CARBON MONOXIDE RELEASING MOLECULES AND PHARMACEUTICAL COMPOSITIONS AND USES THEREOF

BACKGROUND OF THE INVENTION

Carbon monoxide (CO) holds promise as a therapeutic agent (see, e.g., Motterlini et al., Nat Rev Drug Discov 2010, 9:728-743). However, although proved safe, the therapeutic efficacy of administering carbon monoxide gas in the clinic remains challenging. CO-releasing molecules (CORMs) have been put forward as a potential alternative to inhaled CO gas.

Among the known CORMs the ones with the more extensive in vivo use are the lipid- soluble CORM-2, [Ru(CO)3Ch]2, and its water-soluble CORM-3 derivative, [RU(CO) ₃ C12(H ₂ NCH ₂ CO)2]. However, the half-lives of CORM-2 and CORM-3 are too short for therapeutic use. The half-life of CO released from CORM-3 is 2.14 ± 0.17 minutes (Fizan et al., Materials, 2019, 12, 1643). The half-life of CORM-2 is about 1 minute in phosphate buffered saline at 37 °C with pH~7.4 (Nguyen et al., Macromol. Rapid Comm. 2016, 37, 739-744). Such short half-lives reflect the labile nature of those CORMs which rapidly decompose in aqueous media and blood plasma (Johnson, T. R.et al., Dalton Trans. 2007, 1500-1508; Santos-Silva, T.et al., J. Am. Chem. Soc. 2011, 133, 1192-1195; Chaves-Ferreira, M.et al., Angew. Chemie Int. Ed. 2015, 54, 1172-1175) thereby losing scaffold dependent tissue specificity. It would be beneficial to provide CORM types with structural handles, enabling tissue targeting and CO release rate control while avoiding large and potentially toxic increases in carboxyhemoglobin (COHb) levels.

Thus, there remains a need for CORMs having molecular properties and CO release profiles suitable or desirable for therapeutic use.

SUMMARY OF THE INVENTION

The present application provides molybdenum CORM compounds, pharmaceutical compositions thereof, and methods of their use and treatment. In various embodiments, the CORM compounds and pharmaceutical compositions comprising the same allow for a sustained or slow release of CO under physiological conditions. Upon administration to a patient, the CORMs can avoid large and potentially toxic increases in COHb levels, and in some embodiments have the potential to deliver CO in a significantly local manner to target tissues and/or provide a sustained level of therapeutic CO over time.

In some embodiments, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (I) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, and a pharmaceutically acceptable carrier: wherein Ri is Ci-6 alkyl, halo, or Ci-6 alkoxy;

R2 is H or Ci-6 alkyl; each R3 is independently -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; k is 0, 1, or 2; q is 0, 1, 2, 3, or 4; and n is 0, 1, or 2.

In some embodiments, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, and a pharmaceutically acceptable carrier:

wherein Ri is Ci-6 alkyl, halo, or Ci-6 alkoxy;

R3 is -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; and k is 0, 1, or 2.

In some embodiments, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (III) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof and a pharmaceutically acceptable carrier: wherein Ri is Ci-6 alkyl, halo, or Ci-6 alkoxy;

R3 is -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; and k is 0, 1, or 2. In some embodiments, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (IV) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof and a pharmaceutically acceptable carrier: wherein Ri is Ci-6 alkyl, halo, or Ci-6 alkoxy;

R3 is -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; and k is 0, 1, or 2.

In some embodiments, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof and a pharmaceutically acceptable carrier:

wherein Ri is Ci-6 alkyl, halo, or Ci-6 alkoxy;

R3 is -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; and k is 0, 1, or 2.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VI) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof and a pharmaceutically acceptable carrier: each R4 is independently H, Ci-6 alkyl, halo, or Ci-6 alkoxy;

R5 is H or Ci-6 alkyl; each Re is independently OH, -CH2O(C=O)Rn, -O(C=O)Rn, halo, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl; m is 0, 1, 2, 3, 4, or 5; q is 0, 1, 2, 3, or 4; and at least four of Xi, X2, X3, X4, X5, and Xe are selected from CH and CH2, and where one or two of Xi, X2, X3, X4, X5, and Xe may be selected from null, O, N, or NH.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is Ci-6 alkyl; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is methyl; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is methyl; R3 is COORn; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is methyl; R3 is COORn; and Rn is H, methyl, or ethyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is methyl; and R3 is COOH. In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is halo; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is halo; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is halo; R3 is COORn; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is halo; R3 is COORn; and Rn is H, methyl, or ethyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is Br; and R3 is COOH.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl. In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; R3 is COORn; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; R3 is COORn; and Rn is H, methyl, or ethyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; and R ₃ is COOH.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof

wherein Ri is H, Ci-6 alkyl, halo, or Ci-6 alkoxy; each R3 is independently -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; k is 0, 1, or 2.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is H, Ci-6 alkyl, halo, or Ci-6 alkoxy; each R3 is independently COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is Ci-6 alkyl; each R3 is independently COORn; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is methyl; each R3 is independently COORn; and Rn is H or Ci-6 alkyl. In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is halo; each R3 is independently COORn; and Rn is H or Ci-6 alkyl.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is methyl; and each R3 is COOH.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is methyl; and each R3 is COOEt.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is Br; and each R ₃ is COOH.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is Br; and each R3 is COOEt.

In one embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VIII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof and a pharmaceutically acceptable carrier: each R4 is independently H, Ci-6 alkyl, halo, or Ci-6 alkoxy;

Rs is H or Ci-6 alkyl;

R14 and R15 are independently H, halo, Ci-6 alkoxy, or Ci-6 alkyl;

Ri6 is H or CI-6 alkyl; q is 0, 1, 2, 3, or 4.

The pharmaceutical compositions will comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated for local or systemic delivery. In some embodiments, the composition is formulated for parenteral delivery (e.g., intravenous delivery) or oral delivery (e.g., enteral delivery). In various embodiments, the compositions are formulated for intravenous delivery or other administration routes such as transdermal.

In one aspect, the present application provides a method for treating or preventing an inflammatory or immunological disease in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Exemplary inflammatory or immunological conditions may affect any organ or tissue, including but not limited to liver, lungs, gastrointestinal tractjoints and/or bones, kidneys, heart, muscle, and skin. Exemplary inflammatory diseases include non-alcoholic steatohepatitis (NASH) and arthritis. An exemplary immunological disease is cancer, and may include administration of a compound or composition described herein in connection with an immunotherapy, chemotherapy, or radiotherapy regimen.

Other aspects and embodiments will be apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

FIG. 1 illustrates the gradient method used in HPLC for stability and quantification of pyridine-2-carboxaldehyde CO-releasing molecules (PyCa-CORM), wherein water is solvent A and acetonitrile is solvent B.

FIG. 2 illustrates CO release of PyCa-CORMs with aromatic amines in Hepes 7.4 buffer at room temperature, in the dark at a concentration of 50pM.

FIG. 3 illustrates CO release profile of PyCa-CORMs with aromatic amines in sheep blood diluted in Alsever’s solution, at 37°C, at a concentration corresponding to an in vivo dose of 50 mg/kg.

FIG. 4 illustrates CO release profile of PyCa-CORMs with aryl amines as determined by incubation with deoxy-Myoglobin at room temperature and a 20pM concentration.

FIG. 5 illustrates %COHb and amount of CO in blood (pmol/mg) in systemic circulation in mice treated with aryl derived PyCa-CORMs over time.

FIG. 6 illustrates %COHb and amount of CO in blood (pmol/mg) in systemic circulation in mice treated with amino acid derived PyCa-CORMs (ALF 819 and ALF 843) over time, and profiles recorded for both i.v. and i.p administration for ALF843 at 30 mg/kg.

FIG. 7 illustrates HPLC chromatograms of selected PyCa-CORMs in 50% NCMe/ftO.

FIG. 8 illustrates the clearance curve of ALF843 in mice, showing rapid clearance from plasma in the first 5 minutes to a concentration of 106 pg/mL in the plasma (28% of theoretical Cmax).

FIG. 9 illustrate the clearance curve of ALF826 in mice, showing rapid clearance from plasma in the first 5 minutes to a concentration of 89 pg/mL in the plasma (24% of theoretical Cmax) with a terminal half-life of 26.5 minutes.

FIG. 10 illustrate a graphic representation of the Arthritic Score along the time of the execution of the K/BxN mouse model with 5 CORMs (ALF821, ALF826, ALF828, ALF843 and ALF844). Dexamethasone (Dexa) was used as positive control. Daily i.p. dose was 30 mg/kg for 36 days. Time points for blood analysis are also marked.

FIG. 11 illustrate histological analysis of joints from front and hind paws of the animal treated with ALF826 in the K/BxN mice model for 36 days. Top left is a normal, control histological analysis of a healthy mouse.

DETAILED DESCRIPTION OF THE INVENTION

The present application is based on the discovery that CORMs, including molybdenum carbonyl pyridine carboxaldehyde complex compounds, function as CO-releasing molecules (CORMs) under physiological conditions. Compounds and pharmaceutical compositions described herein can provide CO-release kinetics suitable for therapy, including parenteral therapy. Thus, in one aspect, the present application provides compounds and pharmaceutical compositions of compounds of the Formulas (I)-(VIII) (described herein). In another aspect, the present application provides a pharmaceutical composition comprising the compounds of the Formulas (I)-(VIII) and the use thereof.

Unless the context suggests otherwise, certain terms have the following definitions.

The articles “a” and “an” as used herein refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, “an element” means one element or more than one element.

The term “H” denotes a single hydrogen atom. This radical may be attached, for example, to an oxygen atom to form a hydroxyl radical.

Where the term “alkyl” is used, either alone or within other terms such as “haloalkyl” or “alkylamino”, it embraces linear or branched radicals having one to about twelve carbon atoms. More preferred alkyl radicals are “lower alkyl” radicals having one to about six carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, ec-butyl, tert-butyl, pentyl, isoamyl, hexyl and the like. Even more preferred are lower alkyl radicals having one or two carbon atoms. The term “alkylenyl” or “alkylene” embraces bridging divalent alkyl radicals such as methylenyl or ethylenyl. In some embodiments, alkyl radicals can have substituents, for example, wherein one or more carbon atoms in the chain is substituted with a heteroatom selected from halo, oxygen, nitrogen, or sulfur. Such substituted alkyl radicals are described further below.

The term "halo" means halogens such as fluorine, chlorine, bromine or iodine atoms.

The term “alkoxy” embraces linear or branched oxy-containing radicals each having alkyl portions of one to about ten carbon atoms. More preferred alkoxy radicals are “lower alkoxy” radicals having one to six carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and Zc/V-butoxy. Even more preferred are lower alkoxy radicals having one to three carbon atoms. Alkoxy radicals may be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide “haloalkoxy” radicals. Even more preferred are lower haloalkoxy radicals having one to three carbon atoms. Examples of such radicals include fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy and fluoropropoxy.

The term “comprising” is meant to be open ended, including the indicated component but not excluding other elements.

The term “conjugate” means a conjugate made from the CORMs orPyCa-CORMs described herein conjugated (covalently) to another molecule, such as both not limited to a ligand (e.g., GalNac), protein (e.g., albumin, or antibody or antigen-binding fragment), a peptide, an aptamer, a polymer (e.g., PEG) or combinations thereof. Exemplary conjugates include albumin, an antibody or antigen-binding fragment thereof, and polyethylene glycol polymers (PEG). Conjugates can be through any point of attachment of Formulas (I)-(VIII), as can be selected by one of skill in the art.

A group or atom that replaces a hydrogen atom is also called a substituent.

Any particular molecule or group can have one or more substituent depending on the number of hydrogen atoms that can be replaced.

The symbol represents a covalent bond and can also be used in a radical group to indicate the point of attachment to another group. In chemical structures, the symbol is used to represent a methyl group in a molecule. The term “therapeutically effective amount” means an amount of a compound that ameliorates, attenuates or eliminates one or more symptom of a particular disease or condition, or prevents or delays the onset of one of more symptom of a particular disease or condition.

The terms “patient” and “subject” may be used interchangeably and mean animals, such as dogs, cats, cows, horses, sheep and humans. In some embodiments, patients are mammals (e.g., human).

The terms “treating”, “treat” or “treatment” and the like include preventative (e.g., prophylactic) and palliative treatment.

The term “excipient” or “carrier” means any pharmaceutically acceptable additive, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API), which is typically included for formulation and/or administration to a patient.

The term “about” as used herein, means ±10% of the associated numerical value.

The present application provides the molybdenum carbonyl pyridine carboxaldehyde CO- releasing molecules (PyCa-CORMs). In some embodiments, the CORMs described herein provide for slow and/or sustained release of CO upon administration. The CO release kinetics of compounds described herein allow for release of CO in a more local fashion in target tissue (including in connection with targeting moieties) and/or avoid dangerous increases in CO-Hb levels upon administration.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (I) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is Ci-6 alkyl, halo, or Ci-6 alkoxy;

R2 is H or Ci-6 alkyl; each R3 is independently -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; k is 0, 1, or 2; q is 0, 1, 2, 3, or 4; and n is 0, 1, or 2.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof,: wherein Ri is Ci-6 alkyl, halo, or Ci-6 alkoxy;

R3 is -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; and k is 0, 1, or 2.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (III) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is Ci-6 alkyl, halo, or Ci-6 alkoxy;

R3 is -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; and k is 0, 1, or 2.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VI) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: each R4 is independently H, Ci-6 alkyl, halo, or Ci-6 alkoxy;

R5 is H or Ci-6 alkyl; each Re is independently OH, -CH2O(C=O)Rn, -O(C=O)Rn, halo, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl; m is 0, 1, 2, 3, 4, or 5; q is 0, 1, 2, 3, or 4; and at least four of Xi, X2, X3, X4, X5, and Xe are selected from CH and CH2, and where one or two of Xi, X2, X3, X4, X5, and Xe may be selected from null, O, N, or NH. In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is Ci-6 alkyl; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is methyl; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is methyl; R3 is COORn; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is methyl; R3 is COORn; and Rn is H, methyl, or ethyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is methyl; and R3 is COOH.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is halo; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is halo; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl. In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is halo; R3 is COORn; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is halo; R3 is COORn; and Rn is H, methyl, or ethyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is Br; and R3 is COOH.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; R3 is COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; R3 is COORn; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH3; R3 is COORn; and Rn is H, methyl, or ethyl. In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (II), (III), (IV), or (V) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Ri is - OCH ₃ ; and R ₃ is COOH.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VI) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Xi is N; X4 is O; X2, X3, X5, or Xe is CH2.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VI) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Xi is N; X4 is N; X2, X3, X5, or Xe is CH2; Re is Ci-6 alkyl linked to X4; m is 1.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VI) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Xi is N; X4 is N; X2, X3, X5, or Xe is CH2; Re is methyl linked to X4; m is 1.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VI) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Xi is CH; X2 is O; X3, X4, X5, and Xe each is CH; each Rs is -CH2O(C=O)Rn or O(C=O)Rn; Rn is Ci-6 alkyl; m is 4.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VI) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: wherein Xi is CH; X2 is O; X3, X4, X5, and Xe each is CH; each Rs is -CH2O(C=O)Rn or O(C=O)Rn; Rn is methyl; m is 4.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof

wherein Ri is H, Ci-6 alkyl, halo, or Ci-6 alkoxy; each R3 is independently -(C(Ri2Ri3))kCOORn, halo, Ci-6 alkoxy, or Ci-6 alkyl;

R11 is H or Ci-6 alkyl;

R12 is H or Ci-6 alkyl;

R13 is H or Ci-6 alkyl; k is 0, 1, or 2.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is H, Ci-6 alkyl, halo, or Ci-6 alkoxy; each R3 is independently COORn, halo, or Ci-6 alkyl; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is H, Ci-6 alkyl; each R3 is independently COORn; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is methyl; each R3 is independently COORn; and Rn is H or Ci-6 alkyl.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is halo; each R3 is independently COORn; and Rn is H or Ci-6 alkyl. In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is methyl; and each R3 is COOH.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is methyl; and each R3 is COOEt.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is Br; and each R ₃ is COOH.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, wherein Ri is Br; and each R3 is COOEt.

In some embodiments, the present application provides a molybdenum carbonyl pyridine carboxaldehyde complex of Formula (VIII) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof: each R4 is independently H, Ci-6 alkyl, halo, or Ci-6 alkoxy;

Rs is H or Ci-6 alkyl;

R14 and R15 are independently H, halo, Ci-6 alkoxy, or Ci-6 alkyl; Ri6 is H or C I-6 alkyl; q is 0, 1, 2, 3, or 4.

In various embodiments, the CORM compound is selected from Table 1.

Table 1: Examples of CORMs

The CORMs described herein have a CO-releasing kinetic profile desirable for therapeutic use, including in some embodiments for parenteral administration. In various embodiments, the CORM compositions described herein will release CO in blood, serum or plasma (ex vivo) for at least about 30 minutes, or at least about 60 minutes, or at least about 90 minutes, or at least about 120 minutes or at least about 180 minutes. In various embodiments, the CORMs described herein have a half-life (ex vivo) in blood, serum, or plasma of at least about 10 minutes, or at least about 20 minutes, or at least about 30 minutes. In various embodiments, the half-life of the compounds in plasma or serum is not more than about two hours, or not more than about one hour. In exemplary embodiments, the CORM has a half-life in a mouse model of about 10 to about 120 minutes, or about 15 to about 120 minutes, or about 20 to about 120 minutes, or about 30 to about 120 minutes, or about 10 to about 90 minutes, or about 10 to about 60 minutes, or about 10 to about 30 minutes. In some embodiments, the half-life in a mouse model is from about 15 to about 60 minutes or from about 15 to about 45 minutes.

In some embodiments, the CORMs described herein have the ability to release CO under physiological conditions in a sustained fashion, thereby avoiding a sharp increase in COHb. For healthy individuals (and non-smokers) CO-Hb levels will typically be under about 5% (e.g., about 3%). However, for smokers, COHb levels can be as high as about 10%. COHb levels around 20% are generally considered toxic and dangerous. In various embodiments, upon administration of the compositions described herein, COHb levels will not rise by more than about 7% from the baseline level, and in various embodiments, will not rise by more than about 5%, or more than about 3%, or more than about 2%, from the baseline level. In various embodiments, during administration and several (e.g., 1-3) hours thereafter, COHb levels will not rise above about 15%, or above about 12%, or above about 10%, or above about 8% In some embodiments, the compositions described herein allow for release of CO in a target tissue, for example, by accumulating the CORM in target tissue. In some embodiments, the target tissue is a tissue with inflammation or an undesirable immunological environment or reaction. In various embodiments, the CORM will accumulate in target tissue (e.g., liver, lungs, kidneys, muscle joints, and/or skin) without the aid of a targeting moiety. In other embodiments, a targeting moiety or ligand is employed with the composition or compound (e.g., in the form of a particle or conjugate) to drive accumulation in selected tissues. Without limitation, targeted tissues or organs include liver, lung, kidney, gastrointestinal tract, bone, joints, heart, brain, skin, muscle, lymph nodes, spleen, bladder, and pancreas. In some embodiments, targeted tissue is tumor tissue. In some embodiments, the CORM will accumulate in tumor tissue at least in part due to the enhanced permeability and retention (EPR) effect.

In various embodiments, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a molybdenum carbonyl pyridine carboxaldehyde complex of Formulas (I) to (VIII) (described above) or a pharmaceutically acceptable salt, ester, conjugate, amide, solvate, or hydrate thereof, or combination thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated for local or systemic delivery. In some embodiments, the composition is formulated for parenteral delivery (e.g., intravenous delivery) or oral delivery (e.g., enteral delivery). The pharmaceutically acceptable carrier includes any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and/or manufacture of pharmaceutical compositions agents can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980); and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

The pharmaceutical composition can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the compound of the present application (the “active ingredient”) into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient, which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and/or any additional ingredients in a pharmaceutical composition of the disclosure can vary depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 90% (w/w) active ingredient, such as about 1% to about 50%, or about 1% to about 40%, or about 1% to about 20%, or about 1% to about 10% (w/w) active ingredient (e.g., CORM). In some embodiments, the composition comprises the active ingredient from about 5% to about 50%, or from about 5% to about 40%, or about 5% to about 30%, or about 5% to about 20% (w/w).

Pharmaceutically acceptable excipients/carriers used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients/carriers such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof. Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Pol oxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and/or combinations thereof. Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, NeoIone, Kathon, and Euxyl. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and combinations thereof. Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the compounds of the disclosure are mixed with solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable carrier or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The active ingredients can be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a compound of the instant disclosure may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and/or any needed preservatives and/or buffers as can be required. Additionally, the present application contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the disclosure can be a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles, which may comprise the active ingredient (CORM), and which have a diameter in the low nanometer range. Such compositions are conveniently in the form of dry powders for administration, and may optionally employ a propellant. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Pharmaceutical compositions of the disclosure formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

In some embodiments, the formulation is useful for intranasal delivery. An exemplary formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Compositions may be formulated in a dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the dosage regimen will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease, disorder, or condition being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, of the subject; route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

Still further encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound. In some embodiments, the pharmaceutical composition or compound provided in the container and the second container are combined to form one unit dosage form.

Optionally, a single container may comprise one or more compartments for containing a pharmaceutical composition or compound, and/or a pharmaceutically acceptable carrier for suspension or dilution. In some embodiments, a single container can be appropriate for modification such that the container may receive a physical modification so as to allow combination of compartments and/or components of individual compartments. For example, a foil or plastic bag may comprise two or more compartments separated by a perforated seal which can be broken so as to allow combination of contents of two individual compartments once the signal to break the seal is generated. A kit may thus comprise such multi-compartment containers providing a pharmaceutical composition or compound and one or more pharmaceutically acceptable carriers.

Optionally, instructions for use are additionally provided in such kits of the disclosure. Such instructions may provide, generally, for example, instructions for dosage and administration. In other embodiments, instructions may further provide additional detail relating to specialized instructions for particular containers and/or systems for administration. Still further, instructions may provide specialized instructions for use in conjunction and/or in combination with an additional therapeutic agent.

In some embodiments, the composition comprises the CORM described herein encapsulated in a particle, such as a microparticle or nanoparticle. The particle chemistry can be selected for sustained release of the CORM, or release of the CORM in certain environments to affect local release. Encapsulation within non-toxic host molecules is one way to achieve sustained release of the active drug in vivo. This strategy minimizes unwanted effects that can result from a sharp increase in concentration and/or availability of potentially toxic drugs. The conjugates, particles, and micelles described herein can extend the half-life of the CORMs or PyCa-CORMs in circulation and improve delivery and/or accumulation to the target tissue (e.g. as described in Yin et al., Journal of Controlled Release 2014, 187 (C), 14-21).

The particles can include the CORM or PyCa-CORM in a core surrounded by a coating including, without limitation, an enteric coating. The CORM or PyCa-CORM may also be dispersed throughout the particles. The CORM or PyCa-CORM may also be adsorbed within the particles. The particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof. The particles can include, in addition to the CORM or PyCa-CORM, any material routinely used in the pharmaceutical and medical fields, including erodible, non- erodible, biodegradable, or non-biodegradable materials or combinations thereof. The particles may be microcapsules containing the CORM or PyCa-CORM in solution or in a semi-solid state. The particles can be of virtually any shape. In embodiments, particles comprising the CORM with an enteric coating can be used for enteral delivery to the gastrointestinal tract.

Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering CORMs or PyCa-CORMs. Such polymers may be natural or synthetic polymers. In particular for enteral delivery, the polymer is selected for the desired release profile for delivery to the GI. Bioadhesive polymers of particular interest include bioerodible hydrogels as described in H.S. Sawhney, C.P. Pathak and J. A. Hubell in Macromolecules, 1993, 26: 581-587. These include: polyhyaluronic acid, casein, gelatin, glutin, polyanhydride, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate).

In some embodiments, the nanoparticle or microparticle comprises a polymeric matrix that comprises two or more polymers, and can be selected for parenteral delivery. The polymeric matrix may comprise polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, or polyamines, or combinations thereof. In one embodiment of the matrix, at least one polymer is a polyalkylene glycol, such as polyethylene glycol. In another embodiment of the matrix, at least one polymer is a polyester, such as poly(lactic-co-glycolic acid) (PLGA). In another embodiment, the polymeric matrix comprises a copolymer of two or more polymers, such as a copolymer of a polyalkylene glycol and a polyester, e.g., a copolymer of PLGA and polyethylene glycol (PEG).

In some embodiments, the polymeric matrix comprises a lipid-terminated polyalkylene glycol and a polyester, such as lipid-terminated PEG, and PLGA. In one embodiment, the lipid can be 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), or salts thereof. The lipid of the lipid-terminated PEG can self-assemble with PLGA.

In various embodiments, that particles are nanoparticles, and the nanoparticles may be about 40 nm to about 500 nm in size (mean diameter). In other embodiments, the nanoparticles are about 40 nm to about 250 nm in size. In still another embodiment, the nanoparticles are about 40 nm to about 150 nm in size. In another embodiment, the nanoparticle is about 40 to about 100 nm in size. In still other embodiments, the particles are microparticles, which may be in the range of 500 nm to about 2 pm in size.

In some embodiments, the CORMs or PyCa-CORMs described herein are conjugated to a ligand, protein (e.g., albumin), peptide, vitamin, or polymer (including PEG) to improve half-life and tissue selectivity. The conjugated CORMs or PyCa-CORMs enable local carbon monoxide delivery, targeting specific tissues in need thereof. In various embodiments, polymeric conjugates (including with polymers described above) can reduce clearance of the CORMs from circulation, thereby increasing half-life and accumulation in target tissue. In some embodiments, the conjugate comprises a ligand having binding affinity for target tissue. Such ligands include antibodies or antigen-binding fragments or other ligands, such as GalNac. GalNac ligands can be useful for targeting hepatocytes. Other targeting moieties that may be employed include nucleic acid aptamers, growth factors, hormones, cytokines, interleukins, antibodies, integrins, fibronectin receptors, and p-glycoprotein receptors. In some embodiments, the nanoparticle or microparticle is covalently bound to the targeting moiety via a maleimide functional group at the free terminus of PEG.

In some embodiments, the CORMs or PyCa-CORMs described herein are conjugated to a hydrophilic polymer at one end and a hydrophobic polymer at the other end. Such conjugates form micelles, enabling the delivery to the CORMs or PyCa-CORMs described herein to the tissues in need thereof. In one embodiment, the hydrophilic polymer is polyethylene glycol) (PEG). In one embodiment, the hydrophobic polymer is poly (n-butylacrylamide). In one embodiment, the hydrophilic polymer is selected from the group consisting of polyurethane, copolyester, polyesteramide block copolymer, poly etheramide block copolymer, polyetheresteramide block copolymer, and poly etherester block copolymer, and combinations thereof. In one embodiment, the hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefin polymer, a polysiloxane, a polycarbonate, and combinations thereof. In some embodiments, the nanoparticle or microparticle comprising a CORM or PyCa- CORM as described herein further comprises a targeting moiety. In one embodiment, the targeting moiety is covalently attached to the nanoparticle or microparticle. In one embodiment, the targeting moiety is covalently attached to the outer surface of the nanoparticle or microparticle.

The present application is based at least in part on the discovery that compounds and pharmaceutical compositions described herein release an effective amount of carbon monoxide (CO) once administered with a therapeutically desirable release profile.

In one aspect, the present application provides a method for treating or preventing an inflammatory or immunological disease in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

In some embodiments, the targeted tissue or organ includes liver, lung, kidney, gastrointestinal tract, bone, joints, heart, brain, skin, muscle, lymph nodes, spleen, bladder, prostate, and pancreas. In some embodiments, targeted tissue is tumor tissue. In some embodiments, the subject in need of treatment has an inflammatory heart disease, such as endocarditis, myocarditis, pericarditis, or combinations thereof. In some embodiments, the subject in need of treatment has an inflammatory liver disease, which may be selected from fatty liver disease, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), cirrhosis of the liver, alcoholic steatohepatitis (ASH), alcoholic liver diseases (ALD), HCV-associated cirrhosis, viral hepatitis, drug induced liver injury, and hepatocellular necrosis. In some embodiments, the subject in need of treatment has an inflammatory kidney disease, which may be selected from glomerulonephritis, membranoproliferative glomerulonephritis (MPGN), interstitial nephritis, IgA nephropathy (Berger's disease), pyelonephritis, lupus nephritis, goodpasture's syndrome, Wegener's granulomatosis, chronic kidney disease, and acute kidney injury. In some embodiments, the subject in need of treatment is a transplant recipient (e.g., kidney, liver, or lung transplant recipient), having or at risk of graft versus host disease or transplant rejection or transplant failure.

In some embodiments, the subject in need of treatment has an inflammatory lung disease, such as a condition selected from asthma, chronic obstructive pulmonary disease (COPD), interstitial lung tissue fibrosis (e.g., IPF), sarcoidosis, allergic pneumonitis (HP), chronic allergic pneumonitis, acute lung injury, viral pneumonia (e.g., COVID-19), and bronchiolitis obliterans with organizing pneumonitis (BOOP).

In some embodiments, the subject in need of treatment has an inflammatory pancreas disease, which may be selected from acute pancreatitis, chronic pancreatitis, and hereditary pancreatitis.

In some embodiments, the subject in need of treatment has an inflammatory disease of the bones or joints, such as arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, osteoarthritis, and psoriatic arthritis. In some embodiments, the inflammatory disease is rheumatoid arthritis.

Other inflammatory diseases include, but are not limited to, inflammation associated with arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu's arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis and Reiter's arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, cardiovascular disease, chronic prostatitis, conjunctivitis, chronic obstructive pulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., type I diabetes mellitus, type 2 diabetes mellitus), a skin condition (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itch)), endometriosis, Guillain-Barre syndrome, infection, ischaemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g., migraine headaches, tension headaches), ileus (e.g., postoperative ileus and ileus during sepsis), idiopathic thrombocytopenic purpura, interstitial cystitis (painful bladder syndrome), gastrointestinal disorder [e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD), Crohn's disease, Behcet's syndrome, colitis (e.g., ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, indeterminate colitis, microscopic colitis, chemical colitis, infectious colitis, fulminant colitis), and inflammatory bowel syndrome (IBS)], celiac disease, lupus (i.e., SLE), multiple sclerosis, morphea, myeasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus vulgaris, pernicious aneaemia, peptic ulcers, polymyositis, primary biliary cirrhosis, neuroinflammation associated with brain disorders (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, reperfusion injury, regional enteritis, rheumatic fever, schleroderma, scierodoma, sarcoidosis, spondyloarthopathies, Sjogren's syndrome, thyroiditis, transplantation rejection, tendonitis, trauma or injury, vasculitis, vitiligo and Wegener's granulomatosis.

In some embodiments, the present application provides a method for treating or preventing compartment syndrome in a subject in need thereof. The method comprises administering to the subject transdermally, intravenously or via local injection of a therapeutically effective amount of a pharmaceutical composition described herein. For example, the composition can be delivered by local injection to the site or areas surrounding an injury. In some embodiments, compartment syndrome is acute or chronic compartment syndrome.

In some embodiments, the present application provides a method for promoting wound healing in a subject in need thereof. The method comprises administering to the subject via topical or intradermal administration to a wound of a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the wound is a chronic wound (e.g., diabetic ulcer or amputation wound) or a burn (e.g., 2nd or 3rd degree bum). In some embodiments, the composition is applied to the area of a skin graft and/or a donor site.

In some embodiments, the present application provides a method for treating or preventing an inflammatory skin condition in a subject in need thereof. The method comprises administering to the subject via topical or intradermal administration of a therapeutically effective amount of a pharmaceutical composition described herein. Exemplary skin conditions include psoriasis, allergic reaction, autoimmune condition, rash, hives, eczema, pruritus, and dermatitis.

In some embodiments, the subject to be treated has a cancer, which may be a solid tumor or hematological malignancy. In some embodiments, the target tissue is a solid tumor (which may be a primary tumor or may comprise tumor metastases). In various embodiments, the cancer is selected from bladder cancer, head and neck cancer, pancreatic cancer, colon carcinoma, breast cancer, fibrosarcoma, mesothelioma, renal cell carcinoma, lung carcinoma, lymphoma, prostate cancer, colorectal cancer, ovarian cancer, brain cancer, squamous cell cancer, skin cancer, eye cancer, retinoblastoma, intraocular melanoma, oral cavity and oropharyngeal cancers, gastric cancer, stomach cancer, cervical cancer, kidney cancer, liver cancer, esophageal cancer, testicular cancer, gynecological cancer, thyroid cancer, glioblastoma multiforme, nonsmall-cell lung cancer, hepatocellular carcinoma, small-cell lung cancer, melanoma, and combinations thereof.

In these embodiments, the compounds and compositions described herein may provide immunological benefits to potentiate cancer immunotherapy, or in some embodiments, chemotherapy or radiation therapy. In some embodiments, the compounds or compositions described herein are administered with an immune checkpoint inhibitor, such as an immune checkpoint inhibitor therapy that provides for PD-1 blockade and/or targets CTLA-4. In some embodiments, the checkpoint inhibitor is selected from ipilimumab, tremelimumab, nivolumab, averumab, durvalumab, atezolizumab, penbrolizumab and any combination thereof. In various embodiments, therapy with the CORM or compositions thereof is initiated at least one week prior to immune checkpoint inhibitor therapy.

In some embodiments, the subject to be treated is receiving a therapeutically effective amount of an immunotherapy, such as an immunotherapy selected from checkpoint blockade, adoptive cell therapy, CAR-T cell therapy, marrow-infiltrating lymphocytes, A2aR blockade, KIR blockade, vaccines (e.g., tumor vaccines), passive immunotherapy antibodies, and combinations thereof.

In some embodiments, the subject in need of treatment is receiving cancer chemotherapy. Exemplary chemotherapy may comprise an agent selected from antimetabolites, platinum-based drugs, alkylating agents, tyrosine kinase inhibitors, anthracycline antibiotics, vinca alkloid, proteasome inhibitors, and topoisomerase inhibitors (among others). In some embodiments, the chemotherapy comprises 5-fluorouracil, etoposide, cisplatin, paclitaxel, carboplatin, and combinations thereof.

In some embodiments, the subject in need of treatment may receive a therapeutically effective amount of radiation, such as whole body radiation therapy, external beam radiation therapy, and internal beam radiation therapy. The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site.

The CORMs or PyCa-CORMs or the pharmaceutical compositions as described herein can be administered to a subject in need thereof in any suitable dose. In some embodiments, the therapeutically effective dose is in a range of about 1 to about 50 mg/kg, or about 10 to about 50 mg/kg, about 10 to about 40 mg/kg, or about 10 to about 30 mg/kg, or about 15 to about 40 mg/kg, or about 20 to about 40 mg/kg. In one embodiment, the COHb concentration of the subject after administration remains less than about 15%, or less than about 12%. In various embodiments, the COHb levels in the patient do not rise more than about 7% or more than about 5% from their baseline levels.

The dosage can vary depending on the needs of the patient, the particular formulation being administered, and other factors. The dose administered to the patient should be sufficient to produce a beneficial therapeutic response in the patient. The size of the dose will also be determined by the presence, nature and extent of any adverse side effects associated with the administration of the drug in a particular patient. The total dose can be divided and administered in portions over an appropriate period to address the need for carbon monoxide.

Administration of the CORMs or PyCa-CORMs or the pharmaceutical compositions as described herein can occur for a period that varies depending on the nature of the need for the particular carbon monoxide, its severity, and the overall condition of the patient. Administration can be, for example, about twice a day, about daily, several times per week, or about weekly. After treatment, the patient can be monitored for changes in the patient's condition and relief of symptoms of the need for carbon monoxide. The dose of carbon monoxide releasing compound can be increased in situations where the patient is not significantly responsive to a particular dose level, or if the need for carbon monoxide disappears or if unacceptable side effects are observed at a particular dose, the dose can be reduced.

The various aspects and embodiments of the invention will be understood by one of skill in the art in connection with the following non-limiting examples.

EXAMPLES

Compounds of the present application generally can be prepared beginning with commercially available starting materials and using synthetic techniques known to those of skill in the art. Outlined below are some reaction schemes suitable for preparing compounds of the present application. Further exemplification is found in the specific examples provided.

Example 1: Synthesis of ALF523

A transparent solution of Mo(CO)e (6 mmol; 1.58 g) in THF (125 ml) was treated with trimethyl Y-oxide (12 mmol = 0.901 g) added in portions. The solution becomes immediately bright yellow. After 60 minutes the solution containing the ligand (obtained by mixing 1-amino- 4-methylpiperazin with Pyridine-2-aldehyde in methanol with some drops of formic acid and stirring for 18 h) is added and the solution turns immediately red-brown. The solution was stirred for 18 h to complete the reaction. The red THF solution was evaporated to one third of its volume and then filtered. The solution was further evaporated to about 10 ml and hexane (about 50 ml) was added. A precipitate formed immediately. The suspension was filtered and the solid washed three times with 15 ml of dry hexane and dried in vacuum to give an orange-red solid. Yield ca. 80%. Infrared (IR) vCO (KBr; cm' ¹ ): 2022, 1933, 1875, 1810. Elemental analysis: Calc: %C 43,7%; %N 13,59; %H 3,91. Found: %; % C 44.00; %N 13.52 % H 3.88.

Example 2: Synthesis of ALF 529

The ligand was prepared as follows: A methanolic transparent solution ofN- aminomorpholine (12 mmol; 1.157 ml) was treated by dropwise addition of 1 equivalent of pyridine-2-aldehyde (12 mmol; 1.152 ml). The yellow solution was treated with a few drops of acetic acid and stirred overnight under air. The solvent was evaporated to give an orange oil which upon long drying in vacuum became whitish and solidified. This compound was found pure by ¹ H NMR (CDCh) and used for the synthesis of ALF529. A transparent solution of Mo(CO)e (1.89 mmol; 500 mg) in THF (125 ml) was treated with trimethyl A-oxide (4 mmol; 300 mg) added in portions. The solution becomes immediately bright yellow. After 60 minutes the ligand was added (1.89 mmol; 379 mg). The solution immediately turned red-brown and was stirred overnight for 18 hrs. The solvent was evaporated to ca. 40 ml and the suspension filtered. Hexane (ca. 80-90 ml) was added to the filtered solution to induce precipitation. An abundant dark red solid formed. It was filtered, washed twice with hexane and dried under vacuum. Yield ca. 80%. IR vCO (KBr; cm' ¹ ): 2015, 1912, 1880, 1829. Elemental analysis: Calc: %C 42,12; %N 10,53; %H 3,28. Found: % C 42.70; % N 10.97; %H 3.41.

Example 3: Synthesis of ALF554 via ALF552

Synthesis of ALF552

A transparent solution of Mo(CO)e (1.89 mmol; 500 mg) in THF was treated with trimethyl N- oxide (3.79 mmol; 300 mg) added in portions. The solution becomes immediately bright yellow. After 1 hour the ligand (Ferrara et al., Eur. J. Inorg. Chem. 1999, 1939-1947; Borriello et al., J. Chem. Soc., Dalton Trans. 2000, 2545 - 2550) (1.89 mmol; 824 mg) was added in portions. The solution became progressively purple and was stirred overnight at r.t. The next day TLC shows a purple spot (Rf = 0.5 in AcOEt). THF was evaporated to 1/3 of its initial volume and hexane was added. A purple solid precipitates immediately. The solid is washed twice with hexanes and the sample dried under vacuum. IR vCO (KBr/cm' ¹ ): 2019, 1911, 1881, 1836). Elemental analysis: Calc: %C: 44.73; %H 3.75; %N: 4.35 Found: %C 44.60; %H 3.71; %N 4.61.

Synthesis of ALF554

ALF552 (100 mg) were added to a dry schlenk tube to which methanol carefully dried and previously bubbled with N2 for at least i an hour was added. The purple solution was treated with an excess of sodium methoxide (3 or 4 spatula tips) and the mixture was stirred. After 30 minutes the initial suspension became a transparent solution but TLC (50% AcOEt in MeOH) indicated some starting material. The reaction was left stirring under nitrogen overnight. The next day TLC indicated total consumption of starting material and a single, polar and red spot was observed. Dowex (two small spatulas) was added to the mixture until the pH became neutral. The suspension was filtered and washed. The solvent is evaporated giving a dark red oily material which is analytically pure. Yield 70%. IRvCO (KBr; cm' ¹ ): 2016(w), 1898(s), 1870(s), 1826. Elemental analysis: Calc: %C 40.35; %N 5.88; % H 3.39. Found: %C 40.40; %N: 5.80;

%H: 3.44.

Example 4: Synthesis of ALF 560

MO(CO) ₆ (422 mg, 1.60 mmol) was dissolved in anhydrous THF (170 mL), trimethylamine A-oxide (240 mg, 3.20 mmol) was added and the resulting bright yellow mixture was stirred under N ₂ (trimethylamine-A-oxide is not completely soluble in THF and the solution goes increasingly cloudier over time). After 1 h, the ligand 4-OAc-P-GalN=CHPyr (700 mg, 1.60 mmol) (Borriello et al. J. Chem. Soc., Dalton Trans., 2000, 2545 - 2550) was added and the reaction goes from an initially green to a dark red color. The mixture was then left to stir under N ₂ overnight. The solvent was reduced to 1/5 of its initial volume and precipitation of the product was induced by addition of hexane (150 mL) and a ‘dark wine colored’ precipitate started to form. The mixture was filtered out and the precipitate dried to afford Mo(CO) ₄ (4-OAc- p-GalN=CHPyr) (0.65 g, 65 % yield). IR vCO (KBr; cm' ¹ ): 2017(s), 1912(s), 1890(s), 1838(s). Elemental analysis: Calc: %C:44.45; %H:4.35; %N:4.32. Found: %C:44.52; %H:4.37; %N:4.27.

Example 5: Synthesis of ALF821

To a solution of pyridine-2-carbaldehyde (0.085 g, 0.794 mmol, d = 1.126 g/ml, 0.075 mL) and dimethyl-5-aminoisophtalate (0.794 mmol, MW = 209.20 gmol-1, 0.166 g) in 20 mL of ethanol was added Mo(CO) ₄ (pip)2 (0.3 g, 0.794 mmol), and the mixture was heated at 50°C, with stirring, for 30 min. The color changed quickly from yellow to dark purple. The solvent was evaporated in vacuo and the residue was washed twice with hexane.

The residue was extracted several times with diethyl ether and the combined extracts filtered through kieselgur. The solution was concentrated, and hexane was added to precipitate the purple solid. IR vCO (KBr; cm' ¹ ): 2012(s), 1883(br,s), 1839 (s). Elemental analysis: Calc: %C 47,45; %N 5,53; %H 2,79. Found: %C:47.56, %H:2.75, %N:5.52.

Example 6: Synthesis of ALF822

To a solution of 6-methyl-2-pyridinecarboxaldehyde (0.096 g, 0.794 mmol) and ethyl 3- aminobenzoate (0.794 mmol, 0.131 g) in 20 ml of ethanol was added Mo(CO) ₄ (pip)2 (0.3 g, 0.794 mmol), and the mixture was heated at 50 °C, with stirring, for 30 min. The color change quickly from yellow to dark purple. The solvent was evaporated in vacuo and the residue was washed two times with hexane. The residue was extracted several times with diethyl ether and the combined extracts filtered through kieselgur. The solution was concentrated and hexane was added to precipitate the purple solid. Yield 0.195 g; 52%. IR vCO (KBr; cm' ¹ ): 2015(s), 1876(br,s), 1827 (s). Elemental analysis: Calc: %C 50,43; %N 5,88; %H 3,39; Found: %C 50.48; %N 5.77; %H 3.26.

Example 7: Synthesis of ALF823

302.4 mg (2 mmol) 4-aminophenylacetic acid was dissolved in a minimum amount of EtOH in 40 ml toluene, 224 ml (2mmol) acetylpyridine added and heated to reflux with a Dean- Stark apparatus for 3h. 528 mg (2mmol) Mo(CO)e were added and the solution heated to reflux for more 2 hours. The solvents were evaporated in vacuum. The residue was washed with hexane and Et2O, dissolved in CH2Q2, filtered and extracted 3 times with water/NaOH at pH=12. The collected aqueous phases were filtered, HC1 was added and the resulting precipitate centrifuged, decanted and washed 3 times with water, once with hexane and dried in vacuum. vCO (KBr; cm' ^x ): 2011, 1885 (split), 1827. 'H NMR: 6H (CD2CI2, 400MHz) 9.16 (d, 1H), 7.9-8.0 (m, 2H), 7.49 (t, 1H), 7.42 (d, 2H), 7.04 (d, 2H), 3.75 (s, 2H, CH ₂ ), 2.36 (s, 3H, CH ₃ ); 6C (MeOD, 100MHz) 207.0 (CO), 63.6, 59.3, 45.2 (CH ₂ ), 56.3 (CH ₃ ). Elemental analysis: Calc: %C 49,36; %N 6,06; %H 3,05. Found: %C, 39.40; %H, 5.44; % N, 11.57.

Example 8: Synthesis of ALF826

0.242 g (2 mmol) 6-methyl-2-pyridinecarboxaldehyde and 0.274 g (2 mmol) 4- aminobenzoic acid were dissolved in 20 ml THF, 0.9 g (2.39 mmol) [Mo(CO)4(pip)2] added and stirred under N2 at room temperature for 3 hours. The colour changed from yellow to dark purple. The solvent was evaporated under vacuum and the residue was washed with hexane and diethyl ether and dried under vacuum. The residue was chromatographed over silica gel with ethyl acetate and ethyl acetate:isopropanol:water (7:2: 1). The product obtained from the strongly colored band was dried and washed again with diethyl ether to remove solvent contamination. The final yield was 0.42 g (47%). IR vCO (KBr; cm' ¹ ) 2018, 1914, 1878 (split), 1824. 'H NMR 6H (Acetone-d6, 400MHz) 8.97 (s, 1H), 8.20 (d, 2H), 8.11 (m, 2H), 7.77 (dd, 1H), 7.69 (d, 2H), 3.02 (s, 3H, CH ₃ ). ¹³ C NMR 8C (Acetone-d6, 100MHz) 204.6 (M-CO), 172.5 (COOH), 167.0 (C-CH ₃ ), 155.7+155.6 (Ctert), 154.0(C6py), 139.0 (C4py), 131.9 (2Cbenz), 128.4+128.0 (C3py+C5py), 121.9 (2Cbenz), 18.3 (CH ₃ ). Elemental analysis: Calc: %C 48,23; %H 2,70; %N 6,25. Found: %C 48.34, %H 2.74, %N 6.21.

Example 9: Synthesis of ALF827

To a solution of 6-methyl-2pyridinecarboxaldehyde (0.242 g, 2.0 mmol) and dimethyl 5- aminoisophtalate (2 mmol, 0.330 g) in 20 ml of ethanol was added Mo(CO)4(pip)2 (0.9 g, 2.39 mmol), and the mixture was stirred at 50 °C for 2 h. The color changed quickly from yellow to dark purple. The solvent was reduced in vacuo and the compound started to precipitate. The abundant solid precipitate was filtered off and evaporated to dryness. The solid was washed with hexane and diethyl ether. The fraction that is insoluble in diethyl ether is the product, and the soluble fraction contains unreacted dimethyl 5-aminoisophtalate. Yield 0.752 g; 72%). IR vCO (KBr; cm' ¹ ): 2015(s), 1904(br,s), 1832 (s). Elemental analysis: %C 48,47; %N 5,38; %H 3,10; found: %C 48.33; %H 2.98; %N 5.32.

Example 10: Synthesis of ALF828

0.242 g (2 mmol) 6-methyl-2-pyridinecarboxaldehyde and 0.33 g (2 mmol) benzocaine were dissolved in 20 ml Ethanol, 0.9 g (2.39 mmol) [Mo(CO)4(pip)2] added and stirred under N2 at 50°C for 2 hours. The color changed from yellow to dark purple. The solvent was evaporated under vacuum and the residue was washed with hexane and diethyl ether and dried under vacuum to give 0.632 g (yield 66%). IR vCO (KBr; cm' ¹ ): 2018, 1914, 1878 (split), 1824. NMR 6H (Acetone-d6, 400MHz) 8.97 (s, 1H), 8.20 (d, 2H), 8.11 (m, 2H), 7.77 (dd, 1H), 7.69 (d, 2H), 3.02 (s, 3H, CH ₃ ); ¹³ C NMR 6C (Acetone-d6, 100MHz) 204.6 (M-CO), 172.5 (COOH), 167.0 (C-CH ₃ ), 155.7+155.6 (Ctert), 154.0(C6py), 139.0 (C4py), 131.9 (2Cbenz), 128.4+128.0 (C3py+C5py), 121.9 (2Cbenz), 18.3 (CH ₃ ). Elemental analysis: %C 50,43; %N 5,88; %H 3,39. Found %C 50.55; %H 3.44; %N 5.74.

Example 11: Synthesis of ALF829

0.242 g (2 mmol) 6-methyl-2-pyridinecarboxaldehyde and 0.302 g (2 mmol) 4- aminophenylacetic acid were dissolved in 25 ml THF, 0.9 g (2.39 mmol) [Mo(CO)4(pip)2] added and stirred under N2 at room temperature for 4 hours. The colour changed from yellow to dark purple. The solvent was evaporated under vacuum and the residue was washed with diethylether. The residue was chromatographed over silica gel first with ethyl acetate to remove impurities and then with ethyl acetate: isopropanol: water (7:2: 1). The product obtained from the strongly colored band was dried and washed with diethylether to remove residual solvent contamination. Yield 0.303 g; 33%. IR vCO (KBr; cm' ¹ ): 2015, 1897, 1872, 1823. 'H NMR 6H (Acetone-d6, 400MHz) 8.94 (s, 1H), 8.22 (dt, 1H), 8.18 (d, 2H), 7.90 (d, 1H), 7.68 (d, 2H), 7.42 (d, 2H), 4.22 (s, 3H, CH3). ¹³ C NMR 8C (Acetone-d6, 100MHz) 204.6 (M-CO), 172.5 (COOH), 167.0 (C- CH ₃ ), 155.7+155.6 (Ctert), 154.0(C6py), 139.0 (C4py), 131.9 (2Cbenz), 128.4+128.0 (C3py+C5py), 121.9 (2Cbenz), 18.3 (CH ₃ ). Elemental analysis: %C 49,36; %N 6,06; % H 3,05; %C 49.48; %H 2.90; %N 5.98.

Example 12: Synthesis ofALF840

To a solution of pyridine-2-carbaldehyde (2 mmol, d = 1.126 g/mL, 0.19 mL) and 4- aminophenylacetic acid (2 mmol, 0.302 g) in 25 ml of THF was added Mo(CO)4(pip)2 (0.9 g, 2.39 mmol), and the mixture was stirred at room temperature for 4,5 h. The color changed quickly from yellow to dark purple. The solvent was evaporated in vacuo and the residue was washed with diethyl ether. The residue was chromatographed in a silica-gel column and eluted first with ethyl acetate to remove the impurities and after with a mixture of ethyl acetate : isopropanol : water, 7:2: 1, to remove the product which was dried in vacuum. Yield: 0.766 g; 85.5 %. IR vCO (KBr; cm' ¹ ): 2010(s), 1872(br,s), 1823 (s). Elemental analysis: Calc: %C 48.23; %H 2.70; %N 6.25. Found: %C 45.36; %H 2.45; %N 5.68.

Example 13: Synthesis of ALF841

0.24 ml (2 mmol) 6-methoxy-2-pyridinecarboxaldehyde and 0.274 g (2 mmol) 4- aminobenzoic acid were dissolved in 20 ml THF, 0.9 g (2.39 mmol) [Mo(CO)4(pip)2] added and stirred under N2 at room temperature for 2 hours. The color changed from yellow to dark purple. The solvent was evaporated under vacuum and the residue was chromatographed over silica gel with ethylacetate and ethylacetate:isopropanol: water (7:2: 1). The product obtained from the strongly colored band was dried and washed with diethyl ether to remove solvent contamination. Yield: 0.671 g; 72%. IR vCO (KBr; cm' ¹ ): 2012, 1904, 1868, 1821. 'H NMR 6H (Acetone-d6, 400MHz) 8.94 (s, 1H), 8.22 (dt, 1H), 8.18 (d, 2H), 7.90 (d, 1H), 7.68 (d, 2H), 7.42 (d, 2H), 4.22 (s, 3H, CH ₃ ). ¹³ C NMR 8C (Acetone-d6, 100MHz) 204.6 (M-CO), 172.5 (COOH), 167.0 (C- CH ₃ ), 155.7+155.6 (Ctert), 154.0(C6py), 139.0 (C4py), 131.9 (2Cbenz), 128.4+128.0 (C3py+C5py), 121.9 (2Cbenz), 18.3 (CH ₃ ). Elemental analysis: Calc: %C 47,71; %N 5,86; %H 2,95; Found: %C 46.50; %H 2.54; %N 5.91.

Example 14: Synthesis of ALF846

0.5 g (2.69 mmol) 6-Bromo-2-pyridinecarboxaldehyde and 0.369 g (2.69 mmol) 4- aminobenzoic acid were dissolved in 30 ml EtOH and heated to 60°C under N2 for 48 h. 0.71 g (2.69 mmol) [Mo(CO)e] were added and the solution heated to reflux at 90°C. The color changed to dark purple and CO was released. The reaction was stopped when the aldehyde proton became undetectable in ^T H NMR spectra. The solvent was evaporated under vacuum and the residue was chromatographed over silica gel with ethyl acetate to elute the remaining aldehyde and aniline, and the product was collected with a mixture of ethylacetate, 2-propanol, water (7:2: 1) and dried under vacuum. Yield: 0.96 g; 69%. IR vCO (KBr; cm' ¹ ): 2019, 1931, 1905, 1874 (split), 1828. 'H NMR 6H (Acetone-d6, 400MHz) 9.08 (s, 1H), 8.28 (dd, 1H), 8.20 (d, 2H), 8.11 (m, 2H), 7.68 (d, 2H); ¹³ C NMR 6C (Acetone-d6, 100MHz) 203.1 (M-CO), 167.7 (C(H)=N), 166.8 (COOH), 156.7+156.3 (Ctert), 148.3(C-Br), 140.6 (C4py), 133.9 (C5py), 131.6 (2Cbenz), 131.0 (C- COOH), 129.8 (C3py), 123.0 (2Cbenz). Elemental analysis: Calc: %C 39,79; %N 5,46; %H 1,77. Found: %C, 40.40; %H, 2.16; %N, 5.32.

Example 15: Synthesis of ALF858

Synthesis of the ligand: 544 mg (3 mmol) aminophtalic acid and 285.4 ml pyridinecarboxaldehyde (3 mmol) were heated at 60° in 30 ml EtOH overnight. The yellow precipitate was filtered off, washed with EtOH and hexane and dried.

Synthesis of the complex: The ligand obtained was suspended in 50 ml THF, 3 mmol (792 mg) [Mo(CO)e] added and the solution heated to reflux at 90°C for 4 h. The colour changed to dark purple and CO was released. The solvent was evaporated under vacuum and the residue was washed with 10 ml CH2Q2 and ether. The dried product was finely ground and washed twice with 10 ml of IM HC1 followed by two times 10 ml water and pentane. Yield: 0.96 g ; 69%. IR vCO (KBr; cm' ¹ ): 2019, 1931, 1905, 1874 (split), 1828. 'H NMR 6H (Acetone-d6, 400MHz) 9.08 (s, 1H), 8.28 (dd, 1H), 8.20 (d, 2H), 8.11 (m, 2H), 7.68 (d, 2H). ¹³ NMR 6c (Acetone-d6, 100MHz) 204.6 (M-CO), 172.5 (COOH), 167.0 (C-CH ₃ ), 155.7+155.6 (C _tert ), 154.0(C ⁶ _py ), 139.0 (C ⁴ py), 131.9 (2C _be nz), 128.4+128.0 (C ³ _py +C ⁵ _py ), 121.9 (2C _be nz), 18.3 (CH ₃ ).

Elemental analysis: Calc: C, 45.21; H, 2.11; N, 5.86; Found: C, 45.30; H, 2.53; N, 5.40.

Example 16: Methods for quantification of CO release

Several methods have been used to quantify the CO release of each CORM in different conditions, in vitro and in vivo and establish their corresponding profile. These methods are described in the following.

CO release in the dark in Hepes buffer.

The CO release kinetics of all compounds was evaluated in 50 mM HEPES buffer (pH 7.4) in a sealed 8-ml vial. Stock solutions (5 mM) of the compounds were prepared in DMSO and 10 pl were added to 990 pl of 50 mM HEPES buffer (final concentration in buffer was 50 pM) in a 8-ml vial which was sealed with a septum, wrapped in aluminum foil and kept in the dark inside a cardboard box. Gas samples (from 10 pl up to 500 pl) of the vial headspace (7 ml volume) were removed with a gas-tight syringe at 15, 30, 60, 120, 180 min after start of the incubation. The gas samples were injected into sealed vials containing air (8 ml) for dilution. The entire gas volume of the vials (8 ml) was transferred with carrier gas to the RCP-GC and analyzed for CO. The GC-RCP chromatograph(Peak Performer RCP, Peak Laboratories LLC, Mountain View, CA) had been calibrated with gas containing a known amount of CO. The calibration curve had been established starting with gas from a cylinder which contained synthetic air with 30 ppm CO (Linde, Cat. No. 14960013) and preparing dilutions in 8-ml vials as described above (Vreman, H. J. et al. Current Protocols in Toxicology 1999,9.2.1-9.2.10).

Myoglobin assay

This method is described by Motterlini and coworkers (Motterlini et al., Circulation Research, 2002, 90(2), 17e - 24).

The release of CO from the [Mo(CO)4(pyridine-imine)] complexes was determined in triplicates spectrophotometrically by measuring the conversion of deoxy-myoglobin (deoxy -Mb) to carbonmonoxy myoglobin (CO-Mb) as previously reported with small variations. 10 pl of a freshly prepared 2 mM solution of the complex in DMSO was added to 990 pl of the horse skeletal myoglobin solution in buffer previously reduced to deoxy -Mb by sodium dithionite. The final concentration was 20 pM of complex and changes in the Mb spectra were recorded between 500 and 600 nm over time at 23°C. Due to the colour of the complexes, an equally concentrated solution (20 pM) was prepared in buffer as a reference. The concentration of Mb was established between 50 and 60 pM and was determined for every experiment from the absorbance of the deoxy-Mb solution at 555 nm (a = 9.2 1 mM' ¹ cm' ¹ ). Two controls were recorded in duplicates: 0% CO-Mb (deoxy-Mb) and 100% CO-Mb, obtained by bubbling pure CO gas into the Mb.

CO release in sheep blood

Since compounds for pharmaceutical application will generally enter the blood stream, the stability was studied in whole sheep blood in Alsever’s solution. The CO release was determined by measuring the CO-haemoglobin levels with a whole blood oximeter (Avoximeter® 4000, Instrumentation Laboratory). In a single experiment, the Avoximeter provides the total amount of hemoglobin (g dl' ¹ ) in the sample and the percentage of O2-Hb, CO- Hb and Met-Hb thereof. The oximeter has an accuracy of ±0.3 g dl' ¹ in the total amount of hemoglobin and ± 2% of the %COHb value measured (Bailey et al., Journal of Clinical Monitoring, 1997, 13, 191-198). This experiment is the closest to a biological or pharmaceutical study of the CO release in vitro. The concentrations were chosen as a dose of either 20 or 50 mg kg' ¹ of body weight which corresponds between 0.6 and 1.5 mM. With these doses, COHb levels between 30 and 50% could be obtained after 1 h incubation at 37°C. In this experiment, 0.5 CO equivalents correspond to a COHb level of around 15% of the total amount of hemoglobin, which is around 8 g/dl and therefore, the measured values are out of the error of the oximeter.

COHb in systemic circulation in mice

For each CORM, four CD-I mice were injected in the tail vein with a 30 mg/kg dose dissolved in 4.5% DMSO/ 9% Cremophor/saline. Blood was taken from the tail vein of the mice according to the following protocol. At 5 min and 60 min post injection blood was taken from mouse 1; at 10 and 90 min blood was taken from mouse 2; at 20 and 150 min blood was taken from mouse 3; at 30 and 240 min blood was taken from mouse 4. The value of %COHb was measured with the Avoximeter 4000 for each blood sample. An aliquot of this blood sample was weighed and treated with sulfosalicylic acid to liberate all the CO in the blood, including the CO from intact CORM, and quantifying the CO released with a GC-RCP chromatograph as described by Vreman and coworkers (Vreman, et al., Analytical Biochemistry 2005, 37/, 280-289).

Example 17: Methods of quantification of CORMs in vitro and in vivo.

Stability and quantification by HPLC

1 mg/ml solution of PyCa-CORM was prepared in 50% acetonitrile/water. Samples were taken and analyzed by HPLC method every 15 minutes for 60 minutes using the gradient method described in Figure 1 where water is Solvent A and acetonitrile is Solvent B. Small variations in the NCMe/Water ratio or addition of 0.1% TFA to the aqueous solvent were rarely necessary to improve the method. Peak areas can be used to quantify compound’s decay and retention times are used for peak identification.

Pharmacokinetics: clearance curve of PyCa-CORMs in vivo

Six mice were injected i.v. with 30 mg/kg of PyCa-CORM in 4.5%DMSO/9%Cremophor/Saline. Blood was collected through retroorbital bleeding into heparin-coated tubes at 1, 5, 15, 30, 45, and 60 minutes after dosing. Each mouse was bled once. Plasma was collected by centrifugation at 17,000 x g and 4°C for 5 minutes. The plasma was transferred to fresh tubes and treated with 1 ml of acetone to precipitate proteins. The samples were then centrifuged using the same conditions as described above. Supernatants were transferred to new tubes and evaporated to dryness under a gentle stream of nitrogen gas. The linear range of calibration curve and the dose administered made dilutions unnecessary. The resulting pellets were re-suspended in the original volume of 50% acetonitrile/water solution and analyzed by HPLC using the gradient described above and the appropriate calibration curves.

Example 18: Method to determine cytotoxicity

The cytotoxicity of the PyCa CORMs was evaluated in the murine macrophage RAW264.7 (EC ACC 91062702) cell line; A cell suspension of approximately 1.5 x 10 ⁶ cells per ml in a culture medium [DMEM (GIBCO, Cat. no. 41966) for RAW264.7 supplemented with 10% FBS (GIBCO, Cat. no. 10500) was seeded (100 pl) into the wells of a 96-well plate. The cytotoxicity was evaluated by incubating the cells for 24 h at 37 °C, 5% CO2, in the presence of 1, 3, 10, 30 and 100 pM of PyCa CORM dissolved in 10%DMSO in NaHCO ₃ O,1M (pH=8,4). Cell survival was determined using the colorimetric MTT assay, which involves the metabolization of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a yellow tetrazole, into the purple formazan crystals. After 24 h, the culture medium was removed and replaced by a fresh medium supplemented with 1 mg mL-1 MTT. The cells were incubated for 1 h at 37 °C, 5% CO2. The formazan crystals produced were solubilized with DMSO and the absorbance of the final solution was determined at 550 nm (BioRad microplate reader). Toxicity is indicated by a reduced purple color compared to the control.

Results

As shown in Figure 2 a series of PyCa CORMs release CO in a spontaneous manner at room temperature in the dark. The presence of different substituents on either the pyridine ring or the aromatic ring or in both can significantly change the amount of CO released as measured by the number of CO equivalents quantified in the headspace.

The ability of the same group of PyCa-CORMs to release CO to sheep blood at 37°C shows a very similar profile as represented in Figure 3. This strongly suggests that the rate of CO release is largely independent of effects of blood components, namely plasma proteins.

In both these experiments, the corresponding esters of the same PyCa-CORMs precipitated and delivered only < 0.5 equivalents of CO at 180 min post incubation.

Using the myoglobin assay to measure of the CO release profile of a similar series of PyCa-CORMs with aromatic amines, yields the results depicted in Figure 4. As can be seen, the number of CO equivalents released after 60 minutes incubation with deoxy -Mb falls within the same range observed in the spontaneous release in Hepes 7.4 in the dark. Again this suggests the existence of very small interactions between the PyCa-CORMs and proteins. Of course, small differences are present, since the order of the CO release values is not exactly the same for the tests in Figures 1-3. However, the overall picture does not change, namely the range of CO equivalents released by these CORMs in vitro.

For their therapeutic use it is important to see how these CO release profiles translate to an in vivo setting. Following the protocol described above, the evolution of systemic values of COHb after administration of a 30 mg/kg dose of a number of Pyca-CORMs were recorded. Table 2: Values of COHb in systemic circulation after i.v. administration of PyCa- CORMs at 30 mg/kg (measured by blood oximetry)

The data in Table 2 display three types of CO release profiles in vivo corresponding to three different ligand structures. ALF523 is a very stable compound in circulation which barely raises COHb levels above basal levels. The structure of its ligand contains a Py-CH=N-N-R function. The CORMs with a Py-CR=N-Ar (Ar = Aryl) function (R = H, CH3) are better CO releasers in vivo and at the therapeutically effective concentration (30 mg/kg) do not raise systemic COHb values above 10%. Lastly, the CORMs with amino acid groups in the structure Py-CH=N-C(R’)COOR (R = H, CH3) produce a higher elevation of systemic COHb namely up to Ih post administration. The values in Table 2 were taken from a larger study which has produced the data in Figures 5 and 6. Figure 5 shows the time evolution of the systemic COHb levels for four different arylamine derivatives. All of them result in COHb < 10% up to 4h post administration. The figure also reports the amount of CO per milligram of fresh blood contained in the blood sample that provided the COHb reading at the same time point. In this curve, the total CO measured by GC-RCP corresponds to that contained in COHb plus CO contained in intact CORM still present in the blood sample. Both curves are largely parallel. However, in the case of ALF826 there is a wider gap between the curves at early time points (<60 min) suggesting that this compound has a lower initial CO release rate than the other congeners, that is, there is more intact CORM in the blood samples. Figure 6 reveals the different in vivo CO release profile of the amino acid derivatives which, at the same concentration (30 mg/kg) raise the values of COHb well above the 10% level. This threshold has been taken as a reference since it has been considered safe by regulatory authorities in human trials. The therapeutic utility of these different profiles may depend on the indication under treatment with CO since it may demand different rates of CO exposure. Unexpectedly, the alanine derivatives ALF819 and ALF843 performed in essentially the same manner either as the ester or the carboxylic acid, respectively. The impact of the mode (i.p. vs. i.v. ) is also displayed in Figure 6 using ALF843. The initially different COHb values become leveled after 10 minutes post-administration.

In vitro half-life determinations

The half-life of the CORMs can be determined by any of the methods that enable the quantification of the CO released over time. In the case of compounds like the ones described here, which have four CO ligands per molecule, the half-life is taken as the time needed for the release of 0.5 equivalents of CO, to the headspace or to the scavenger which is being used to quantify CO release (Zhang, et al., Dalton Transactions, 2009, 4351-4358).

A comparative analysis of a group of aryl substituted PyCa-CORMs chosen to attempt obtain fine structure-activity relationships determined the half-life values of these CORMs under several in vitro different media, as shown in Table 3.

Table 3: Half-lives (ti/2) of PyCa-aryl CORMs incubated in several media allowing for the detection and quantification of the amounts of released CO. ti/2 is the time necessary for the release of 0.5 equivalents of CO.

Myo (pH 6.4) = Myoglobin assay; 20 mM complex in PBS pH=6.4 (1% DMSO) Myo (pH 7.4) = Myoglobin assay; 20 mM complex in PBS pH=7.4 (1% DMSO) GC-RCP (CO in headspace); in HEPES buffer, pH=7.4, dark (1% DMSO);

GC-RCP (CO in headspace); in fetal bovine serum (1% DMSO) COHb = Oximetry; sheep blood in Alsever’s solution (~1% DMSO)

As can be seen, half-life values depend on the medium used for their determination. The deoxy-myoglobin assay reveals a small dependence of the half-life with pH. At pH 6.4 most, but not all half-lives of the compounds tested are slightly shorter than those measured at physiologic pH 7.4. The ti/2 values determined by this method are clearly shorter than those obtained in Hepes at pH 7.4. Such acceleration of the initial decomposition rate can be most likely due to the scavenging of CO by deoxy -Mb. Interestingly, the ti/2 values measured in sheep blood are comparable to those measured in the Mb assay although somewhat longer.

The consistently short half-life of ALF846 agrees with expectations based on the bond weakening derived from the electron-withdrawing effect of the bromide substituent in the pyridine ring. On the contrary, the strong electron donating effect of the methoxy substituent at the same position in ALF841, does not result in a longer half-life. No electronic correlation of this type is apparent for this group of aryl substituted PyCa-CORMs.

The origin of the initial slower CO release rate of ALF826 in vivo (Figure 5) in these in vitro tests is not evident and reveals that the in vivo behavior of similar CORMs may be different enough to eventually distinguish their therapeutic efficacy.

Stability of CORMs by HPLC

For purposes of purity control as well as the quantification of PyCa-CORMs in studies like blood clearance, logP or biodistribution, the stability of the PyCa-CORMs was studied by HPLC. Since decomposition is medium dependent and takes off within minutes of incubation, a medium was sought that prevented such fast decomposition. A mixture of 50% NCMe /water was found to be adequate for these purposes. The stability of several PyCa-CORMs in this medium is depicted in some examples in Figure 7.

Pharmacokinetics: Plasma clearance curve of ALF843 in vivo

The plasma clearance curve for ALF843 in mice at the dose of 30 mg/kg is shown in Figure 8. The plasma clearance curve for ALF826 at the dose of 30 mg/kg is shown in Figure 9.

Cytotoxicity The CORMs featuring the PyCa ligands are almost non-toxic to RAW264.7 cells at a concentration of lOOpM (survival between 90-100%) as can be seen for the examples in Table 4.

Table 4: Cytotoxicity of PyCa CORMs measured with RAW.264.7 cells (MTT assay). Medium: 10%DMSO in NaHCO ₃ 0,lM (pH=8,4).

Example 19: Study of the protective effect of PyCa CORMs in the K/BxN serum-transfer model of Rheumatoid Arthritis

A set of CORMs were selected for testing in an animal model of chronic arthritis, the K/BxN mouse model. This model also enables the study of the anti-inflammatory properties of the 5 selected compounds in vivo. The molecules tested were ALF821, ALF826, ALF828, ALF843 and ALF844. Dexamethasone was used as a positive control in all experiments. The experiments were performed through the administration of each molecule in a daily dose of 30mg/Kg intraperitoneally, in groups of 5 mice. The administration of each molecule was maintained for a period of 36 days, and then the treatment was stopped for an additional period of 24 days after which all animals were sacrificed. This pause in the administration of CORMs allowed the study of the arthritis relapse. The tests performed included blood analysis, histological analysis of the paws, as well as spleen and lymph nodes analysis in different time points throughout the 60 days of this study. The results showed that ALF826 in particular pronouncedly reduced the inflammatory score and delayed the appearance of symptoms after stopping the administration of the compound (Figure 10). ALF826 also shows high drug efficiency.

Interestingly, this CORM (ALF826) was also the only molecule that was able to preserve the articular structure of front and hind paws, without any signs of inflammation, cellular infiltration and bone degradation, comparing with the other CORMs and also with Dexamethasone, as revealed by histological analysis (Figure 11). These results do not result from any dosing and administration protocol optimisation.