WO2017153977A1

WO2017153977A1 - Solid formulations of resolvins and uses thereof

Info

Publication number: WO2017153977A1
Application number: PCT/IL2017/050245
Authority: WO
Inventors: Philip KUEHL
Original assignee: Salzman Lovelace Investments, Ltd.
Priority date: 2016-03-08
Filing date: 2017-02-27
Publication date: 2017-09-14

Abstract

The present invention relates to a solid formulation of a resolvin molecule selected from a mono- or poly-hydroxylated eicosapentaenoic acid, docosahexaenoic acid, or n-3 docosapentanoic acid, or a pharmaceutically acceptable salt, ester or amide thereof; and uses thereof. Particular such resolvin molecules are tri-hydroxylated EPA such as Rv E1, or pharmaceutically acceptable salts thereof.

Description

SOLID FORMULATIONS OF RESOLVINS AND USES THEREOF

TECHNICAL FIELD

[0001] The present invention relates to a solid formulation of a resolvin molecule selected from a mono- or poly-hydroxylated eicosapentaenoic acid, docosahexaenoic acid, or n-3 docosapentanoic acid, or a pharmaceutically acceptable salt, ester or amide thereof; and uses thereof.

BACKGROUND ART

[0002] Resolvins are endogenous picomolar-potent small molecules derived from cellular metabolism of dietary omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA; C20:5) and docosahexaenoic acid (DHA; C22:6), that activate a complex intracellular mechanism by which tissue inflammation is modulated and ultimately resolved. These compounds are, in fact, multi-focal-acting mediators acting via limiting polymorphonuclear (PMN) cell/neutrophil transendothelial migration in vitro and infiltration in vivo (Pluess et al., 2007; Rittirsch et al., 2009), as well as enhancing pro- inflammatory chemokine scavenging (Buras et al., 2005), non-phlogistic recruitment on monocytes and phagocytosis, and phagocyte clearance via the lymphatics (Spite et al., 2009).

[0003] A particular such compound, resolvin El (RvEl), is an enzymatically-oxygenated lipid mediator that functions as a specialized pro-resolution mediator and actively "turns off" the inflammatory response (Farolan et al., 1996). RvEl acts via its binding to the two discrete G-protein coupled receptors (GPCRs) ChemR23 and BLT-1 (Levy, et al. 2012; Seki et al 2010), which transduce the resolution of inflammation via the removal ("efferocytosis") of neutrophils (PMNs) and the reduction of a broad array of proinflammatory cytokines and chemokines. The relevance of these receptors is demonstrated in genetic deletion models in mice. For instance, ChemR23-/- mice challenged with pneumonia virus of mice display higher mortality/morbidity, alteration of lung function, delayed viral clearance, and increased neutrophil infiltration (Bondue, et al. 2011).

[0004] RvEl has been shown to be effective in various rodent models of inflammatory disease, e.g., asthma, colitis, pneumonia, acute lung injury (ALI), peritonitis, periodontitis, and renal fibrosis (Haworth et al., 2008; Gilroy et al., 2004; Flierl et al., 2008; Serhan et al., 2008; Kurihara et al., 2013; Schwartz et al., 1994; Schwab et al., 2007; Busse et al., 2001; Bettelli et al, 2007; Langrish et al, 2005), at doses as low as 4 μg/kg. As shown in models of inflammation such as allergic asthma, RvEl reduces IL-6, IL-17 and IL-23, and increases IFN-y and LXA4 in lungs to dampen allergic airway inflammation (Gilroy et al., 2004); and decreases eosinophil and lymphocyte recruitment in a murine model of asthma (Flierl et al., 2008; Serhan et al., 2008). These effects on inflammation were associated with a reduction in airway hyperresponsiveness (Buras et al., 2005; Spite et al., 2009; Seki et al., 2010). Administration of RvEl stimulates murine dermal healing, reducing neutrophilic infiltration and stimulating re-epithelialization (Allard et al., 2011; Fredman et al, 2011; Gao et al, 2013; Hasturk et al, 2007; Herrera et al, 2008). A further study has shown that RvEl is also protective in periodontal disease, a chronic inflammatory disease in which infection leads to PMN-mediated tissue injury around the tooth, as demonstrated by its effect on stimulation of bone regeneration in lapine models of periodontitis (Amin et al., 2013).

[0005] With respect to more neutrophilic disease models, in both E. coli and Staphylococcus aureus infections, administration of RvD2 limited neutrophil (PMN) infiltration, enhanced phagocyte clearance of bacteria, and accelerated resolution. These actions were lost in GPR18-deiIcient mice (Chiang et al., 2015).

[0006] In mammals, alpha-linolenic acid is converted via elongation and desaturation to EPA and subsequently to DHA. An intermediate in the conversion of EPA to DHA is n-3 docosapentaenoic acid (n-3 DP A), which carries 22 carbons and contains five double bonds, with the first double bond being found on carbon 7 (Dalli et al, 2013).

[0007] RvEl has been supplied as a liquid that is kept refrigerated or frozen due to chemical instability under ambient conditions. The results of accelerated stability studies demonstrated that the concentration of refrigerated RvEl was still within specified values after three months of storage; however, when stored at 25°C and 60% relative humidity, the concentration of RvEl decreased by approximately 13% during that period. The change in the concentration of RvEl was reflected in a concomitant change in RvEl- related impurities. These results demonstrate that there is a need for a formulation that will enable cold chain-independent storage of resolvin. SUMMARY OF INVENTION

[0008] It has now been found, in accordance with the present invention, that spray-dried formulations of RvEl with various excipients, in particular, sodium citrate, dextran, leucine, trehalose and/or lactose, prepared from an aqueous:organic solutions, are both physically and chemically stable, as well as respirable.

[0009] Various such formulations were evaluated for chemical and physical integrity of RvEl and for chemical and physical stability. The particles were determined to be crystalline, wrinkled spheres when lactose was not used; and amorphous, smooth spheres when lactose was present, and particle size were all between 1-5 μιη mass median aerodynamic diameter. Initial analysis of all formulations confirmed that RvEl was chemically identical following spray drying with a high purity and glass transition temperatures above 100°C. Further analyses following storing for about eight months at ambient temperature have indicated changes in neither the chemical stability of the formulations nor their aerosol performance.

[0010] In one aspect, the present invention thus provides a solid formulation comprising a resolvin molecule and an excipient, wherein said resolvin molecule is selected from a mono- or poly-hydroxylated eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or n-3 docosapentanoic acid (n-3 DPA), or a pharmaceutically acceptable salt, ester or amide thereof; and said excipient is selected from a carbohydrate, an amino acid, a salt, an alditol, or a combination thereof. In particular embodiments, said resolvin molecule is a tri- hydroxylated EPA such as 5S,12R,18R-trihydroxy EPA (RvEl) or a pharmaceutically acceptable salt, ester or amide thereof. In certain embodiments, the solid formulation disclosed is in the form of a powder comprising discrete particles, e.g., wherein said particles are inhalable, and said excipient is suitable for respiratory and pulmonary administration.

[0011] In another aspect, the present invention relates to a method for inhibiting, reducing or ameliorating inflammation or infection, thereby treating a disease, disorder, or condition associated with an inflammation of infection in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a solid formulation as defined above, e.g., a formulation comprising a tri-hydroxylated EPA such as RvEl or a pharmaceutically acceptable salt, ester or amide thereof. Particular such diseases, disorders or conditions include, e.g., an inflammatory bowel disease such as Crohn's disease, or colitis; an arthritis such as rheumatoid arthritis, or osteoarthritis; pancreatitis; meningitis; pneumonia; a local infection; or sepsis.

[0012] In a further aspect, the present invention provides a delivery system, comprising a dry powder inhaler and a solid formulation as defined above, wherein said formulation is formulated for inhalation, i.e., in the form of a powder comprising discrete inhalable particles, and said excipient is suitable for respiratory and pulmonary administration.

BRIEF DESCRIPTION OF DRAWINGS

[0013] Figs. 1A-1C show scanning electron micrograms of spray-dried RvEl powder particle of each one of the formulations BREC-1380-026A (1A), BREC-1380-026B (IB), and BREC-1380-026C (1C), prepared in Example 1.

[0014] Fig. 2 shows the powder X-ray diffraction (PXRD) patterns of the RvEl formulations prepared in Example 1 (BREC-1380-026A, BREC-1380-026B, and BREC- 1380-026C) in comparison with that of L-leucine.

[0015] Fig. 3 shows mDSC chromatograms (reversing heat flow and non-reversing heat flow as functions of the temperature) of the three RvEl formulations prepared in Example 1 (BREC-1380-026A, BREC-1380-026B, and BREC-1380-026C).

[0016] Fig. 4 shows aerosol performance of the three RvEl formulations prepared in Example 1 (BREC-1380-026A, BREC-1380-026B, and BREC-1380-026C), as determined by next generation impactor (NGI). Stages 1-8 represent the depth of particle penetration expected in the pulmonary and respiratory tract.

DETAILED DESCRIPTION

[0017] In one aspect, the present invention provides a formulation comprising a resolvin molecule (herein also referred to as "the active/therapeutic agent/ingredient") and an excipient, wherein said resolvin molecule is selected from a mono- or poly-hydroxylated EPA, DHA, or n-3 DPA, or a pharmaceutically acceptable salt, ester or amide thereof; said excipient is selected from a carbohydrate, an amino acid, a salt, an alditol, or a combination thereof; and said formulation is solid.

[0018] In certain embodiments, the resolvin molecule comprised within the solid formulation of the invention is a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri- hydroxylated, EPA, or a pharmaceutically acceptable salt, ester or amide thereof.

[0019] In other embodiments, the resolvin molecule comprised within the solid formulation of the invention is a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri- hydroxylated, DHA, or a pharmaceutically acceptable salt, ester or amide thereof. [0020] In further embodiments, the resolvin molecule comprised within the solid formulation of the invention is a mono-hydroxylated or poly-hydroxylated, e.g., di- or tri- hydroxylated, n-3 DPA, or a pharmaceutically acceptable salt, ester or amide thereof.

[0021] The resolvin molecule has one or more asymmetric centers at each one of the hydroxyl groups thereof, 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, including the endpoints of the stated range of optical purity) and as diastereoisomers. Accordingly, each one of the hydroxyl groups of the resolvin molecule or pharmaceutically acceptable salt, ester, or amide thereof independently has either an R or S configuration, or is a racemic mixture.

[0022] Optically active forms of the resolvin molecules may be obtained 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.

[0023] In certain embodiments, the resolvin molecule comprised within the solid formulation of the invention is selected from a mono- or poly-hydroxylated EPA, DHA, or n-3 DPA, or a pharmaceutically acceptable salt or ester thereof. In particular such embodiments, said resolvin molecule is a compound having a carboxyl group of the formula -COOR, wherein R is H, (Ci-C₈)alkyl, (C₃-Ci₀)cycloalkyl, -CH₂-CHOH-CH₂OH, or -CH-(CH₂OH)₂; and one or more hydroxyl groups each independently of the formula - OP, wherein P is H or a hydroxyl protecting group, or a pharmaceutically acceptable salt thereof.

[0024] The term "alkyl" as used herein typically means a linear or branched saturated hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec -butyl, isobutyl, ie/ -butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n- heptyl, n-octyl, and the like. In certain embodiments, the term "alkyl" refers to (Ci-C6)alkyl groups, e.g., (Ci-C₄)alkyl groups such as methyl, ethyl and isopropyl.

[0025] The term "cycloalkyl" as used herein means a cyclic or bicyclic hydrocarbyl group having 3-10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like. Particular such cycloalkyls are (C5-Cio)cycloalkyls, e.g., (C5-C7)cycloalkyls.

[0026] The term "hydroxyl protecting group" as used herein refers to any hydroxyl protecting group known in the art. An artisan skilled in the art can readily determine which protecting group(s) may be useful for the protection of the hydroxyl group(s), and standard methods are known in the art and are described in the literature. For example, suitable protecting groups are described in Green and Wuts, "Protective Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991. Preferred protecting groups include methyl and ethyl ethers, silyl ethers such as trimethylsilyl ether (TMS) and triisopropylsilyl ether (TIPPS) groups, acetate or proprionate groups, and glycol ethers such as ethylene glycol and propylene glycol derivatives.

[0027] The mono- or poly-hydroxylated EPA, DHA or n-3 DPA, or pharmaceutically acceptable salt or ester thereof, comprised within the solid formulation of the invention, may be protected in one or more of the hydroxyl groups thereof, wherein this can be accomplished by the stoichiometric choice of reagents used to protect the hydroxyl groups. In order to separate the various protected compounds, e.g., the mono, di- or tri-protected hydroxylated EPA or DHA, methods known in the art such as high-performance liquid chromatography (HPLC), liquid chromatography (LC), flash chromatography, gel permeation chromatography, crystallization, and distillation, can be utilized.

[0028] In more particular such embodiments, the resolvin molecule is a mono-, di- or tri- hydroxylated EPA of the formula (l)-(5), a mono- or tri-hydroxylated DHA of the formula (6)-(13), or a mono- or di-hydroxylated n-3 DPA of the formula (14)-(24) in Table 1, wherein R is H, (Ci-C₈)alkyl, (C₃-Ci₀)cycloalkyl, -CH₂-CHOH-CH₂OH, or -CH- (CH₂OH)₂; and P each independently is H or a hydroxyl protecting group, or a pharmaceutically acceptable salt thereof. Table 1: Resolvin molecules 1-24 described herein

[0029] According to the present invention, (i) in the mono-hydroxylated EPA of the formula (1), the hydroxyl linked to the carbon atom at position 15 has either R or S configuration, or is an R/S racemic mixture; (ii) in the mono-hydroxylated EPA of the formula (2), the hydroxyl linked to the carbon atom at position 18 has either R or S configuration, or is an R/S racemic mixture; (iii) in the tri-hydroxylated EPA of the formula (3), the hydroxyl linked to the carbon atom at position 5 has S configuration, the hydroxyl linked to the carbon atom at position 12 has R configuration, and the hydroxyl linked to the carbon atom at position 18 has R configuration; or each one of the hydroxyls linked to the carbon atoms at positions 5, 12 and 18 independently has R/S configuration; (iv) in the di-hydroxylated EPA of the formula (4), the hydroxyl linked to the carbon atom at position 5 has S configuration, and the hydroxyl linked to the carbon atom at position 18 has R configuration; or each one of the hydroxyls linked to the carbon atoms at positions 5 and 18 independently has R/S configuration; (v) in the tri-hydroxylated EPA of the formula (5), the hydroxyl linked to the carbon atom at position 5 has S configuration, the hydroxyl linked to the carbon atom at position 6 has R configuration, and the hydroxyl linked to the carbon atom at position 15 has R configuration; or each one of the hydroxyls linked to the carbon atoms at positions 5, 6 and 15 independently has R/S configuration; (vi) in the mono-hydroxylated DHA of the formula (6), the hydroxyl linked to the carbon atom at position 13 has either R or S configuration, or is an R/S racemic mixture; (vii) in the mono-hydroxylated DHA of the formula (7), the hydroxyl linked to the carbon atom at position 14 has either R or S configuration, or is an R/S racemic mixture; (viii) in the mono-hydroxylated DHA of the formula (8), the hydroxyl linked to the carbon atom at position 16 has either R or S configuration, or is an R/S racemic mixture; (ix) in the mono- hydroxylated DHA of the formula (9), the hydroxyl linked to the carbon atom at position 17 has either R or S configuration, or is an R/S racemic mixture; (x) in the mono- hydroxylated DHA of the formula (10), the hydroxyl linked to the carbon atom at position

19 has either R or S configuration, or is an R/S racemic mixture; (xi) in the mono- hydroxylated DHA of the formula (11), the hydroxyl linked to the carbon atom at position

20 has either R or S configuration, or is an R/S racemic mixture; (xii) in the tri- hydroxylated DHA of the formula (12), the hydroxyl linked to the carbon atom at position

7 has S configuration, the hydroxyl linked to the carbon atom at position 8 has R configuration, and the hydroxyl linked to the carbon atom at position 17 has S configuration; or each one of the hydroxyls linked to the carbon atoms at positions 7, 8 and 17 independently has R/S configuration; (xiii) in the tri-hydroxylated DHA of the formula (13), the hydroxyl linked to the carbon atom at position 7 has S configuration, the hydroxyl linked to the carbon atom at position 16 has R configuration, and the hydroxyl linked to the carbon atom at position 17 has S configuration; or each one of the hydroxyls linked to the carbon atoms at positions 7, 16 and 17 independently has R/S configuration; (xiv) in the mono-hydroxylated n-3 DPA of the formula (14), the hydroxyl linked to the carbon atom at position 17 has either R or S configuration, or is an R/S racemic mixture; (xv) in the mono-hydroxylated n-3 DPA of the formula (15), the hydroxyl linked to the carbon atom at position 14 has either R or S configuration, or is an R/S racemic mixture; (xvi) in the mono-hydroxylated n-3 DPA of the formula (16), the hydroxyl linked to the carbon atom at position 7 has either R or S configuration, or is an R/S racemic mixture; (xvii) in the di- hydroxylated n-3 DPA of the formula (17), each one of the hydroxyls linked to the carbon atoms at positions 7 and 8 independently has either R or S configuration, or is an R/S racemic mixture; (xviii) in the tri-hydroxylated n-3 DPA of the formula (18), each one of the hydroxyls linked to the carbon atoms at positions 7, 16 and 17 independently has either R or S configuration, or is an R/S racemic mixture; (xix) in the di-hydroxylated n-3 DPA of the formula (19), each one of the hydroxyls linked to the carbon atoms at positions 7 and 17 independently has either R or S configuration, or is an R/S racemic mixture; (xx) in the di-hydroxylated n-3 DPA of the formula (20), each one of the hydroxyls linked to the carbon atoms at positions 10 and 17 independently has either R or S configuration, or is an R/S racemic mixture; (xxi) in the di-hydroxylated n-3 DPA of the formula (21), each one of the hydroxyls linked to the carbon atoms at positions 16 and 17 independently has either R or S configuration, or is an R/S racemic mixture; (xxii) in the di-hydroxylated n-3 DPA of the formula (22), each one of the hydroxyls linked to the carbon atoms at positions 7 and 14 independently has either R or S configuration, or is an R/S racemic mixture; (xxiii) in the mono-hydroxylated n-3 DPA of the formula (23), the hydroxyl linked to the carbon atom at position 14 has either R or S configuration, or is an R/S racemic mixture; and (xxiv) in the di-hydroxylated n-3 DPA of the formula (24), each one of the hydroxyls linked to the carbon atoms at positions 14 and 21 independently has either R or S configuration, or is an R/S racemic mixture.

[0030] More specific such resolvin molecules for use in the solid formulation of the invention are tri-hydroxylated EPAs of the formula (3) in Table 1, wherein R is H, or (Q- C₈)alkyl such as methyl, ethyl, or isopropyl; and P each independently is H or a hydroxyl protecting group, or a pharmaceutically acceptable salt thereof, e.g., a tri-hydroxylated EPA of the formula (3), wherein R is H; and P is H, such as 5S,12R,18R-trihydroxy EPA (RvEl) or a pharmaceutically acceptable salt thereof.

[0031] In certain embodiments, the resolvin molecule comprised within the solid formulation of the invention is a pharmaceutically acceptable salt of a mono- or poly- hydroxylated EPA, DHA, or n-3 DPA as defined above. 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 /?-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 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; as well as salts of ammonium (NH₄ ⁺) or an organic cation derived from an amine of the formula R₄N⁺, wherein each one of the Rs independently is selected from H, Q-C22, e- -, Ci-C₆ alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, ie/t-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. [0032] 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.

[0033] 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: C5₂Hi₀₆N₆O₄4CF₃CO₂H, C₈₈Hi₇₈N₈0₄S₂4CF₃C0₂H, C₄₀H₈₄NO₃P CF₃CO₂H, C₅oH₁₀ N₇0₃-4CF₃C0₂H, C₅5H₁₁₆N₈0₂ 6CF₃C0₂H,

C₄₉H₁₀₂N₆O₃4CF₃CO₂H, C₄₄H₈₉N₅0₃-2CF₃C0₂H, C₁₀oH₂o₆N₁₂0₄S₂-8CF₃C0₂H, C₁₆₂H₃₃₀N₂₂O₉ 13CF₃CO₂H, C₄₃H₈₈N₄0₂-2CF₃C0₂H, C₄₃H₈₈N₄0₃-2CF₃C0₂H, C₄iH₇₈N0₈P); 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). [0034] Pharmaceutically acceptable salts of the resolvin molecule for use in the solid formulations of the present invention may be formed by conventional means, e.g., by reacting the free base form of the resolvin molecule 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 on a suitable ion exchange resin.

[0035] The solid formulation of the invention comprises one or more, e.g., a combination of two or three, excipients each independently selected from a carbohydrate, an amino acid, a salt, or an alditol.

[0036] The term "carbohydrate" refers to a molecule containing carbon, hydrogen and oxygen atoms, which can be cyclic or linear, saturated or unsaturated, and substituted or unsubstituted. As used herein, this term particularly refers to a monosaccharide such as fructose, galactose, glucose, D-mannose, and sorbose; a disaccharide such as sucrose, maltose, lactose, trehalose, and cellobiose; an oligosaccharide; a polysaccharide such as mannotriose, raffinose, melezitose, a maltodextrin, i.e., a chain consisting of a variable number (e.g., 3-17) of D-glucose units linked with a(l→4)glycosidic bonds, or a dextran, i.e., a complex branched glucan (polysaccharide of D-glucose monomers linked by glycosidic bonds); or a cyclodextrin.

[0037] 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.

[0038] 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

A ° 2. 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 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.

[0039] In certain embodiments, the cyclodextrin used as an excipient in the solid formulation of the invention is an hydroxyalkyl-cyclodextrin, more particularly hydroxyalkyl-α-, hydroxyalkyl-β- or hydroxyalkyl-y-cyclodextrin, but preferably hydroxyalkyl-P-cyclodextrin. The term "hydroxyalkyl" as used herein refers to any hydroxyl derivative of a Ci-C₄ alkyl, i.e., a straight or branched saturated hydrocarbon radical having 1-4 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, isobutyl and tert-butyl. In more particular embodiments, said hydroxyalkyl- β- cyclodextrin is hydroxyethyl-P-cyclodextrin, hydroxypropyl-P-cyclodextrin, dihydroxypropyl-P-cyclodextrin, or hydroxybutyl-P-cyclodextrin, preferably hydroxypropyl-P-cyclodextrin, more preferably 2-hydroxypropyl-P-cyclodextrin (HPpCD).

[0040] In certain embodiments, the cyclodextrin used as an excipient in the solid formulation of the present invention is a sulphoalkylether-cyclodextrin, more particularly sulphoalkylether-α-, sulphoalkylether-β- or sulphoalkylether-y-cyclodextrin, preferably sulphoalkylether-P-cyclodextrin. The term "sulphoalkylether" as used herein refers to a group of the general formula -0-(Ci-C₄)alkylene-S0₃H, wherein (Ci-C₄)alkylene typically means a divalent straight or branched hydrocarbon radical having 1-4 carbon atoms such as methylene, ethylene, propylene, butylene and 2-methylpropylene. In more particular embodiments, said sulphoalkylether-P-cyclodextrin is sulphoethylether-P-cyclodextrin, sulphopropylether-P-cyclodextrin, sulphobutylether-P-cyclodextrin, or sulphopentylether- β-cyclodextrin, preferably sulphobutylether-P-cyclodextrin (Captisol^®).

[0041] The hydroxyalkyl or sulphoalkylether (-0-(Ci-C₄)alkylene-S0₃H) 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 or sulphoalkylether groups per cyclodextrin, and it is the preferred manner of describing the substitution. The molecular weight of the hydroxyalkyl- or sulphoalkylether-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 or sulphoalkylether 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.

[0042] The degree of substitution may have an effect on the binding of guests to the hydroxyalkyl- or sulphoalkylether-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.

[0043] In certain embodiments, the cyclodextrin used as an excipient in the solid formulation of the invention is an hydroxyalkyl- or sulphoalkylether-cyclodextrin as defined above, wherein said hydroxyalkyl- or sulphoalkylether-cyclodextrin may have any degree of substitution, i.e., may be either fully or partially modified with hydroxyalkyl or sulphoalkylether groups, wherein each a-D-glucopyranoside units has three hydroxyl groups which can be substituted. In particular embodiments, the hydroxyalkyl-cyclodextrin is an hydroxyalkyl-P-cyclodextrin, preferably HPpCD, having a degree of substitution in a range of 3 to 8, preferably 3.5 to 7; or the sulphoalkylether-cyclodextrin is an sulphoalkylether-P-cyclodextrin, preferably Captisol^®, having a degree of substitution in a range of 3 to 8, preferably 3.5 to 7.

[0044] The term "amino acid" refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either a natural or non-natural amino acid. The twenty two natural amino acids are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Val), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gin), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (He), cysteine (Cys), selenocysteine (Sec), and pyrrolysine (Pyl). Non-limiting examples of non-natural amino acids include diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Orn), aminoadipic acid, β-alanine, 1-naphthylalanine, 3-(l-naphthyl)alanine, 3- (2-naphthyl)alanine, γ-aminobutiric acid (GABA), 3-(aminomethyl) benzoic acid, p- ethynyl-phenylalanine, /?-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine, p- bromophenylalanine, /?-iodophenylalanine, /?-azidophenylalanine, /?-acetylphenylalanine, azidonorleucine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine, p- chlorophenylalanine, a-aminocaprylic acid, O-methyl-L-tyrosine, N-acetylgalactosamine- a-threonine, and N-acetylgalactosamine-a-serine. In certain embodiments, the amino acid used as an excipient in the solid formulation of the present invention is glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, or leucine.

[0045] The salt used as an excipient in the solid formulation of the present invention is preferably a salt of an organic acid and a base such as, without being limited to, sodium citrate, sodium ascorbate, magnesium gluconate, or sodium gluconate.

[0046] The term "alditol" denotes a polyhydroxy alcohol derived by reduction of an aldose, i.e., a monosaccharide containing only one aldehyde (-CH=0) group per molecule, e.g., a diose, triose, tetrose, pentose, hexose, or heptose, containing two, three, four, five, six, or seven carbon atoms, respectively. In certain embodiments, the alditol used as an excipient in the solid formulation of the present invention is mannitol (hexose alcohol) or xylitol (pentose alcohol).

[0047] The weight ratio between the resolvin molecule and the excipient(s) composing the solid formulation of the invention may be determined based on various parameters, e.g., the purpose of the solid formulation and the resolvin molecule, i.e., active agent, dose, and may thus theoretically be in the range of <1:>99 to >99:<1, respectively. Yet, in most embodiments, the weight ratio between said resolvin molecule and said excipient(s) is about 1:99 to about 30:70, more particularly about 1:99 to about 20:80, respectively. In particular embodiments, the weight ratio between said resolvin molecule and said excipient(s) is about 2:98 to about 10:90, e.g., about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, or about 10:90, respectively.

[0048] In certain embodiments, the present invention relates to a solid formulation comprising a resolvin molecule and an excipient, wherein (i) said resolvin molecule is a mono-, di- or tri-hydroxylated EPA of the formula (l)-(5) in Table 1, preferably a tri- hydroxylated EPA of the formula (3), wherein R is H or (Ci-Cs)alkyl; and P each independently is H or a hydroxyl protecting group, or a pharmaceutically acceptable salt thereof; and (ii) said excipient is a monosaccharide such as fructose, galactose, glucose, D- mannose, or sorbose; a disaccharide such as sucrose, maltose, lactose, trehalose, or cellobiose; an oligosaccharide, a polysaccharide such as mannotriose, raffinose, melezitose, a maltodextrin, or a dextran; a cyclodextrin such as 2-hydroxypropyl-P- cyclodextrin; an amino acid such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, or leucine; a salt of an organic acid and a base such as sodium citrate, sodium ascorbate, magnesium gluconate, or sodium gluconate; an alditol such as mannitol, or xylitol; or a combination thereof. In particular such embodiments, said excipient is lactose, trehalose, raffinose, a maltodextrin, leucine, sodium citrate, or a combination thereof, e.g., lactose, a combination of trehalose and L-leucine, or a combination of sodium citrate and L-leucine. Preferred such embodiments are those wherein said resolvin molecule is RvEl or a pharmaceutically acceptable salt thereof, e.g., wherein the weight ratio between said RvEl or pharmaceutically acceptable salt thereof and said excipient is in the range of 1:99 to 20:80, 2:98 to 10:90, or 4:96 to 6:94. Specific such formulations are those wherein (i) said excipient is lactose, and the weight ratio between said RvEl or pharmaceutically acceptable salt thereof and lactose is 5:95, respectively; (ii) said excipient is a combination of trehalose and L-leucine, and the weight ratio between said RvEl or pharmaceutically acceptable salt thereof, trehalose and L-leucine is 5:70:25, respectively; or (iii) said excipient is a combination of sodium citrate and L-leucine, and the weight ratio between said RvEl or pharmaceutically acceptable salt thereof, sodium citrate and L- leucine is 5:70:25, respectively.

[0049] In certain embodiments, the solid formulation of the present invention, in any one of the embodiments defined above, is in the form of a powder comprising discrete, i.e., separate, particles. Such particles might be either crystalline or amorphous, and may have a smooth or wrinkled surface. In particular such embodiments, the solid formulation of the invention is in the form of a powder comprising discrete inhalable particles, wherein said excipient(s) is suitable for respiratory and pulmonary administration. More particular such formulations are those wherein said particles have (i) a water content of about 1% to about 8% by weight, preferably about 1.5% to about 5.5% by weight; or (ii) a mass median diameter (MMD) of 1-10 μιη, preferably 1-5 μιη, more preferably 2, 3, 4 or 5 μιη; or (iii) a mass median aerodynamic diameter (MMAD) under 10 μιη, preferably between about 3 to about 5 μηι; or (iv) a geometric standard deviation (GSD) of about 1 μιη to about 2.5 μιη; or (v) a glass transition temperature (T_g) of about 105°C to about 180°C, or said formulation has a fine particle fraction of about 45% to about 70%.

[0050] The term "inhalable particles" as used herein refers to airborne particles that can be inhaled, i.e., aspirated into the nose or mouth during normal breathing, by a subject, i.e., a mammal such as a human. The human respiratory tract can be divided into three main regions based on size, structure, and function, i.e., the head, tracheobronchial region (also known as the conducting airways), and the gas-exchange region (also known as the parenchymal, alveolar, or pulmonary) region. Thus, while an inhalable fraction is the mass fraction of total airborne particles inhaled through the nose and mouth, an extrathoracic fraction is the mass fraction of inhaled particles failing to penetrate beyond the larynx; a thoracic fraction is the mass fraction of inhaled particles penetrating beyond the larynx; and a respirable fraction is the mass fraction of inhaled particles penetrating to the unciliated airways. Relative to total airborne particles, the particle size (aerodynamic diameter) having 50% penetrations for the thoracic and respirable fractions are 10 μιη and 4.0 μιη, respectively.

[0051] In other embodiments, the solid formulation of the present invention, in any one of the embodiments defined above, is optionally mixed with one or more further excipients suitable for oral administration, and either filled into capsules or sachets, or compressed to tablets (e.g., chewable tablets which disintegrate with saliva in the buccal/mouth cavity) or caplets by any conventional method known in the art. Such tablets or caplets may comprise the active agent in at least two separate layers (bilayer or multilayer tablets or caplets) optionally separated by an intermediate, inactive layer, e.g., a layer comprising one or more disintegrants. Alternatively, the solid formulation may be used for the preparation of films, wafers, or buccal/mucoadhesive patches.

[0052] In further embodiments, the solid formulation of the present invention, in any one of the embodiments defined above, is suspended in a liquid organic dispersion medium to form a suspension.

[0053] Solid formulations as disclosed herein, when in the form of a powder comprising discrete inhalable particles, may further comprise one or more excipients that are suitable for respiratory and pulmonary administration. Similarly, enlisted excipients may be added, suitable for oral, sublingual, otic, buccal, rectal, topical, ocular, or parenteral administration. The additional excipients added to the formulation of the invention may serve simply as bulking agents so as to reduce the resolvin molecule concentration in the powder delivered to a patient, but may also serve to enhance the stability of the formulation and to improve the dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the powder and to improve handling characteristics such as flowability and consistency to facilitate manufacturing and powder filling.

[0054] The solid formulations disclosed herein may be prepared utilizing any suitable technology known in the art, e.g., spray drying or freeze drying (lyophilization). In certain embodiments, such formulations are prepared by spray drying of a solution of said resolvin molecule and said excipient(s) in a spray solvent. Such a spray solvent may comprise, e.g., water and a lower alkanol, i.e., a straight or branched (Ci-C₆)alkanol such as methanol, ethanol, propanol, isopropanol or butanol. In a particular such embodiment exemplified herein, the solid formulation is prepared by spray drying of a solution of said resolvin molecule and said excipient(s) in a spray solvent comprising water and methanol, preferably at a weight ratio of 30:70. In case further ingredients are necessary, such ingredients can be added to the solution of said resolvin molecule and said excipient(s) either prior to or after spray drying or lyophilization.

[0055] Actual dosage levels of the active ingredient in the solid formulation of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject and formulation, without being toxic to the subject. The selected dosage level will depend upon a variety of factors including the activity of the active agent employed and the rate of excretion thereof; the time of administration; the duration of the treatment; other drugs used in combination with said treatment; age, sex and weight of the subject treated; and general health and prior medical history of said subject.

[0056] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the solid formulation, thus therapeutic agent, required. For example, the physician or veterinarian could start doses of the therapeutic agent at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0057] In certain embodiments, the formulation of the present invention is for oral, sublingual, topical, ocular, intramuscular or subcutaneous administration. Such formulations may be in the form of, e.g., tablets (e.g., chewable tablets which disintegrate with saliva in the buccal/mouth cavity), caplets, capsules, sachets, films, wafers, buccal/mucoadhesive patches, or suspensions of said formulation in a liquid organic dispersion medium.

[0058] Formulations for oral or sublingual administration may be prepared by any method known to the art for the manufacture of such formulations and may further comprise one or more ingredients selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Such formulations, e.g., when in the form of tables, capsules, or sachets, may further comprise 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.

[0059] Formulations for oral administration may be formulated for controlled release of the active agent. Such formulations may be in the form of a controlled-release matrix, e.g., a controlled-release matrix tablet 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.

[0060] In certain embodiments, the formulation of the present invention is for intratracheal or inhalation administration, i.e., in the form of a powder comprising discrete inhalable particles, wherein said excipient is suitable for respiratory and pulmonary administration. Such formulations may be administered utilizing any suitable device known in the art, such as metered dose inhalers, dry powder inhalers, sprayers, electrohydrodynamic aerosolizers, and the like. [0061] The solid formulations disclosed herein, in any one of the embodiments defined above, are useful in inhibiting, reducing or ameliorating inflammation or infection, thereby treating a disease, disorder, or condition associated with inflammation or infection. Examples of such diseases, disorders, or conditions include, without being limited to, an inflammatory bowel disease such as Crohn's disease, or colitis; an arthritis such as rheumatoid arthritis, or osteoarthritis; pancreatitis; meningitis; pneumonia; a local infection; or sepsis.

[0062] In particular embodiments, the disease, disorder, or condition treated with the formulations of the invention is pneumonia, i.e., an inflammatory condition of the lung. Such pneumonia may be, e.g., pseudomonal pneumonia, i.e., a pneumonia caused by pseudomonal infection, more particularly primary or secondary pseudomonal pneumonia; or viral pneumonia, i.e., a pneumonia caused by a viral infection, e.g., an infection caused by influenza virus A (IAV) or B, respiratory syncytial virus (RSV), rhinovirus, coronavirus, a human parainfluenza virus, an adenovirus, a metapneumovirus, severe acute respiratory syndrome virus (SARS-coronovirus), Epstein-Barr virus, cytomegalovirus, measles, hantaviruses, bocavirus, or middle east respiratory syndrome virus (MERS).

[0063] In particular embodiments, the disease, disorder, or condition treated with the formulation of the invention is sepsis, i.e., a serious, life-threatening medical condition characterized by an overwhelming systemic infection resulting in vasolidation leading to hypotension, i.e., to septic shock, which may consequently lead to an inadequate tissue perfusion that is insufficient to meet cellular metabolic needs and is also known as hypoperfusional state. Sepsis may be caused by Gram negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Serratia species, Salmonella species, Shigella species, Enterobacter species, Citrobacter species, Proteus species, and Klebsiella species; Gram-positive cocci such as Pneumococcal species, Staphylococcal species, Enterococcal species, and Streptococcal species; fungi and yeast; Rickettsial species Plasmodial species, Clostridial species; viruses, or Gram-positive bacterial toxins, including toxic shock syndrome toxins. In certain embodiments, the formulation of the invention is useful in inhibiting development of sepsis-related coagulopathy.

[0064] In another aspect, the present invention relates to a method for inhibiting, reducing or ameliorating inflammation or infection, thereby treating a disease, disorder, or condition associated with an inflammation of infection in an subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a solid formulation as defined in any one of the embodiments above, e.g., wherein the resolvin molecule is a tri-hydroxylated EPA of the formula (3) in Table 1, wherein R is H or (Ci-Cs)alkyl; and P each independently is H or a hydroxyl protecting group, preferably RvEl, or a pharmaceutically acceptable salt thereof. Examples of diseases, disorders, or conditions associated with an inflammation of infection include, without limiting, an inflammatory bowel disease such as Crohn's disease, or colitis; an arthritis such as rheumatoid arthritis, or osteoarthritis; pancreatitis; meningitis; pneumonia; a local infection; or sepsis.

[0065] The term "subject" as used herein refers to any mammal, e.g., a human, non- human primate, horse, ferret dog, cat, cow, and goat. In a preferred embodiment, the term "subject" denotes a human, i.e., an individual.

[0066] The term "therapeutically effective amount" as used herein with respect to the solid formulation administered according to the method of the invention (or in fact, with respect to the resolvin molecule comprised within said formulation) refers to an amount of said formulation that, upon administration under a particular regimen and during a particular period of time, e.g., days or weeks, is sufficient to inhibit, reduce or ameliorate an inflammatory or infectious process occurring in the body of the subject administered with, thereby treating a disease, disorder, or condition associated with an inflammation of infection. The actual dosage of the formulation administered may be varied so as to obtain an amount of said resolvin molecule that is effective to achieve the desired therapeutic response for a particular subject and mode of administration, without being toxic to the subject. The dosage selected will depend upon a variety of factors including the severity/progression of the disease, disorder or condition treated; the specific resolvin molecule employed, the administration route, and the duration of the treatment; and the age, sex and weight of the subject treated.

[0067] In certain embodiments, the method of the invention is for treating pneumonia, i.e., an inflammatory condition of the lung, and comprises administering by inhalation to said subject a therapeutically effective amount of a solid formulation as defined above, wherein said formulation is in the form of a powder comprising discrete inhalable particles, and said excipient is suitable for respiratory and pulmonary administration.

[0068] In particular embodiments, the method of the invention is for treating pneumonia due to pseudomonal infection, i.e., pseudomonal pneumonia, and may further comprise administering to said subject a therapeutically effective amount of at least one antibiotic agent such as ceftazidime, ciprofloxacin, imipenem, gentamicin, tobramycin, mezlocillin or piperacillin. According to the method of the invention, the resolvin molecule-containing formulation and said at least one antibiotic agent can be administered either concomitantly or sequentially in any order; and said at least one antibiotic agent may be administered to said subject by any administration route.

[0069] In particular embodiments, the method of the invention is for treating pneumonia due to primary pseudomonal pneumonia. The subject treated according to this method is, e.g., a subject having pre-existing lung disease such as bronchiectasis, chronic bronchitis, chronic obstructive pulmonary disease, or cystic fibrosis, or a subject who has experienced an aspiration pneumonia induced by sedation or endotracheal intubation. In a specific embodiment, the subject treated according to the method of the invention has cystic fibrosis, and said method further comprises colistin administration, e.g., by inhalation.

[0070] In particular embodiments, the method of the invention is for treating pneumonia due to secondary pseudomonal pneumonia. The subject treated according to this method is, e.g., a subject currently receiving or having completed a course of antibiotic treatment, or a subject having neutropaenia, e.g., due to cytotoxic chemotherapy.

[0071] In certain embodiments, the method of the invention is for treating pneumonia due to a viral infection, i.e., viral pneumonia, caused, e.g., by influenza virus A (IAV) or B, respiratory syncytial virus (RSV), rhinovirus, coronavirus, a human parainfluenza virus, an adenovirus, a metapneumovirus, severe acute respiratory syndrome virus (SARS- coronovirus), Epstein-Barr virus, cytomegalovirus, measles, hantaviruses, bocavirus, or middle east respiratory syndrome virus (MERS). In particular such embodiments, said method further comprises administering to said subject a therapeutically effective amount of at least one antiviral agent such as oseltamivir, paramavir, zanamivir, rimantadine, aspirin, palivizumab or ribavirin. According to the method of the invention, the resolvin molecule-containing formulation and said at least one antiviral agent can be administered either concomitantly or sequentially in any order; and said at least one antiviral agent may be administered to said subject by any administration route.

[0072] The method of the present invention, when used for treatment of pneumonia, including when further comprising administration of either at least one antibiotic agent (for treatment of lung inflammation due to pseudomonal infection) or at least one antiviral agents (for treatment of lung inflammation due to a viral infection), may further comprise administering to said individual a therapeutically effective amount of at least one anti- inflammatory agent such as a non-steroidal anti-inflammatory drug (NSAID) and/or at least one antioxidant agent. The administration of said NSAID(s) and/or antioxidant agent(s) can be done either concomitantly or sequentially in any order with said resolvin molecule-containing formulation and optionally said at least one antibiotic or antiviral agent, and by any administration route(s).

[0073] The term "non-steroidal anti-inflammatory drug" (NSAID) as used herein refers to any non-steroidal anti-inflammatory drug/agent/analgesic/medicine, and relates to both cyclooxygenase (COX)-2 selective and non-selective inhibitors. Non-limiting examples of NSAIDs include celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, acetaminophen (paracetamol, considered to be an NSAID for the purposes of the present invention), firocoxib, meloxicam, etodolac, aspirin, naproxen, ibuprofen, indomethacin, piroxicam, nabumetone, flurbiprofen, ketoprofen, ketorolac, lornoxicam, droxicam, tenoxicam, diclofenac, meclofenamate, mefenamic acid, diflunisal, sulindac, tolmetin, fenoprofen, suprofen, benoxaprofen, aceclofenac, tolfenamic acid, oxyphenbutazone, azapropazone, and phenylbutazone, oxaprozin, CS-502, JTE-522, L-745,337, and NS398.

[0074] Examples of antioxidant agents that can be used according to the present invention include, without being limited to, ascorbic acid or a salt thereof such as sodium ascorbate, potassium ascorbate, calcium ascorbate, ascorbyl stearate, and ascorbyl palmitate; L-cysteine (L-Cys) or a salt thereof such as cysteine hydrochloride; a cysteine derivative such as N-acetylcysteine (NAC), glutathione, diacetylcystine, S-methyl-N- acetylcysteine amide, acetyl derivatives of S-methyl-N-acetylcysteine methylhydrazide, S- methylcysteine morpholineamide, and S-methyl-N-acetylcysteine morpholineamide, or a salt thereof; a bisulfite such as sodium bisulfite, sodium hydrogen sulfite, or sodium metabisulfite; a tocopherol, and a superoxide dismutase (SOD) mimetic, i.e., a synthetic compound that mimic the native SOD enzyme, e.g., manganese(III)meso-tetrakis(N,N'- diethyl-l,3-imidazolium-2-yl)porphyrin, 3-nitratomethyl-proxyl, l-0-[4-(di-sodium- phosphonoxy)phenyl]acetyl-(2,2,5,5-tetramethyl pyrrolidin-3-yl)methyl nitrate, and 2,2,5, 5-tetramethyl-3-(nitrooxymethyl)pyrrolidin- 1-yl acetate.

[0075] In a further aspect, the present invention provides a delivery system comprising a dry powder inhaler and a solid formulation for inhalation as defined above, i.e., a solid formulation as defined in any one of the embodiments above wherein said formulation is in the form of a powder comprising discrete inhalable particles, and said excipient is suitable for respiratory and pulmonary administration. In particular embodiments, the resolvin molecule comprised within the solid formulation contained within said delivery system is a tri-hydroxylated EPA of the formula (3) in Table 1, wherein R is H or (Ci-Cs)alkyl; and P each independently is H or a hydroxyl protecting group, preferably RvEl, or a pharmaceutically acceptable salt thereof.

[0076] A dry powder inhaler (DPI) is a device that delivers medication to the lungs in the form of a dry powder, and is commonly used to treat respiratory diseases such as asthma, bronchitis, emphysema and chronic obstructive pulmonary disease (COPD). Dry powder inhalers can be classified into single dose-, multiple unit dose-, and multi-dose devices.

[0077] Unless otherwise indicated, all numbers expressing quantities of ingredients and so forth used in the present description and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this description and attached claims are approximations that may vary by up to plus or minus 10% depending upon the desired properties sought to be obtained by the present invention.

[0078] The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Example 1. Preparation of spray-dried RvEl formulations

[0079] Solution formulations of RvEl were spray dried with different excipients, in particular sodium citrate, dextran, leucine, trehalose and/or lactose, from an aqueous: organic solution. The target loading for 0.5 g formulations are presented in Table 1. RvEl was added to the spray solvent (H₂0/MeOH, 30/70 wt%/wt%), and dissolution was confirmed. Excipients were then directly added to the spray solution, in the proportions indicated in Table 2, for a final total solid content of 2%, and dissolution was again confirmed.

Table 2. Spray-dried formulations

[0080] The operational parameters of the spray drying (liquid feed rate of 5 g/min), atomizer (2-fluid), atomizer pressure (10 psi), inlet temperature (110°C), and outlet temperature (55°C) were maintained to enable a high yield of respirable dry powder.

Example 2. Analysis of spray-dried RvEl formulations

[0081] The spray-dried RvEl formulations were analyzed using the following methods: (i) water content by Karl Fischer (KF); (ii) morphology by scanning electron microscopy (SEM); (iii) physical state by powder X-ray diffraction (XRPD); (iv) glass transition temperature (Tg) by modulated differential scanning calorimetry (mDSC); (v) purity (chemical and chiral) by HPLC; and (vi) aerosol performance by next generation impact (NGI) at a single fill weight (N=3 for each formulation).

[0082] The results of the various RvEl formulation analyses are presented in Table 3. Electron microscopy (Fig. 1) results indicate that the process yields particles in the 1-5 μιη range that are free of fusion, and that addition of L-leucine increased particle rugosity.

Table 3. Summary of spray-dried RvEl formulation analyses

* RvEl content; " RvEl and related compounds.

[0083] The crystallinity of the spray-dried RvEl formulations was determined using powder X-ray diffraction (PXRD). The PXRD patterns of the three different formulations are shown in Fig. 2. While the spray-dried formulations containing L-leucine have crystalline peaks, the formulation that contains only lactose as the carrier (BREC-1380- 026C) is amorphous.

[0084] The heat capacity of the spray-dried RvEl formulations was determined using modulated differential scanning calorimetry (mDSC). The mDSC chromatograms of the three different RvEl formulations are presented in Fig. 3. The glass transition range is particularly elevated in the formulation that contains sodium citrate. [0085] Aerodynamic particle size distribution was assessed using next generation impactor (NGI) that classifies aerosol particles into size fractions. 10 mg of powder was loaded into Capsugel Vcaps size 3 capsules and actuated through the NGI at 601/min, 4 seconds, 3 capsules per replicate. Individual deposition sites were weighed before and after actuation to determine deposition mass gravimetrically. Three replicates were performed per each formulation, and the results are shown with the standard deviations in Table 4 and in Fig. 4. All three formulations delivered a similar fine particle fraction (FPF), a measure of mass deposited. Addition of leucine improved dispersibility and consequently the mass of drug emitted from the device, as determined by emitted fraction.

Table 4. Aerosol performance of spray-dried RvEl formulations

MMAD - mass median aerodynamic diameter; GSD - geometric standard deviation

Example 3. Stability of spray-dried RvEl formulations

[0086] The three formulations were analyzed for their chemical stability out to ~3 months when stored at ambient temperature. Analysis included chemical purity and aerosol performance. As found, the aerosol performance was unchanged from the initial testing; and the chemical purity analysis indicated that the purity of the formulations was not adversely affected during storage.

REFERENCES

Allard M.; Barnes K.; Chen X.; Cheung Y.-Y.; Duffy B.; Heap C; Inthavongsay J.; Johnson M.; Krishnamoorthy R.; Liu X.; Manley C; Shan Z.; Steffke S.; Varughese D.; Wang Y.; Yang Q.; Schwartz C.E., Total synthesis of Resolvin El. Tetrahedron Letters 2011, 52, 2623-2626

Amin R.; Chen J.-X.; Cotterill I.C.; Emerich D.; Ganley D.; Kjmelnitsky T.L.; McLaws M.D.; Michels P.C.; Schwartz C.E.; Thomas D.; Yan J.; Yang Q., Improved Synthesis of the C16-C20 Segment of Resolvin El Using Enantioselective Ketone Reduction and Lipase-Catalyzed Resolution. Organic Process Research Developments 2013, 7, 915-920

Bettelli E.; Oukka M.; Kuchroo V.K., T(H)-17 cells in the circle of immunity and autoimmunity. Nature immunology 2007, 8, 345-50

Buras J. A.; Holzmann B.; Sitkovsky M., Animal models of sepsis: setting the stage. Nature reviews. Drug discovery 2005, 4, 854-65

Busse W.W.; Lemanske R.F. Jr., Asthma. N Engl J Med 2001, 344, 350-362

Chiang N.; Dalli J.; Colas R.A.; Serhan C.N., Identification of resolvin D2 receptor mediating resolution of infections and organ protection. Exp Med. 2015, 212(8), 1203- 1217

Dalli J.; Colas R.A.; Serhan C.N., Novel n-3 immunoresolvents: structures and actions, Sci. Rep. 2013, 3, 1940

Farolan L.R.; Goto M.; Myers T.F.; Anderson C.L.; Zeller W.P., Perinatal nutrition enriched with omega-3 polyunsaturated fatty acids attenuates endotoxic shock in newborn rats. Shock 1996, 6, 263-6

Flierl M.A.; Rittirsch D.; Gao H.; Hoesel L.M.; Nadeau B.A.; Day D.E.; Zetoune F.S.; Sarma J.V.; Huber-Lang M.S.; Ferrara J.L.; Ward P. A., Adverse functions of IL-17A in experimental sepsis. FASEB J 2008, 22, 2198-205

Fredman G.; Oh S.F.; Ayilavarapu S.; Hasturk H.; Serhan C.N.; Van Dyke T.E., Impaired phagocytosis in localized aggressive periodontitis: rescue by Resolvin El. PLoS One. 2011, 6(9), e24422

Gao L.; Faibish D.; Fredman G.; Herrera B.S.; Chiang N.; Serhan C.N.; Van Dyke

T.E.; Gyurko R., Resolvin El and chemokine-like receptor 1 mediate bone preservation. Immunol. 2013, 190(2), 689-694 Gilroy D.W.; Lawrence T.; Perretti M.; Rossi A.G., Inflammatory resolution: new opportunities for drug discovery. Nature reviews. Drug discovery 2004, 3, 401-16

Hasturk H.; Kantarci A.; Goguet-Surmenian E.; Blackwood A.; Andry C; Serhan C.N.; Van Dyke T.E., Resolvin El regulates inflammation at the cellular and tissue level and restores tissue homeostasis in vivo. J Immunol. 2007, 179(10), 7021-7029

Haworth O.; Cernadas M.; Yang R.; Serhan C.N.; Levy B.D., Resolvin El regulates interleukin 23, interferon-gamma and lipoxin A4 to promote the resolution of allergic airway inflammation. Nature immunology 2008, 9, 873-9

Herrera B.S.; Ohira T.; Gao L.; Omori K.; Yang R.; Zhu M.; Muscara M.N.; Serhan C.N.; Van Dyke T.E.; Gyurko R., An endogenous regulator of inflammation, resolvin El, modulates osteoclast differentiation and bone resorption. Br J Pharmacol. 2008, 155(8), 1214-1223

Kurihara T.; Jones C.N.; Yu Y.M.; Fischman A.J.; Watada S.; Tompkins R.G.; Fagan S.P.; Irimia D., Resolvin D2 restores neutrophil directionality and improves survival after burns . FASEB J 2013, 27, 2270-81

Langrish C.L.; Chen Y.; Blumenschein W.M.; Mattson J.; Basham B.; Sedgwick J.D.; McClanahan T.; Kastelein R.A.; Cua D.J., IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. Exp Med 2005, 201, 233-40

Pluess T.T.; Hayoz D.; Berger M.M.; Tappy L.; Revelly J.P.; Michaeli B.; Carpentier Y.A.; Chiolero R.L., Intravenous fish oil blunts the physiological response to endotoxin in healthy subjects. Intensive Care Med 2007, 33, 789-97

Rittirsch D.; Huber-Lang M.S.; Flierl M.A.; Ward P.A., Immunodesign of experimental sepsis by cecal ligation and puncture. Nature protocols 2009, 4, 31-6

Schwab J.M.; Chiang N.; Arita M.; Serhan C.N., Resolvin El and protectin Dl activate inflammation-resolution programmes. Nature 2007, 447, 869-74

Schwartz J.; Weiss S.T., The relationship of dietary fish intake to level of pulmonary function in the first National Health and Nutrition Survey (NHANES I). Eur Respir J 1994, 7, 1821-4

Seki H.; Fukunaga K.; Arita M.; Arai H.; Nakanishi H.; Taguchi R.; Miyasho T.; Takamiya R.; Asano K.; Ishizaka A.; Takeda J.; Levy B.D., The anti-inflammatory and proresolving mediator resolvin El protects mice from bacterial pneumonia and acute lung injury. Immunol 2010, 184, 836-43 Serhan C.N.; Chiang N.; Van Dyke T.E., Resolving inflammation: dual antiinflammatory and pro-resolution lipid mediators. Nature reviews. Immunology 2008, 8, 349-61

Spite M.; Norling L.V.; Summers L.; Yang R.; Cooper D.; Petasis N.A.; Flower R.J.; Perretti M.; Serhan C.N., Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis. Nature 2009, 461, 1287-91

Claims

1. A solid formulation comprising a resolvin molecule and an excipient, wherein said resolvin molecule is selected from a mono- or poly-hydroxylated eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or n-3 docosapentanoic acid (n-3 DPA), or a pharmaceutically acceptable salt, ester or amide thereof; and said excipient is selected from a carbohydrate, an amino acid, a salt, an alditol, or a combination thereof.

2. The formulation of claim 1, wherein said resolvin molecule is a mono- or poly- hydroxylated EPA, or a pharmaceutically acceptable salt, ester or amide thereof.

3. The formulation of claim 1, wherein said resolvin molecule is a mono- or poly- hydroxylated DHA, or a pharmaceutically acceptable salt, ester or amide thereof.

4. The formulation of claim 1, wherein said resolvin molecule is a mono- or poly- hydroxylated n-3 DPA, or a pharmaceutically acceptable salt, ester or amide thereof.

5. The formulation of claim 1, wherein each one of the hydroxyl groups of said resolvin molecule or pharmaceutically acceptable salt, ester or amide thereof independently has either R or S configuration, or is a racemic mixture.

6. The formulation of claim 1, wherein in said resolvin molecule or pharmaceutically acceptable salt or ester thereof: the carboxyl group is of the formula -COOR, wherein R is H, (Ci-C₈)alkyl, (C₃-Ci₀)cycloalkyl, -CH₂-CHOH-CH₂OH, or -CH-(CH₂OH)₂; and each one of the hydroxyl groups independently is of the formula -OP, wherein P is H or a hydroxyl protecting group.

7. The formulation of claim 6, wherein said resolvin molecule is a mono-, di- or tri- hydroxylated EPA of the formula (l)-(5), a mono- or tri-hydroxylated DHA of the formula (6)-(13), or a mono- or di-hydroxylated n-3 DPA of the formula (14)-(24), or a pharmaceutically acceptable salt thereof:

/=\ ^^COOR

1

1 z

OP OP

31

8. The formulation of claim 7, wherein said resolvin molecule is a tri-hydroxylated EPA of the formula (3), wherein R is H, or (Ci-C₈)alkyl such as methyl, ethyl, or isopropyl; and P is H or a hydroxyl protecting group, or a pharmaceutically acceptable salt thereof.

9. The formulation of claim 8, wherein R is H; and P is H, preferably wherein said resolvin molecule is 5S,12R,18R-trihydroxy EPA (RvEl) or a pharmaceutically acceptable salt thereof.

10. The formulation of claim 1, wherein said carbohydrate is a monosaccharide such as fructose, galactose, glucose, D-mannose, or sorbose, a disaccharide such as sucrose, maltose, lactose, trehalose, and cellobiose, an oligosaccharide, a polysaccharide such as mannotriose, raffinose, melezitose, a maltodextrin, or a dextran, or a cyclodextrin such as 2-hydroxypropyl-P-cyclodextrin; said amino acid is glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, or leucine; said salt is a salt of an organic acid and a base such as sodium citrate, sodium ascorbate, magnesium gluconate, or sodium gluconate; or said alditol is mannitol, or xylitol.

11. The formulation of claim 1, wherein the weight ratio between said resolvin molecule and said excipient is in the range of 1:99 to 20:80, 2:98 to 10:90, or 4:96 to 6:94, respectively.

12. The formulation of claim 1, wherein (i) said resolvin molecule is a tri-hydroxylated EPA of the formula (3), wherein R is H or (Ci-C₈)alkyl; and P is H or a hydroxyl protecting group, or a pharmaceutically acceptable salt thereof; and (ii) said excipient is a monosaccharide; a disaccharide; an oligosaccharide; a polysaccharide; a cyclodextrin; an amino acid selected from glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, or leucine; a salt of an organic acid and a base; an alditol selected from mannitol, or xylitol; or a combination thereof.

13. The formulation of claim 12, wherein said excipient is lactose, trehalose, raffinose, a maltodextrin, leucine, sodium citrate, or a combination thereof.

14. The formulation of claim 13, wherein said excipient is lactose, a combination of trehalose and L-leucine, or a combination of sodium citrate and L-leucine.

15. The formulation of any one of claims 12 to 14, wherein said resolvin molecule is RvEl or a pharmaceutically acceptable salt thereof.

16. The formulation of claim 15, wherein the weight ratio between said RvEl or pharmaceutically acceptable salt thereof and said excipient is in the range of 1:99 to 20:80,

2:98 to 10:90, or 4:96 to 6:94, respectively.

17. The formulation of claim 16, wherein (i) said excipient is lactose, and the weight ratio between said RvEl or pharmaceutically acceptable salt thereof and lactose is 5:95, respectively; (ii) said excipient is a combination of trehalose and L-leucine, and the weight ratio between said RvEl or pharmaceutically acceptable salt thereof, trehalose and L- leucine is 5:70:25, respectively; or (iii) said excipient is a combination of sodium citrate and L-leucine, and the weight ratio between said RvEl or pharmaceutically acceptable salt thereof, sodium citrate and L-leucine is 5:70:25, respectively.

18. The formulation of any one of claims 1 to 17, wherein said formulation is in the form of a powder comprising discrete particles.

19. The formulation of claim 18, wherein said particles are crystalline or amorphous particles, having a smooth or wrinkled surface.

20. The formulation of claim 18 or 19, wherein said particles are inhalable, and said excipient is suitable for respiratory and pulmonary administration.

21. The formulation of claim 20, wherein said particles have (i) a water content of about 1% to about 8% by weight, preferably about 1.5% to about 5.5% by weight; (ii) a mass median diameter (MMD) of 1-10 microns, preferably 1-5 microns; (iii) a mass median aerodynamic diameter (MMAD) under 10 microns, preferably between about 3 to about 5 microns; (iv) a geometric standard deviation (GSD) of about 1 micron to about 2.5 micron; or (v) a glass transition temperature (T_g) of about 105°C to about 180°C, or said formulation has a fine particle fraction of about 45% to about 70%.

22. The formulation of any one of claims 1 to 17, wherein said formulation is filled into capsules or sachets; or compressed to tablets such as chewable tablets which disintegrate with saliva in the buccal/mouth cavity, or caplets.

23. The formulation of any one of claims 1 to 17, wherein said formulation is suspended in a liquid organic dispersion medium to form a suspension.

24. The formulation of any one of claims 1 to 23, for inhibiting, reducing or ameliorating inflammation or infection, thereby treating a disease, disorder or condition associated with inflammation or infection.

25. The formulation of claim 24, wherein said disease, disorder or condition associated with inflammation or infection is an inflammatory bowel disease such as Crohn's disease, or colitis; an arthritis such as rheumatoid arthritis, or osteoarthritis; pancreatitis; meningitis; pneumonia; a local infection; or sepsis.

26. A method for inhibiting, reducing or ameliorating inflammation or infection, thereby treating a disease, disorder or condition associated with inflammation or infection in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a solid formulation according to any one of claims 1 to 23.

27. The method of claim 26, for treatment of pneumonia, comprising administering by inhalation to said subject a therapeutically effective amount of a solid formulation according to claim 20.

28. A delivery system comprising a dry powder inhaler and a solid formulation according to claim 20.