown that, for a given ratio of R-100 to HPCD, higher concentrations of R-100 can be achieved by preparing solutions as described above, filtering and freeze drying the filtrate, either as is or diluted further with water (containing other additives or excipients as desired), and then dissolving the resulting lyophilizate in a volume of water that is less than that originally used to prepare the solution prior to lyophilization.

[00105] In a particular experiment, 13 mg of R-100 was stirred with 221 mg of HPCD in 2 ml water for 30 minutes. The remaining solids were filtered off and the filtrate was diluted to 13 ml with more water. The solution obtained was frozen and freeze dried; and the resulting solid cake was then dissolved in 0.5 ml water to give a solution of R-100 at approximately 26 mg/ml. Concentrations before and after filtration and upon reconstitution were compared by HPLC.

[00106] A wide range of concentration of the R-100 solutions can be lyophilyzed to produce solid cakes that can be dissolved to give clear solutions of greater than 20 mg/ml. For example: (i) a solution of 13 mg R-100 and 221 mg HPCD stirred in 2 ml water produces a turbid solution. This was diluted and filtered and further diluted to 8 ml. The solution was lyophilyzed and reconstituted in 0.5 ml water to give a clear solution of 26 mg/ml with respect to R-100; (ii) 13.4 mg R-100 and 221 mg HPCD stirred in 2 ml water (6.7 mg/ml R-100/ml) for several hours still retained some turbidity, even when diluted to 3 ml. The solution was filtered and the filtrate was lyophilyzed and reconstituted in 0.5 ml water to give a clear solution of approximately 27 mg/ml with respect to R-100.

Example 2. R-100 resuscitation of a rat RIRI model - first study

[00107] Anesthetized Sprague-Dawley rats (n=10 per experimental group), exposed to bilateral occlusion of the renal pedicles for 30 minutes, were treated three times a day (TID) via an IP route with compound la (R-100; 80 mg/kg/day; formulated with HPCD) or vehicle control, with the initial dose given 5 minutes prior to the onset of reperfusion. Tissues and sera were collected at 24 hours for analysis. Renal function was assessed by measurement of serum creatinine (expressed as micromolar) and NGAL (expressed as ng/ml). PMN infiltration into the renal parenchyma was assessed by measuring MPO activity (expressed as U/g wet tissue). Histological assessment of tubular necrosis was determined semiquantitatively using a method modified from McWhinnie (McWhinnie et at, 1987): Random cortical fields were observed using a 20X objective. A graticule grid (25 squares) was used to determine the number of line intersects involving tubular profiles. 100 intersections were examined for each kidney, and a score from 0 to 3 was given for each tubular profile involving an intersection: 0 = normal histology; 1 = tubular cell swelling, brush border loss, nuclear condensation, with up to one third of tubular profile showing nuclear loss; 2 = same as for score 1, but greater than one third and less than two thirds of tubular profile shows nuclear loss; 3 = greater than two thirds of tubular profile shows nuclear loss. The total score for each kidney was calculated by addition of all 100 scores.

[00108] Despite its administration after the conclusion of the 30 minute period of ischemia, R-100 inhibited renal dysfunction, PMN infiltration, and histological damage by 75%-90% (p<10^~14 for all 4 endpoints; 2-tailed, Student's T-test), as shown in Table 5. As shown in Fi<*« 1 A-1 R while no histological alterations were observed in the kidney section from sham-operated rats, animals that underwent renal ischemia/reperfusion demonstrated the recognized features of severe acute injury, including brush border loss, nuclear condensation, cytoplasmic swelling, and consistent loss of significant numbers of nuclei from tubular profiles. On the other hand, R-100 significantly reduced the ischemia/reperfusion-induced histological alteration, as clearly shown in Fig. 1C. Taken together, these results constitute dramatic affirmation that R-100 is protective in resuscitating RIRI.

Table 5: The effect of R-100 in a rat RIRI model

Example 3. R-100 resuscitation of a rat RIRI model - second study

[00109] The aim of this study was to evaluate the effect of compound la (R-100) at the dose of (80 mg/kg) in the well characterized model of kidney ischemia and reperfusion in rats described in Example 2 above.

Animal preparation

[00110] Male Sprague-Dawley rats (300-350 g; Charles River) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).

[00111] The animals were placed onto a thermostatically-controlled heating mat (Harvard Apparatus Ltd., 2Biol, Milan Italy, and body temperature was maintained at 38+1 °C by means of a rectal probe attached to a homeothermic blanket. A tracheotomy was performed to maintain airway patency and to facilitate spontaneous respiration. A midline laparotomy was performed, and the bladder was cannulated (PP90, 1.D. 0.76 mm; Portex). Both kidneys were located, and the renal pedicles, containing the artery, vein, and nerve supplying each kidney, were carefully isolated.

Renal ischemia/reperfusion

[00112] Rats (Groups 2 and 3) were allowed to stabilize for 30 minutes before they were subjected to bilateral renal occlusion for 30 minutes using artery clips to clamp the renal pedicles. Reperfusion commenced once the artery clips were removed (control animals). Occlusion was verified visually by change in the color of the kidneys to a paler shade and reperfusion by a blush. The other rats (Group 1), which underwent identical surgical procedures similar to control animals but did not undergo bilateral renal clamping, were subjected to sham operation (sham operated) and were maintained under anesthesia for the duration of the experiment. At the end of all experiments, animals were killed by an overdose of sodium thiopentone.

Experimental protocol

[00113] Upon completion of surgical procedures, the animals were randomly allocated (n=6 rats per experimental group) as followed described: Group 1 : Sham (no ischemia), Group 2: (ischemia-reperfusion, I/R, and HPCD vehicle control), and Group 3: (ischemia-reperfusion, I/R, and R-100 therapy). Urine was collected from the rats during the following periods: 1. -2 hours prior to ischemia until -30 minutes prior to ischemia; 2. from the onset of reperfusion until 1.5 hours after the onset of reperfusion; 3. from 1.5 hours after the onset of reperfusion until 3.0 hours after the onset of reperfusion; 4. from 3 hours after the onset of reperfusion until 4.5 hours after the onset of reperfusion; and 5. from 4.5 hours after the onset of reperfusion until 6.0 hours after the onset of reperfusion. Arterial blood samples were obtained at 0, 1.5 hours, 3.0 hours, 4.5 hours, and 6.0 hours after the onset of reperfusion. R- 100 (80 mg/kg; formulated with HPCD; Group 3) or HPCD vehicle control (Group 2) was administered as a 10 minute IV infusion beginning 20 minutes after the onset of ischemia. Measurement of biochemical parameters

[00114] At the end of the reperfusion period, blood (1 ml) samples were collected via the carotid artery into Sl/3 tubes containing serum gel. The samples were centrifuged (6000 rpm for 3 minutes) to separate plasma. All plasma samples were analyzed for biochemical parameters within 24 hours after collection. Urine samples were collected during the reperfusion period, and the volume of urine produced was recorded. Urine concentrations of Na⁺ were measured and were used in conjunction with plasma Na⁺ concentrations to calculate FE_Na using standard formulae, which was used as an indicator of tubular function. Plasma and urine concentrations of creatinine were measured as indicators of impaired glomerular function. Creatinine clearance was calculated using the following formula (=UV/P), where U refere to Creatinine concentration in urine, V to urine volume/min and P to serum creatinine. Plasma concentration of NGAL levels were evaluated as indicated by the commercial kit. Urine concentration of NGAL was evaluated as indicated by the commercial kit.

Determination of myeloperoxidase (MPO) activity

[00115] MPO activity in kidneys was used as an indicator of PMN cell infiltration using a method previously described. Briefly, at the end of the experiments, kidney tissue was weighed and homogenized in a solution containing 0.5% (wt/vol) hexadecyltrimethylammonium bromide dissolved in 10 mmol/1 potassium phosphate buffer (pH 7.4) and centrifuged for 30 minutes at 20,000 g at 4°C. An aliquot of supernatant was then removed and added to a reaction mixture containing 1.6 mmol/1 tetramethylbenzidine and 0.1 mmol/1 hydrogen peroxide (H₂O₂). The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme required to degrade 1 mmol of H₂O₂ at 37°C and was expressed in U/g wet tissue.

Determination of malondialdehye (MDA) levels

[00116] Levels of MDA in kidneys were determined as an indicator of lipid peroxidation following a protocol described previously. Briefly, kidney tissue was weighed and homogenized in a 1. 1 Cwt/vnl KCl solution. A 100 ml aliquot of homogenate was then removed and added to a reaction mixture containing 200 ml 8.1% (wt/vol) lauryl sulfate, 1.5 ml 20% (vol/vol) acetic acid (pH 3.5), 1.5 ml 0.8% (wt/vol) thiobarbituric acid, and 700 ml distilled water. Samples were then boiled for one hour at 95°C and centrifuged at 3000 x g for 10 minutes. The absorbance of the supernatant was measured spectrophotometrically at 650 nm. MDA levels were expressed as μΜ/100 mg wet tissue.

Histologic evaluation

[00117] At post-mortem, a 5 μπι section of kidney was removed and placed in formalin and processed through to wax. Five millimeter sections were cut and stained with hematoxylin and eosin. Histologic assessment of tubular necrosis was determined semiquantitatively using a method modified from McWhinnie et al. (1987). Random cortical fields were observed using a x20 objective. A graticule grid (25 squares) was used to determine the number of line intersects involving tubular profiles. One hundred intersections were examined for each kidney, and a score from 0 to 3 was given for each tubular profile involving an intersection, as described in Example 2. The total score for each kidney was calculated by addition of all 100 scores.

Materials

[00118] Unless otherwise stated, all compounds were obtained from Sigma- Aldrich Company Ltd. (Milan, Italy). All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Italy) or 10% DMSO.

Statistical evaluation

[00119] All values in the figures and text are expressed as mean ± standard error of the mean (SEM) of N observations. For the in vivo studies N represents the number of animals studied. In the experiments involving histology, the figures shown are representative of at least three experiments performed on different experimental days. A p- value of less than 0.05 was considered significant. BBB scale data were analyzed by the Mann-Whitney test and considered significant when p- value was less than 0.05. Results

[00120] The effect of R-100 on ischemia/reperfusion-mediated tubular dysfunction and injury is shown in Figs. 2-5. In particular, Fig. 2 shows the plasma concentrations of urea in the different groups, Fig. 3 shows the plasma concentration of creatinine in the different groups, Fig. 4 shows the creatinine clearance in the R-100 treated group vs. the vehicle group, and Fig. 5 shows the FE_Na in the R-100 treated group vs. the vehicle group, calculated using plasma Na⁺ concentrations, urine production (urine flow, ml/min) and urinary concentrations of Na⁺. Figs. 6 and 7 show the effect of R-100 on plasma and urine NGAL levels, respectively, and Fig. 8 shows the effect of R-100 on kidney MPO activity and MDA levels. The effects of R-100 on ischemia/reperfusion-mediated renal histopathology is shown in Figs. 9-10, demonstrating the representative hematoxylin/eo sin- stained section of the kidney tissues (Fig. 9) and the histological evaluation (Fig. 10), 6 hours after reperfusion.

[00121] Figs. 2 and 3 show that R-100 therapy entirely inhibited the RIRI-induced elevation in serum urea and partially inhibited the RIRI-induced elevation in serum creatinine, both markers of renal function. Taken together, these two figures provide strong evidence that R-100 therapy profoundly blocks RIRI-induced renal dysfunction. This conclusion is further supported by Fig. 4, which depicts the effect of R-100 therapy on creatinine clearance, a classic proxy of glomerular filtration rate. As noted in Fig. 4, the RIRI-induced reduction in creatinine clearance was partially inhibited by R-100 therapy, indicating that R-100 administration is highly protective against RIRI in this model. As a consequence of RIRI, the ability of the kidney to reabsorb filtered Na⁺ is impaired, as shown in Fig. 5B by the RIRI- induced increase in the FE_Na. Administration of R-100 reduced the elevation in FE_Na almost back to the level in the sham animals (Group 1). RIRI induces significant damage to the renal tubules, as reflected by serum and urinary levels of NGAL that are depicted in Figs. 6 and 7, respectively. Administration of R-100 partially inhibited elevations of both serum and urinary NGAL, indicating that R-100 blocks RIRI-induced renal tubular damage. RIRI is associated with an increase in PMN infiltration into renal tissue, as shown in Fig. 8A, an effect that was partially reversed by administration of R-100. RIRI also induces redox stress within the renal parenchyma, as reflected by an increase in MDA formation in the renal tissue. As shown in Fig. 8B, administration of R-100 partially blocked this elevation in MDA. RIRI also produces renal damage that may be appreciated at the level of histologic examination, as shown in Fig. 9. Administration of R-100 profoundly reduced histologic injury, as reflected by the reduction in histologic score.

[00122] Taken together, the data illustrated in Figs. 2-9 are consistent with a massive reduction in renal injury, at the levels of morphology, function, and biochemical damage. In each instance, treatment with R-100 partially or entirely inhibited these manifestations of renal injury.

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Claims

1. A method for prevention or treatment of renal ischemia-reperfusion injury in an individual in need thereof, comprising administering to said individual a prophylactic ally or therapeutically effective amount of a compound of the general formula I:

wherein

Ri each independently is selected from H, -OH, -COR₃, -COOR₃, -OCOOR₃, -OCON(R₃)₂, -(C C₁₆)alkylene-COOR₃, -CN, -NO₂, -SH, -SR₃, -(C C₁₆)alkyl, -O- (C Ci₆)alkyl, -N(R₃)₂, -CON(R₃)₂, -SO₂R₃, -S(=O)R₃, or a nitric oxide donor group of the formula -X X₂-X₃, wherein Xi is absent or selected from -O-, -S- or -NH-; X₂ is absent or is (CrC₂₀)alkylene optionally substituted by one or more -ONO₂ groups and optionally further substituted by a moiety of the general formula D:

R₂ each independently is selected from (CrC₁₆)alkyl, (C₂-C₁₆)alkenyl, or (C₂-C₁₆)alkynyl; R₃ each independently is selected from H, (C₁-C₈)alkyl, (C₃-C₁₀)cycloalkyl, 4-12-membered heterocyclyl, or (C₆-C₁₄)aryl, each of which other than H may optionally be substituted with -OH, -COR₄, -COOR₄, -OCOOR₄, -OCON(R₄)₂, - (C C₈)alkylene-COOR₄, -CN, -NO₂, -SH, -SR₄, -(C C₈)alkyl, -O-(C C₈)alkyl, - N(R₄)₂, -CON(R₄)₂, -SO₂R4, or -S(=O)R₄;

R₄ each independently is selected from H, (C C₈)alkyl, (C₃-C₁₀)cycloalkyl, 4-12-membered heterocyclyl, or (C₆-C₁ )aryl; and

n and m each independently is an integer of 1 to 3.

2. The method of claim 1, wherein Ri each independently is selected from H, - COOR₃, -CON(R₃)₂, or a nitric oxide donor group; and R₃ is H.

3. The method of claim 1, wherein R₂ each independently is (C C₈)alkyl, preferably (CrC )alkyl, more preferably (CrC₂)alkyl, most preferably methyl.

4. The method of claim 3, wherein R₂ are identical.

5. The method of claim 1, wherein in said nitric oxide donor group, X is absent or -O-; X₂ is absent or (C₁-C₂₀)alkylene, preferably (C₁-C₆)alkylene, more preferably (CrC₃)alkylene, most preferably methylene; X₃ is -NO or -ONO₂, preferably -ONO₂; and said alkylene is optionally substituted by one or more - ONO₂ groups and optionally further substituted by a moiety of the general formula D.

6. The method of any one of claims 1 to 5, wherein (i) n is 1 ; and one or two of the carbon atoms at positions 3 or 4 of the pyrrolidine ring are linked to a nitric oxide donor group; (ii) n is 2; and one or more of the carbon atoms at positions 3 to 5 of the piperidine ring are linked to a nitric oxide donor group; or (iii) n is 3; and one or more of the carbon atoms at positions 3 to 6 of the azepane ring are linked to a nitric oxide donor group.

7. The method of claim 6, wherein said compound comprises more than one identical or different nitric oxide donor groups.

8. The method of claim 6, wherein each one of said nitric oxide donor groups independently is of the formula -(Ci-C₆)alkylene-ONO₂, preferably -(C C₃)alkylene-ONO₂, more preferably -CH₂-ONO₂, or -O-(C C₆)alkylene-ONO₂, wherein said alkylene is optionally substituted by one or more -ONO₂ groups; or is - ONO₂.

9. The method of claim 8, wherein n is 1 ; R₂ each is methyl; and

(i) Ri linked to the carbon atom at position 3 of the pyrrolidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and Ri linked to the carbon atom at position 4 of the pyrrolidine ring is H (herein identified compounds la and lb, respectively); or

(ii) each one of Ri linked to the carbon atoms at positions 3 and 4 of the pyrrolidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂ (herein identified compounds 2a and 2b, respectively).

10. The method of claim 8, wherein n is 2; R₂ each is methyl; and

(i) Ri linked to the carbon atom at position 3 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and each one of Ri linked to the carbon atoms at positions 4 and 5 of the piperidine ring is H (herein identified compounds 3a and 3b, respectively);

(ii) Ri linked to the carbon atom at position 4 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and each one of Ri linked to the carbon atoms at positions 3 and 5 of the piperidine ring is H (herein identified compounds 4a and 4b, respectively);

(iii) each one of Ri linked to the carbon atoms at positions 3 and 4 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and Ri linked to the carbon atom at position 5 of the piperidine ring is H (herein identified compounds 5a and 5b, respectively);

(iv) each one of Ri linked to the carbon atoms at positions 3 and 5 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and Ri linked to the carbon atom at position 4 of the piperidine ring is H (herein identified compounds 6a and 6b, respectively);

(v) each one of Ri linked to the carbon atoms at positions 3 to 5 of the piperidine ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂ (herein identified compounds 7a and 7b, respectively).

11. The method of claim 8, wherein n is 3; R₂ each is methyl; and

(i) Ri linked to the carbon atom at position 3 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and each one of Ri linked to the carbon atoms at positions 4 to 6 of the azepane ring is H (herein identified compounds 8a and 8b, respectively);

(ii) Ri linked to the carbon atom at position 4 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and each one of Ri linked to the carbon atoms at position 3, 5 and 6 of the azepane ring is H (herein identified compounds 9a and 9b, respectively);

(iii) each one of Ri linked to the carbon atoms at positions 3 and 4 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and each one of Ri linked to the carbon atoms at positions 5 and 6 of the azepane ring is H (herein identified compounds 10a and 10b, respectively);

(iv) each one of Ri linked to the carbon atoms at positions 3 and 5 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and each one of Ri linked to the carbon atoms at positions 4 and 6 of the azepane ring is H (herein identified compounds 11a and lib, respectively);

(v) each one of Ri linked to the carbon atoms at positions 3 and 6 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and each one of Ri linked to the carbon atoms at positions 4 and 5 of the azepane ring is H (herein identified compounds 12a and 12b, respectively); (vi) each one of Ri linked to the carbon atoms at positions 3 to 5 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and Ri linked to the carbon atom at position 6 of the azepane ring is H (herein identified compounds 13a and 13b, respectively);

(vii) each of Ri linked to the carbon atoms at positions 3, 4 and 6 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂; and Ri linked to the carbon atom at position 5 of the azepane ring is H (herein identified compounds 14a and 14b, respectively); or

(viii) each of Ri linked to the carbon atoms at positions 3 to 6 of the azepane ring is the nitric oxide donor group -CH₂-ONO₂ or -ONO₂ (herein identified compounds 15a and 15b, respectively).

12. The method of claim 8, wherein n is 1; R₂ each is methyl; Ri linked to the carbon atom at position 3 of the pyrrolidine ring is the nitric oxide donor group - CH₂-ONO₂ or -ONO₂; and Ri linked to the carbon atom at position 4 of the pyrrolidine ring is -CONH₂ (herein identified compounds 16a and 16b, respectively).

13. The method of claim 8, wherein n is 2; R₂ each is methyl; Ri linked to the carbon atom at position 3 of the piperidine ring is the nitric oxide donor group - CH₂-ONO₂ or -ONO₂; Ri linked to the carbon atom at position 4 of the piperidine ring is -COOH; and Ri linked to the carbon atoms at position 5 of the piperidine ring is H (herein identified compounds 17a and 17b, respectively).

14. The method of claim 8, wherein n is 2; R₂ each is methyl; Ri linked to the carbon atom at position 4 of the piperidine ring is the nitric oxide donor group -O- CH₂-CH(ONO₂)CH₂-ONO₂; and each one of Ri linked to the carbon atoms at positions 3 and 5 of the piperidine ring is H (herein identified compound 18).

15. The method of claim 6, wherein each one of said nitric oxide donor groups independently is of the formula -O-(Ci-C₆)alkylene-ONO₂, wherein said alkylene is substituted by a moiety of the general formula D and optionally further substituted by one or more -ONO₂ groups.

16. The method of claim 15, wherein n is 2; each one of R linked to the carbon atoms at positions 3 and 5 of the piperidine ring is H; and (i) linked to the carbon atom at position 4 of the piperidine ring is the nitric oxide donor group -O-CH₂- CH₂-CH(CH₃)-ONO₂, wherein the 1,3 butane diyl is substituted at position 2 with the -ONO₂ group and at position 4 with a moiety of the general formula D, wherein m is 2, and the oxygen atom is linked to the carbon atom at position 4 of the piperidine ring in the formula D; and R₂ each is methyl (herein identified compound 19); or (ii) linked to the carbon atom at position 4 of the piperidine ring is the nitric oxide donor group -O-CH₂-CH(CH₃)-ONO₂, wherein the 1,2 propane diyl is substituted at position 3 with a moiety of the general formula D, wherein m is 2, and the oxygen atom is linked to the carbon atom at position 4 of the piperidine ring in the formula D; and R₂ each is methyl (herein identified compound 20).

17. The method of claim 9, wherein compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, is administered.

18. The method of any one of claims 1 to 17, wherein said compound, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, is formulated as an inclusion complex with an hydroxyalkyl-cyclodextrin.

19. The method of claim 18, wherein said hydroxyalkyl-cyclodextrin is hydroxyalkyl-α-, β- or γ-cyclodextrin, preferably hydroxyalkyl- -cyclodextrin.

20. The method of claim 19, wherein said hydroxyalkyl- -cyclodextrin is selected from hydroxyethyl- -cyclodextrin, hydroxypropyl- -cyclodextrin, dihydroxypropyl- -cyclodextrin, or hydroxybutyl- -cyclodextrin, preferably hydroxypropyl- -cyclodextrin, more preferably 2-hydroxypropyl- -cyclodextrin.

21. The method of claim 20, wherein an inclusion complex of compound la and 2-hydroxypropyl- -cyclodextrin is administered.

22. The method of claim 21, wherein the ratio of compound la to 2- hydroxypropyl- -cyclodextrin is between 1: 15 (w/w) to 1:20 (w/w).

23. The method of any one of claims 1 to 22, wherein said compound is administered prior to the onset of renal ischemia; following the onset of renal ischemia but prior to reperfusion; or after reperfusion.

24. A pharmaceutical composition for prevention or treatment of renal ischemia- reperfusion injury comprising a compound of the general formula I, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, wherein said compound is selected from compounds la, lb, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11a, lib, 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 18b, 18, 19 or 20, preferably compound la, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof.

26. The pharmaceutical composition of claim 24, wherein said compound, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, is formulated as an inclusion complex with an hydroxyalkyl-cyclodextrin.

27. The pharmaceutical composition of claim 26, wherein said hydroxyalkyl- cyclodextrin is hydroxy alkyl- α- , β- or γ-cyclodextrin, preferably hydroxyalkyl-β- cyclodextrin.

28. The pharmaceutical composition of claim 27, wherein said hydroxyalkyl-β- cyclodextrin is selected from hydroxyethyl- -cyclodextrin, hydroxypropyl-β- cyclodextrin, dihydroxypropyl- -cyclodextrin, or hydroxybutyl- -cyclodextrin, preferably hydroxypropyl-P-cyclodextrin, more preferably 2-hydroxypropyl-P- cyclodextrin.

29. The pharmaceutical composition of claim 28, comprising an inclusion complex of compound la and 2-hydroxypropyl- -cyclodextrin.

30. The pharmaceutical composition of claim 29, wherein the ratio of compound la to 2-hydroxypropyl- -cyclodextrin is between 1: 15 (w/w) to 1:20 (w/w).

31. The pharmaceutical composition of any one of claims 24 to 30, for intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, or subcutaneous administration.

32. A compound of the general formula I, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, for use in prevention or treatment of renal ischemia-reperfusion injury.

33. Use of a compound of the general formula I, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, for the preparation of a pharmaceutical composition for prevention or treatment of renal ischemia- reperfusion injury.

34. An inclusion complex of a compound of the general formula I, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt or solvate thereof, and an hydroxyalkyl-cyclodextrin.

35. A pharmaceutical composition comprising an inclusion complex according to claim 34, and a pharmaceutically acceptable carrier.

36. A process for the preparation of a solution of an inclusion complex according to claim 34 in distilled water or a physiological solution, comprising the steps of:

37. The process of claim 36, wherein compound la and 2-hydroxypropyl- - cyclodextrin are dissolved in step (i), and the ratio of compound la to 2- hydroxypropyl- -cyclodextrin is between 1: 15 (w/w) to 1:20 (w/w).