Pharmaceutical formulations and uses thereof

Abstract

The present disclosure relates generally to pharmaceutical formulations (e.g., lyophilized formulations or topical formulations) comprising a perforin inhibitor (e.g., a benzenesulfonamide). In particular embodiments, the pharmaceutical formulations of the present disclosure are useful for treating diseases or disorders associated with undesirable perforin activity.

Description

PHARMACEUTICAL FORMULATIONS AND USES THEREOFRelated Applications

[0001] This application claims priority from Australian Provisional Patent Application No. 2024902868 filed on 11 September 2024, the entire content of which is incorporated by reference.Field

[0002] The present disclosure relates generally to pharmaceutical formulations (e.g., lyophilized formulations or topical formulations) comprising a perforin inhibitor (e.g., a benzenesulfonamide). In particular embodiments, the pharmaceutical formulations of the present disclosure are useful for treating diseases or disorders associated with undesirable perforin activity.Background

[0003] Robust T cell-mediated immunity is critical for the health of all mammals, as it provides protection against intracellular pathogens, especially viruses, and some cancers. However, adverse clinical outcomes can arise when the immune system becomes overstimulated following infection (post-infectious immunopathology) or attacks normal selfantigens (autoimmunity). Similarly, transplant rej ection (e.g., solid organ or bone marrow stem cells across a histocompatibility barrier) is another setting in which cellular immunity results in adverse clinical outcomes. Currently, such adverse clinical outcomes are typically treated with broad spectrum immunosuppressant drugs, such as corticosteroids. However, these drugs are associated with a broad range of off-target effects, which cause considerable morbidity and, in some cases, can lead to mortality.

[0004] Perforin inhibitors have been developed to block perforin, the key mediator of tissue damage inflicted by CD8+cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Both cell types are able to kill target cells perceived to be dangerous by the immune system through a contact-dependent mechanism. In the case of CTLs, clonotypic receptors on the surface of the cell recognize a foreign or mutant peptide antigen sampled from the interior of the cell and presented on major histocompatibility complex (MHC) proteins of the target cell surface.

[0005] If the binding is sufficiently avid, receptor clustering and subsequent signalling result in rearrangement of the CTL actin cytoskeleton, and a stable immune synapse is formed with the target cell. Preformed, highly specialized cytotoxic secretory vesicles (CSVs) in the CTL cytoplasm are then recruited, migrate along the microtubular apparatus to the site of cellcell contact, and release a cocktail of cytotoxins into the immune synapse by exocytosis. As explained below, perforin sits at the apex of a complex molecular signaling cascade that then rapidly induces the target cell to undergo programmed cell death via apoptosis.

[0006] Benzenesulfonamides are a class of perforin inhibitors and potential therapeutics for a range of diseases and disorders associated with undesirable perforin activity. More specifically, benzenesulfonamides exhibit the ability to block granule exocytosis cell death path way in vivo (Spicer, J.A. et al. J. Med. Chem. 2022, 65, 21, 14305-14325). However, benzenesulfonamides may, in certain circumstances, exhibit low solubility and / or low stability towards ultraviolet (UV) radiation, thereby presenting challenges in the development of pharmaceutically suitable formulations and compositions.

[0007] Accordingly, there remains a need for the development of improved formulations comprising a perforin inhibitor (e.g., a benzenesulfonamide), which are adapted for use in clinical settings, including in the treatment of diseases and disorders associated with undesirable perforin activity.Summary

[0008] Provided herein are pharmaceutical formulations comprising a perforin inhibitor (e.g., a benzenesulfonamide). In particular embodiments, the pharmaceutical formulations of the present disclosure are designed for, as an example, use in the treatment of diseases and disorders associated with undesirable perforin activity.

[0009] Accordingly, in an aspect, the present disclosure provides a lyophilized formulation comprising Compound (I):(I), or a pharmaceutically acceptable salt, solvate, or hydrate thereof;an albumin; and a stabilizer, wherein the stabilizer is a surfactant.

[0010] In some embodiments, surfactant is a lipid or a polymer conjugated lipid.

[0011] In some embodiments, the stabilizer is a polymer conjugated lipid, and the polymer conjugated lipid is l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), l,2-distearoyl-rac-glycerol-3-methoxypoly ethylene glycol-2000 (DSG-PEG), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG), or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the surfactant is DSPE-PEG.

[0012] In some embodiments, the lyophilized formulation comprises a plurality of particles, wherein the plurality of particles each comprise the Compound (I), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, encapsulated by the stabilizer and / or the albumin.

[0013] In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 200 nm.

[0014] In some embodiments, the lyophilized formulation is prepared from an aqueous formulation.

[0015] In some embodiments, the aqueous formulation comprises Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and the albumin at a weight ratio of from about 1 : 1 to about 1 :20 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin).

[0016] In some embodiments, the aqueous formulation comprises Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and the stabilizer at a weight ratio of from about 10: 1 to about 1 :40 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : stabilizer). In some embodiments, the aqueous formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1 : 15 (stabilizer : albumin).

[0017] In another aspect, the present disclosure provides a pharmaceutical formulation comprising a lyophilized formulation, wherein lyophilized formulation has been reconstituted in one or more pharmaceutically acceptable carriers or diluents.

[0018] In some embodiments, the pharmaceutical formulation has a pH of from about 4 to about 10. In some embodiments, the pharmaceutical formulation has a pH of from about 4 to about 9. In some embodiments, the pharmaceutical formulation has a pH of from about 6 to about 10.

[0019] In some embodiments, the pharmaceutical formulation has an osmolality of from about 200 mOsm / L to about 900 mOsm / L. In some embodiments, the pharmaceutical formulation has an osmolality of from about 200 mOsm / L to about 500 mOsm / L.

[0020] In some embodiments, the pharmaceutical formulation comprises one or more pharmaceutically acceptable excipients or adjuvants.

[0021] In some embodiments, the pharmaceutical formulation is configured for parenteral administration.

[0022] In some embodiments, the pharmaceutical formulation is configured for intravenous, topical, or ophthalmic administration. In some embodiments, the pharmaceutical formulation is configured for intravenous administration. In some embodiments, the pharmaceutical formulation is configured for topical administration. In some embodiments, the pharmaceutical formulation is configured for ophthalmic administration.

[0023] In some embodiments, the pharmaceutical formulation comprises plurality of particles, wherein the plurality of particles each comprise Compound (I), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, encapsulated by the stabilizer and / or the albumin, and wherein the plurality of particles each have a Z-average particle size of from about 30 nm to about 300 nm following reconstitution.

[0024] In some embodiments, the pharmaceutically acceptable diluent comprises a saccharide (e.g., dextrose, sucrose, a polysaccharide (e.g., Dextran 40, Dextran 70, etc.), etc.), propylene glycol, glycerol, water, or any combination of the foregoing. In some embodiments, the pharmaceutically acceptable diluent comprises dextrose (e.g., 2.5% (w / v) dextrose or 5% (w / v) dextrose), propylene glycol, glycerol, or any combination of the foregoing.

[0025] In another aspect, the present disclosure provides a pharmaceutical formulation comprising Compound (I):or a pharmaceutically acceptable salt, solvate, or hydrate thereof; a solvent, wherein the solvent is 2-(2-ethoxyethoxy)ethan-l-ol; and a gelling agent, wherein the pharmaceutical formulation is a gel, and wherein the pharmaceutical formulation is configured for topical administration.

[0026] In some embodiments, the gelling agent cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxyethyl cellulose (HEC). In some embodiments, the gelling agent is hydroxypropyl methylcellulose (HPMC).

[0027] In some embodiments, the pharmaceutical composition comprises from about 0.1 wt.% to about 20 wt.% of Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the pharmaceutical composition comprises at least about 40 wt.% of the solvent. In some embodiments, the pharmaceutical composition comprises from about 10 wt.% to about 95 wt.% of the solvent. In some embodiments, the pharmaceutical composition comprises from about 0.1 wt.% to about 55 wt.% of the gelling agent. In some embodiments, the pharmaceutical composition comprises from about 1 wt.% to about 55 wt.% of the gelling agent. In some embodiments, the pharmaceutical composition comprises from about 1 wt.% to about 10 wt.% of the gelling agent.

[0028] In some embodiments, the pharmaceutical composition further comprises an ultraviolet (UV) absorption agent. In some embodiments, the pharmaceutical composition comprises from about 0.1 wt.% to about 20 wt.% of the UV absorption agent.

[0029] In some embodiments, the UV absorption agent absorbs UVA rays. In some embodiments, the UV absorption agent is avobenzone.

[0030] In some embodiments, the UV absorption agent is further defined as a first UV absorption agent, and the pharmaceutical formulation further comprises a second ultraviolet(UV) absorption agent, wherein the second UV absorption agent and the first UV absorption agent are different.

[0031] In some embodiments, the pharmaceutical formulation comprises from about 0.1 wt.% to about 20 wt.% of the second UV absorption agent.

[0032] In some embodiments, the second UV absorption agent absorbs UVB rays. In some embodiments, the second UV absorption agent is oxybenzone.

[0033] In some embodiments, the pharmaceutical formulation comprises the first UV absorption agent and the second UV absorption agent at a weight ratio of from about 5: 1 to about 1 :5 (first UV absorption agent : second UV absorption agent).

[0034] In another aspect, the present disclosure provides a pharmaceutical formulation as described herein for use in a method for treating a disease or disorder associated with undesirable perforin activity.

[0035] In another aspect, the present disclosure provides a method for treating a disease or disorder associated with undesirable perforin activity in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical formulation described herein.

[0036] In another aspect, the present disclosure provides use of the pharmaceutical formulation described herein in the manufacture of a medicament for treating a disease or disorder associated with undesirable perforin activity.

[0037] In some embodiments, the disease or disorder associated with undesirable perforin activity is selected from the group consisting of diabetes mellitus, Crohn's disease, colitis, inflammatory bowel disease, fibrosis and fibrotic disorders (e.g., scleroderma), Guillain-Barre syndrome, lupus erythematosus, psoriasis, pancreatitis, rheumatoid arthritis, sepsis, vasculitis and Wegener's granulomatosis, proliferative retinopathies, graft-versus-host disease, chronic or acute allograft rejection, infectious diseases, cancer and conditions associated with cytotoxic T lymphocyte- or natural killer cell-mediated immune pathology.Brief Description of the Drawings

[0038] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the accompanying drawings.

[0039] Figure 1 shows the size distribution of a plurality of particles in a reconstituted pharmaceutical formulation according to Batch 1 of Example 5. A graphical representation ofZ-average particle size (d.nm; x-axis) and intensity (%; y-axis) for a reconstituted pharmaceutical formulation comprising a plurality of particles according to Batch 1 of Example 5 is provided.

[0040] Figure 2 shows the size distribution of a plurality of particles in a microfluidized pharmaceutical formulation according to a Model Batch of Example 5. A graphical representation of Z-average particle size (d.nm; x-axis) and intensity (%; y-axis) before (darker plot) and after (lighter plot) microfluidization is provided.

[0041] Figure 3 shows the size distribution of a plurality of particles in a microfluidized pharmaceutical formulation according to Batch 2 of Example 5. A graphical representation of Z-average particle size (d.nm; x-axis) and intensity (%; y-axis) after microfluidization is provided.

[0042] Figure 4 shows the size distribution of a plurality of particles in reconstituted pharmaceutical formulations. A graphical representation of Z-average particle size (d.nm; x- axis) and intensity (%; y-axis) for reconstituted pharmaceutical formulations of a core of a lyophilized formulation (lighter plot) and a periphery of the lyophilized formulation (darker plot) according to Batch 2 of Example 5 is provided.

[0043] Figure 5 shows the size distribution of a plurality of particles in a microfluidized pharmaceutical formulation according to Batch 4 of Example 5. A graphical representation of Z-average particle size (d.nm; x-axis) and intensity (%; y-axis) after microfluidization is provided.

[0044] Figure 6 shows the size distribution of a plurality of particles in a reconstituted pharmaceutical formulation according to Batch 4 of Example 5. A graphical representation of Z-average particle size (d.nm; x-axis) and intensity (%; y-axis) after reconstitution is provided.

[0045] Figure 7 shows the size distribution of a plurality of particles in a microfluidized pharmaceutical formulation. A graphical representation of Z-average particle size (d.nm; x- axis) and intensity (%; y-axis) for a pharmaceutical formulation after microfluidization for three pass-throughs at 400 mL (darker plot) and after two additional pass-throughs at 600 mL (lighter plot) according to a Batch 5 of Example 5.

[0046] Figure 8 shows the size distribution of a plurality of particles in a reconstituted pharmaceutical formulation according to Batch 5 of Example 5. A graphical representation of Z-average particle size (d.nm; x-axis) and intensity (%; y-axis) after reconstitution is provided.

[0047] Figure 9 shows the average particle size for microfluidized pharmaceutical formulations according to Batches 3-5 of Example 5. A graphical representation of Z-average particle size (nm; y-axis) and derived count (Kcps; x-axis) is provided.

[0048] Figure 10 shows a series of high-performance liquid chromatography plots of a solution of Compound (I) according to Example 6 (A) before irradiation in a photochemical reactor; (B) after irradiation in the photochemical reactor for 1 day; and (C) after irradiation in the photochemical reactor for 3 days.

[0049] Figure 11 is a photographic representation of sample solutions of Compound (I) after ultraviolet (UV) irradiation according to Example 6.

[0050] Figure 12 is a graphical representation of the degradation of Compound (I) for the sample solutions of Compound (I) after ultraviolet (UV) irradiation according to Example 8.

[0051] Figure 13 is a graphical representation of ear thickness (mm; y-axis) and time (weeks post-infection (wpi); x-axis) measured from NOD scid gamma mice infected with Leishmania braziliensis . A first group of the NOD scid gamma mice was treated topically with Compound (I) and a diluent (black circular symbols). A second group of the NOD scid gamma mice was treated only with the diluent (white circular symbols). Error bars indicate mean + / - standard error of the mean (SEM). * p < 0.05.

[0052] Figure 14 is a graphical representation of pathology score (y-axis) and time (weeks post-infection (wpi); x-axis) measured from NOD scid gamma mice infected with Leishmania braziliensis. A first group of the NOD scid gamma mice was treated topically with Compound (I) and a diluent (black circular symbols). A second group of the NOD scid gamma mice was treated only with the diluent (white circular symbols). Error bars indicate mean + / - SEM. * p < 0.05.

[0053] Figure 15 is a photographic representation of a pinna of a NOD scid gamma mouse 5 weeks post-infection with Leishmania braziliensis. The NOD scid gamma mouse was treated with diluent only.

[0054] Figure 16 is a photographic representation of a pinna of a NOD scid gamma mouse five (5) weeks post-infection with Leishmania braziliensis. The NOD scid gamma mouse was treated with Compound (I) and a diluent.

[0055] Figure 17 is a graphical representation of parasite burden (Logio; y-axis) for NOD scid gamma mice 5 weeks post-infection with Leishmania braziliensis. A first group of the NOD scid gamma mice were treated topically with Compound (I) and a diluent (black bar). Asecond group of the NOD scid gamma mice were treated only with the diluent (white bar). Each circular symbol represents a single mouse (4 mice evaluated per group).Detailed Description

[0056] As generally described herein, the present disclosure provides pharmaceutical formulations comprising a perforin inhibitor (e.g., a benzenesulfonamide) designed for, as an example, use in the treatment of diseases and disorders associated with undesirable perforin activity.Definitions

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0058] The articles "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an ultraviolet absorption agent" includes a single ultraviolet absorption agent, as well as two or more ultraviolet absorption agents.

[0059] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0060] The term “about”, as used herein, means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the statedvalue by a variance of 10%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”.

[0061] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment, unless expressly stated otherwise.

[0062] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0063] The term “optionally” is used herein to mean that the subsequent described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiment in which the event or circumstance occurs as well as embodiments in which it does not.

[0064] As used herein, the terms "perforin activity", "biological activity of perforin" and the like refer to the cytolytic activity of a perforin polypeptide; that is, its ability to bind to a target cell membrane and polymerise into pore-like transmembrane channels leading to cell lysis. The activity also includes the capacity to synergise with other toxins such as granule toxins and other molecules to induce apoptosis. The target cell can be any cell that is capable of being lysed by native perforin.

[0065] The biological activity of perforin can be assessed by a person skilled in the art by any number of means known in the art including, but not limited to, the measurement of target cell lysis, the delivery of granzyme B molecules into the target cell, the measurement of target cell membrane disruption (such as by changes in ion transport), the induction of apoptosis in the target cell, the modification of vesicular trafficking and the general assessment of targetcell death. The target cell may be a red blood cell (RBC) and hence a common means of measuring perforin activity is by a RBC lysis test. It may also be any nucleated cell.

[0066] The terms “perforin”, “cytolysin”, “pore-forming protein (pfp)” and “C9-like protein” are used interchangeably herein and encompass perforin polypeptides and fragments thereof in various forms, including naturally occurring or synthetic variants. Examples of perforins encompassed by the present invention include human perforin. Also encompassed by the present invention are mouse and rat perforin isoforms, although perforins derived from other species, including those that may be made by lower organisms such as bacteria, are also envisaged.

[0067] As used herein, the term "native perforin" refers to a perforin polypeptide molecule having an amino acid sequence that occurs in nature (e.g., a natural protein). Native perforin, or naturally occurring perforin, may be identified as one of the main constituents of cytocidal granules, is found to migrate with a molecular mass of approximately 66 kDa upon reduction and SDS-polyacrylamide gel electrophoresis, and migrates more slowly under non-reducing conditions (70-75 kDa), suggestive of a tightly disulphide-bonded structure in its native form. In the presence of calcium ions (Ca2+), perforin monomers aggregate into tubular structures that span the lipid bilayer, producing circular lesions (varying between 6 and 20 nm in diameter) that are thought to grow in diameter through the progressive recruitment of additional monomers.

[0068] The compounds of the present disclosure have been identified by their ability to inhibit perforin activity, and as such, may be referred to herein as “inhibitors”, “perforin inhibitors”, “inhibitors of perforin activity”, and the like.

[0069] The term "pharmaceutically acceptable" as applied to salts of the present invention and / or used in methods of the present invention refers to salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic, or a like negative response that exceeds a reasonable risk / therapeutic benefit ratio. A person skilled in the art will understand that a pharmaceutically acceptable salt is a salt that is suitable for administration to a patient. Accordingly, the present disclosure also extends to a pharmaceutically acceptable salt of any one of the compounds of the present invention.

[0070] Pharmaceutically acceptable salts are generally known in the art, and in the case of the present invention, include relatively non-toxic, organic or inorganic salts of the compoundsof the present invention. Examples of such salts include, but are not limited to, acid addition salts such as hydrochloride salts, sulfate salts, bisulfate salts, borate salts, nitrate salts, acetate salts, phosphate salts, hydrobromide salts, laurylsulfonate salts, glucoheptonate salts, oxalate salts, oleate salts, laurate salts, stearate salts, palmitate salts, valerate salts, benzoate salts, naphthylate salts, mesylate salts, tosylate salts, citrate salts, lactate salts, maleate salts, succinate salts, tartrate salts, fumarate salts, and the like. In addition, pharmaceutically acceptable salts also include basic salts such as alkali metal salts, alkaline earth salts, and ammonium salts. For example, pharmaceutically acceptable basic salts include salts of aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and the like. In addition, organic salts may also be used including, e.g., salts of lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine and tris. The basic nitrogen-containing groups in the compounds of the present invention can be quatemized with various organic agents including, e.g., alkyl halides (such as lower alkyl halide including methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl and diamyl sulfates).

[0071] The pharmaceutically acceptable salts of the compounds of the present invention also can exist in the form of solvates, e.g., with water, methanol, ethanol, dimethylformamide, ethyl acetate, and the like, and mixtures thereof.

[0072] The term "optionally substituted" as used throughout the specification denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more non-hydrogen substituent groups. In certain embodiments the substituent groups are one or more groups independently selected from the group consisting of halogen, hydroxyl, amino, alkylamino, alkenylamino, cycloalkylamino, cycloalkenylamino, arylamino, heteroaryl, heteroarylamino, heterocyclylamino, aminoarylamino, aminoheteroarylamino, aminoheterocyclylamino, tetrahydropyridinylamino, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, azetidinylamino, pyrrolidinylamino, piperidinylamino, piperazinylamino, azetidinylcarbonylamino, pyrrolidinylcarbonylamino, piperidinylcarbonylamino, piperazinylcarbonylamino, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, aminoalkoxy, aminoalkenyloxy, aminoalkynyloxy, aminocycloalkoxy, aminocycloalkenyloxy,aminoaryloxy, aminoheteroaryloxy, azetidinyloxy, pyrrolidinyloxy, piperidinyloxy, or piperazinyloxy.

[0073] "Acyl" means an R-C(=O)- group in which the R group may be an alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group as defined herein. Examples of acyl include acetyl and benzoyl.

[0074] "Alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a Ci-Cioalkyl, more preferably a Ci-Csalkyl, most preferably Ci-Cealkyl unless otherwise noted. Examples of suitable straight and branched Ci-Cealkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like.

[0075] "Alkylene" refers to divalent alkyl groups preferably having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms, and even more preferably 1 to 3 carbon atoms. Examples of such alkylene groups include methylene (-CH2-), ethylene (-CH2CH2-), and the propylene isomers (e.g., -CH2CH2CH2- and -CH(CH3)CH2-), and the like.

[0076] "Alkenyl” means an aliphatic hydrocarbon group containing at least one carboncarbon double bond and which may be straight or branched, preferably a C2-Cioalkenyl, more preferably a C2-Csalkenyl, most preferably C2-Cealkenyl. The group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl.

[0077] " Alkynyl” means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched, preferably a C2-Cioalkynyl, more preferably a C2-Csalkynyl, most preferably C2-Cealkynyl.

[0078] "Alkylamino" includes both mono-alkylamino and dialkylamino, unless specified. "Mono-alkylamino" means an alkyl-NH- group, in which alkyl is as defined herein. "N,N- dialkylamino" means a (alkyl)2N- group, in which each alkyl may be the same or different and are each as defined herein for alkyl. The alkyl group is preferably a Ci-Cioalkyl group. The group is bonded to the remainder of the molecule through the nitrogen atom.

[0079] "Alkenylamino" includes both mono-alkenylamino and dialkenylamino, unless specified. "Mono-alkenylamino" means an alkenyl-NH- group, in which alkenyl is as defined herein. The alkenyl group is preferably a C2-Cioalkenyl group. The group is bonded to the remainder of the molecule through the nitrogen atom.

[0080] "Alkoxy" as a group or part of a group refers to an alkyl-O- group in which alkyl is as defined herein. Preferably the alkoxy is a Ci-Cioalkoxy. Examples include, but are not limited to, methoxy and ethoxy.

[0081] "Aminoalkoxy" refers to an alkoxy group as defined herein, further substituted with at least one amine. Preferred aminoalkoxy groups are Ci-Cioaminoalkoxy groups.

[0082] "Alkenyloxy" refers to an alkenyl-O- group in which alkenyl is as defined herein. Preferred alkenyloxy groups are C2-Cioalkenyloxy groups.

[0083] "Aminoalkenyloxy" refers to an alkenyl-O- group as defined herein, further substituted with at least one amine. Preferred aminoalkenyloxy groups are C2- Cioaminoalkenyloxy groups.

[0084] "Alkynyloxy" refers to an alkynyl-O- group in which alkynyl is as defined herein. Preferred alkynyloxy groups are C2-Cioalkynyloxy groups.

[0085] "Aminoalkynyloxy" refers to an alkynyl-O- group as defined herein, further substituted with at least one amine. Preferred aminoalkynyloxy groups are C2- Cioaminoalkynyloxy groups.

[0086] "Aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (eg., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), preferably having from 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl and the like.

[0087] "Arylamino" includes both mono-arylamino and di-arylamino unless specified. Mono-arylamino means a group of formula aryl-NH-, in which aryl is as defined herein. “N,N- diarylamino” means a group of formula (aryl)iN- where each aryl may be the same or different and are each as defined herein for aryl.

[0088] "Aminoarylamino" refers to a group of formula (NH2)n-aryl-NH-, in which arylamino is as defined herein, further substituted with at least one amine at the ortho-, meta- or para position.

[0089] "Aryloxy" refers to an aryl-O- group in which the aryl is as defined herein. Preferably the aryloxy is a Ce-Cioaryloxy.

[0090] "Aminoaryloxy" refers to a group of formula (NH2)n-aryl-O-, in which aryloxy is as defined herein, further substituted with at least one amine at the ortho-, meta- or para position.

[0091] "Cycloalkyl" refers to a saturated monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 10 carbons per ring, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane.

[0092] "Cycloalkenyl" refers to a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl.

[0093] "Cycloalkylamino" refers to a cycloalkyl-NH- group in which cycloalkyl is as defined herein. Preferably the cycloalkylamino is a Cs-Ciocycloalkylamino.

[0094] "Cycloalkenylamino" refers to a cycloalkenyl-NH- group in which the cycloalkenyl is as defined herein. Preferably the cycloalkenylamino is a C3- Ciocycloalkenylamino.

[0095] "Cycloalkoxy" refers to a cycloalkyl-O- group in which cycloalkyl is as defined herein. Preferably the cycloalkoxy is a Cs-Ciocycloalkoxy. Examples include, but are not limited to, cyclopropanoxy and cyclobutanoxy.

[0096] "Aminocycloalkoxy" refers to a cycloalkoxy group is as defined herein, further substituted on one or more of the available carbon atoms with at least one amine. Preferably the aminocycloalkoxy is a Cs-Cioaminocycloalkoxy. The group is bonded to the remainder of the molecule through the oxygen atom.

[0097] "Cycloalkenyloxy" refers to a cycloalkoxy group defined herein containing at least one carbon-carbon double bond.

[0098] "Aminocycloalkenyloxy" refers to an aminocycloalkloxy group defined herein containing at least one carbon-carbon double bond.

[0099] "Heteroaryl" either alone or part of a group refers to groups containing an aromatic ring (preferably a 5, 6, 9, 10 or 11 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur. Examples of heteroaryl include thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, IH-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-, 3- or 4- pyridyl, 2-, 3-, 4-, 5-, or 8- quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or 3- indolyl, and 2-, or 3-thienyl, and includes benzofused heteroaryl, such as benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothi azole, and naphtho[2,3-b]thiophene.

[0100] "Heteroarylamino" refers to a heteroaryl -NH- group in which the heteroaryl is as defined herein. Preferably the heteroarylamino is a C2-Cioheteroarylamino.

[0101] "Aminoheteroarylamino" refers to a (NH2)n-heteroaryl-NH- group in which the heteroarylamino is as defined herein, further substituted at one or more of the ring members with at least one amine. Preferably the aminoheteroarylamino is a C2-Cioaminoheteroarylamino.

[0102] "Heteroaryloxy" refers to a heteroaryl-O- group in which the heteroaryl is as defined herein.

[0103] "Aminoheteroaryloxy" refers to a (NH2)n-heteroaryl-O- group in which the heteroaryloxy is as defined herein, further substituted at one or more of the ring members with at least one amine.

[0104] "Heteroarylcarbonylamino" refers to a heteroaryl-C(O)-NH- group in which the heteroaryl is as defined herein.

[0105] "Heterocyclyl" or "heterocyclic" refers to a saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 11 membered, more preferably 4 to 7 membered or 9-11 membered. Examples of suitable heterocyclyl substituents include aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thistanyl, pyrrolinyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidinyl, thiazolidinyl, piperazinyl, tetrahydropyridinyl, morpholino, thiomorpholinyl, 1,3- diazapane, 1,4-diazapane, 1,4-oxazepane, and 1,4-oxathiapane, and includes benzofused compounds such as inddinyl, isoindolinyl, oxoisoindolinyl, isoquinolinyl, and quinolinyl.

[0106] "Heterocyclyloxy" refers to a heterocyclyl-O- group in which the heterocycloalkyl is as defined herein.

[0107] "Heterocyclylamino" refers to a heterocyclyl-NH- group in which the heterocycloalkyl is as defined herein.

[0108] "Aminoheterocyclylamino" refers to a NH2-heterocyclyl-NH- group in which the heterocycloalkylamino is as defined herein, further substituted with an amine at one of the ring members.

[0109] "Heterocyclylcarbonylamino" refers to a heterocyclyl-C(O)-NH- group in which the heterocyclyl is as defined herein. Examples of suitable heterocyclylcarbonylamino substituents include azetidinylcarbonylamino, piperidinylcarbonylamino and piperazinylcarbonylamino.

[0110] "Alkylheterocyclyl" refers to an alkyl-heterocyclyl- group in which alkyl and heterocyclyl groups are as defined herein. Preferably the alkyl is a Ci-Cealkyl group bound to the heterocyclyl group via either a carbon or heteroatom. The heterocyclic ring is preferably from 5 to 11 membered.[oni] It is understood that included in the family of compounds of Formula (A) and related formulae are isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers in "E" or "Z" configurational isomer or a mixture of E and Z isomers. It is also understood that some isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and / or chemical methods and by those skilled in the art.

[0112] Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and / or mixtures of enantiomers and / or diastereomers. All such single stereoisomers, racemates and mixtures thereof, are intended to be within the scope of the subject matter described and claimed.

[0113] The terms "lyophilized formulation" and "freeze-dried formulation" are used interchangeably herein to refer to a lyopholisate obtained after lyophilization of a formulation comprising a solvent (e.g., an organic solvent, water, or mixtures thereof). The lyopholisate may be a powder and / or may have a cake-like appearance (i.e., a "lyophilized cake").

[0114] As used herein, the term "topical formulation" refers to a pharmaceutical formulation configured to be applied to a subject's skin, nail, or hair.

[0115] As used herein, "wt.%" refers to weight percent. For particular components of a pharmaceutical formulation: > Weight of a particular component of the pharmaceutical formulation (g)1 nnTotal weight of the pharmaceutical formulation (g)Lyophilized formulations

[0116] In an aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer.

[0117] The perforin inhibitor may be any compound that inhibits perforin activity. In some embodiments, the perforin inhibitor is a compound provided in PCT Publication No. WO 2014 / 028968 Al, the entire contents of which is incorporated herein by reference. In some embodiments, the perforin inhibitor is a compound described in Spicer, J. A. et al. Inhibition of the Cytolytic Protein Perforin Prevents Rejection of Transplanted Bone Marrow Stem Cells in Vivo. J. Med. Chem. 2020, 63, 5, 2229-2239, the entire contents of which is incorporated herein by reference.

[0118] In some embodiments, the perforin inhibitor is a compound of Formula (A):whereinRing A is selected from a 6-10 membered aryl, 5-6 membered cycloalkyl, 5-6 membered heteroaryl or 5-6 membered heterocyclyl, wherein the heteroaryl and heterocyclyl rings comprise at least one heteroatom selected from N, O or S; and wherein the aryl, cycloalkyl, heteroaryl or heterocyclyl rings are optionally substituted with 1 to 3 substituents selected from halo, nitro, cyano, -Ci-Cealkyl, -Ci-Ceaminoalkyl, -Ci-Cehydroxyalkyl, -haloCi- Cealkyl, -Ci-Cealkoxyl, -haloCi-Cealkoxyl, heteroaryl, aryl, hydroxyl, -C(O)Ci-Cealkyl, - OC(O)Ci-C6alkyl, -CH2OC(O)Ci-C6alkyl, -C(O)OCi-C6alkyl, -NHC(O)Ci-C6alkyl, - NHS(O)2Ci-C6alkyl, -S(O)2Ci-C6, -S(O)2NH2, and -C(O)NJJ;Ring B is a 6-10 membered arylene or a 5-6 membered heteroarylene comprising at least one heteroatom selected from N, O or S; and wherein the aryl or heteroaryl is optionally substituted with one or more substituents selected from -NJJ, -OJ, halo, -Ci-Cealkyl, -haloCi- Cealkyl, -Ci-Cealkoxy, -haloCi-Cealkoxy, and -C(O)NJJ;Ring C is selected from a 5-10 membered heteroarylene or a 5-10 membered heterocyclene, each comprising at least one heteroatom selected from N, S and O;Ring D is an optionally substituted 6-11 membered heterocyclyl or optionally substituted 6-11 membered heteroaryl comprising at least one heteroatom selected from N or O;L is a linker selected from branched or unbranched C1-C4 alkylene, -S(0)2-NH-, -C(O)- NH-, -NH-C(O)-NH-, -S(O)2-NH-C(O)-NH-, -S(O)2-NH-C(O) -C(O)-NH-C(S)-NH- and - CH=CH-; wherein Rings B and C, and Rings C and D, are connected to each other via a C-C bond at any of the available C atoms on each respective ring; andJ in each occurrence is independently selected from H, optionally substituted Ci- Cealkyl or optionally substituted haloCi-Cealkyl; and pharmaceutically acceptable salts, solvates, and hydrates thereof.

[0119] In some embodiments, the perforin inhibitor is a compound as set forth in Table 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0120] In some embodiments, the perforin inhibitor is Compound (I):or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0121] In some embodiments, the perforin inhibitor is Compound (I). In other embodiments, the perforin inhibitor is a pharmaceutically acceptable salt of Compound (I). In some embodiments, the perforin inhibitor is a solvate of Compound (I). In other embodiments, the perforin inhibitor is a hydrate of Compound (I).

[0122] As used herein, the term "albumin" refers collectively to the family of globular, water-soluble proteins, and fragments and derivatives thereof. In some embodiments, the albumin is a serum albumin. As used herein, "serum albumin" refers to albumin proteins from vertebrate blood. Suitable examples of serum albumin include, by way of non-limiting example, human serum albumin (HSA), bovine serum albumin (BSA), and rat serum albumin (RSA).

[0123] In some embodiments, the albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RS A), or ovalbumin (OVA). In some embodiments, the albumin is HSA or BSA. In some embodiments, the albumin is HSA. In some embodiments, the albumin is BSA. In some embodiments, the albumin is RS A. In some embodiments, the albumin is OVA.

[0124] In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 : 1 to about 1 :20, from about 1 :5 to about 1 : 15, or from about 1 : 10 to about 1 : 15 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 : 1 to about 1 :20 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 :5 to about 1 : 15 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 : 10 to about 1 : 15 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 : 11 to about 1 : 14 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin).

[0125] Without wishing to be bound by theory or mode of operation, it is believed that the albumin binds and / or encapsulates the perforin inhibitor in its molecular form and as nanoparticles, thereby improving colloidal stability of a reconstituted formulation formed from the lyophilized formulation and also improving the solubility of the perforin inhibitor in the reconstituted formulation.

[0126] The terms "stabilizer" and "stabilizing agent" may be used interchangeably to refer to an agent that promotes colloidal stability of a reconstituted formulation formed from the lyophilized formulation. In some embodiments, the stabilizer is a surfactant (e.g., a non-ionicsurfactant, an ionic surfactant, a zwitterionic surfactant, or any combination thereof). In some embodiments, the surfactant is a lipid, a polymer conjugated lipid, or any combination thereof. In such embodiments, the stabilizer may be amphiphilic. In this manner, the stabilizer may form liposomes and / or micelles that encapsulate the perforin inhibitor and / or the albumin, thereby promoting colloidal stability of a reconstituted formulation formed from the lyophilized formulation.

[0127] Suitable surfactants would be known to persons skilled in the art, illustrative examples of which include sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate, etc.), sorbitan trioleate, glycerine fatty acid esters (e.g., glycerine monocaprylate, glycerine monomyristate, glycerine monostearate, etc.), polyglycerine fatty acid esters (e.g., decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate, etc.), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tri stearate, etc.), polyoxyethylene sorbitol fatty acid esters (e.g., polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate, etc.), polyoxyethylene glycerine fatty acid esters (e.g., polyoxyethylene glyceryl monostearate, etc.), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether, etc.), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether, etc.), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether, etc.), polyoxyethylene hydrogenated castor oils (e.g. polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, etc.), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitol beeswax, etc.), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin, etc.), and polyoxyethylene fatty acid amides (e.g., polyoxyethylene stearic acid amide, etc.), C10-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate, etc.), polyoxyethylene C10-C18 alkyl ether sulfate with an average of 2 to 4 moles of ethylene oxide units added (e.g., sodium polyoxyethylene lauryl sulfate, etc.), and Cl -Cl 8 alkyl sulfosuccinate ester salts (e.g., sodium lauryl sulfosuccinate ester); and natural surfactants such as lecithin, glycerophospholipid, sphingophospholipids (e.g., sphingomyelin), and sucrose esters of C12-C18 fatty acids.

[0128] In some embodiments, the stabilizer is a lipid. In some embodiments, the stabilizer is a phospholipid. Suitable phospholipids would be known to persons skilled in the art, illustrative examples of which include soybean phosphatidylcholine (SC), hydrogenated soybean phosphatidylcholine (HSPC), egg sphingomyelin (ESM), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol (DOPG), distearoyl phosphatidylglycerol (DSPG), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), dioleoyl phosphatidylserine (DOPS), distearoyl phosphatidylethanolamine (DSPE), stearoyl oleoyl phosphatidylcholine (SOPC), l-stearioyl-2- oleoyl-phosphatidy ethanolamine (SOPE), N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), and derivatives of any of the foregoing phospholipids.

[0129] In some embodiments, the stabilizer is a polymer conjugated lipid. As used herein, the term "polymer conjugated lipid" refers to a lipid that is conjugated to a polymer. Suitable polymer conjugated lipids would be known to persons skilled in the art, illustrative examples of which include l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), l,2-distearoyl-rac-glycerol-3-methoxypoly ethylene glycol-2000 (DSG-PEG), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG), and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the stabilizer is DSPE-PEG. In some embodiments, the stabilizer is DSG-PEG. In some embodiments, the stabilizer is DMPE-PEG. In some embodiments, the stabilizer is DMPE-PEG. In some embodiments, the stabilizer is DMG-PEG. In some embodiments, the stabilizer is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0130] In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 10: 1 to about 1 :40, from about 1 : 1 to about 1 : 15, from about 1 : 1 to about 1 : 10, or from about 1 : 1 to about 1 :5 (perforin inhibitor : stabilizer). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weightratio of from about 10: 1 to about 1 :40 (perforin inhibitor : stabilizer). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1 : 1 to about 1 : 15 (perforin inhibitor : stabilizer). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1 : 1 to about 1 : 10 (perforin inhibitor : stabilizer). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1 : 1 to about 1 :5 (perforin inhibitor : stabilizer). In some embodiments, the lyophilized formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1 :2 to about 1 :5 (perforin inhibitor : stabilizer).

[0131] In some embodiments, the lyophilized formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1 : 15, from about 1 : 1 to about 1 : 10, or from about 1 : 1 to about 1 :5 (stabilizer : albumin). In some embodiments, the lyophilized formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1 : 15 (stabilizer : albumin). In some embodiments, the lyophilized formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1 : 10 (stabilizer : albumin). In some embodiments, the lyophilized formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1 :5 (stabilizer : albumin). In some embodiments, the lyophilized formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 :2 to about 1 :5 (stabilizer : albumin).

[0132] In some embodiments, the lyophilized formulation comprises a plurality of particles. The plurality of particles each comprise the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) encapsulated by the stabilizer and / or the albumin. In some embodiments, the plurality of particles have a Z-average particle size of from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, from about 1 nm to about 150 nm, or from about 50 nm to about 150 nm. In some embodiments, the plurality of particles have a Z-average particle size of from about 1 nm to about 300 nm. In some embodiments, the plurality of particles have a Z-average particle size of from about 1 nm to about 200 nm. In some embodiments, the plurality of particles have a Z-average particle sizeof from about 1 nm to about 150 nm. In some embodiments, the plurality of particles have a Z-average particle size of from about 50 nm to about 150 nm.

[0133] In some embodiments, the plurality of particles are each independently a liposome or a micelle. In some embodiments, the plurality of particles are each independently a liposome. In some embodiments, the plurality of particles are each independently a micelle.

[0134] In some embodiments, the lyophilized formulation further comprises a saccharide (e.g., dextrose, sucrose, mannitol, a polysaccharide (e.g., Dextran 40, Dextran 70, etc.), etc.). In some embodiments, the lyophilized formulation comprises mannitol.

[0135] In an aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the stabilizer is a surfactant.

[0136] In another aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RS A), or ovalbumin (OVA). In some embodiments, the stabilizer is a surfactant. In some embodiments, the surfactant is a lipid or a polymer conjugated lipid.

[0137] In a further aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is a compound as set forth in Table 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the stabilizer is a surfactant. In some embodiments, the surfactant is a lipid or a polymer conjugated lipid.

[0138] In yet another aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is a compound as set forth in Table 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RS A), or ovalbumin (OVA). The stabilizer is a polymer conjugated lipid. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0139] In an aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is a compound as set forth in Table 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), or ovalbumin (OVA). The stabilizer is 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE- PEG), l,2-distearoyl-rac-glycerol-3-methoxypoly ethylene glycol-2000 (DSG-PEG), 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3 -methoxypoly ethylene glycol-2000 (DMG- PEG), or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0140] In another aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The stabilizer is a lipid or a polymer conjugated lipid.

[0141] In another aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), or ovalbumin (OVA). The stabilizer is l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), 1,2-distearoyl-rac- glycerol-3 -methoxypoly ethylene glycol-2000 (DSG-PEG), l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), or 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0142] In another aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The albumin is human serum albumin (HSA) or bovine serum albumin (BSA). The stabilizer is l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), l,2-distearoyl-rac-glycerol-3-methoxypoly ethylene glycol-2000(DSG-PEG), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG), or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0143] In an aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The albumin is bovine serum albumin (BSA). The stabilizer is 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG).

[0144] In another aspect, the present disclosure provides a lyophilized formulation. The lyophilized formulation comprises a perforin inhibitor and an albumin. The lyophilized formulation comprises a plurality of particles. The plurality of particles each comprise the perforin inhibitor encapsulated by the albumin. The plurality of particles each have a Z-av erage particle size of from about 1 nm to about 300 nm.

[0145] In some embodiments, the perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0146] In some embodiments, the albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RS A), or ovalbumin (OVA). In some embodiments, the albumin is human serum albumin (HSA) or bovine serum albumin (BSA). In some embodiments, the albumin is BSA.

[0147] In some embodiments, the lyophilized formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 : 1 to about 1 :20, from about 1 :5 to about 1 : 15, or from about 1 : 10 to about 1 : 15 (perforin inhibitor : albumin). In some embodiments, the lyophilized formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 : 1 to about 1 :20 (perforin inhibitor : albumin). In some embodiments, the lyophilized formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 :5 to about 1 : 15 (perforin inhibitor : albumin). In some embodiments, the lyophilized formulation comprises the perforin inhibitor and the albumin at a weight ratio offrom about 1 : 10 to about 1 : 15 (perforin inhibitor : albumin). In some embodiments, the lyophilized formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 : 11 to about 1 : 14 (perforin inhibitor : albumin).

[0148] In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 300 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 200 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 150 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 50 nm to about 150 nm.

[0149] In some embodiments, the lyophilized formulation is free of a stabilizer.

[0150] In an embodiment, the lyophilized formulation disclosed herein is prepared from an aqueous formulation. In accordance with this embodiment, the aqueous formulation comprises the perforin inhibitor, the albumin, and the stabilizer.

[0151] The perforin inhibitor may be any perforin inhibitor described herein. In some embodiments, the perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0152] In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 20 wt.%, from about 0.01 wt.% to about 10 wt.%, from about 0.01 wt.% to about 5 wt.%, or from about 0.01 wt.% to about 1 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 20 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the aqueous formulation comprises from about about 0.01 wt.% to about 10 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 5 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, theaqueous formulation comprises from about 0.01 wt.% to about 1 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the aqueous formulation comprises from about 0.1 wt.% to about 1 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the aqueous formulation comprises from about 0.1 wt.% to about 0.5 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof).

[0153] In an embodiment the albumin is any albumin described herein. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RS A), or ovalbumin (OVA). In some embodiments, the albumin is HSA or BSA. In some embodiments, the albumin is HSA. In some embodiments, the albumin is BSA. In some embodiments, the albumin is RSA. In some embodiments, the albumin is OVA.

[0154] In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 20 wt.%, from about 0.01 wt.% to about 10 wt.%, or from about 0.01 wt.% to about 5 wt.% of the albumin. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 20 wt.% of the albumin. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 10 wt.% of the albumin. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 5 wt.% of the albumin. In some embodiments, the aqueous formulation comprises from about 1 wt.% to about 5 wt.% of the albumin.

[0155] In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 : 1 to about 1 :20, from about 1 :5 to about 1 : 15, or from about 1 : 10 to about 1 : 15 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin). In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 : 1 to about 1 :20 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin). In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 :5 to about 1 : 15 (Compound (I) or a pharmaceutically acceptable salt, solvate,or hydrate thereof : albumin). In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 : 10 to about 1 : 15 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin). In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the albumin at a weight ratio of from about 1 : 11 to about 1 : 14 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin).

[0156] In an embodiment, the stabilizer is any stabilizer described herein. In some embodiments, the stabilizer is a lipid, a polymer conjugated lipid, or any combination thereof. In some embodiments, the stabilizer is a lipid. In some embodiments, the stabilizer is a polymer conjugated lipid. In some embodiments, the stabilizer is l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG).

[0157] In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 20 wt.%, from about 0.01 wt.% to about 10 wt.%, or from about 0.01 wt.% to about 5 wt.% of the stabilizer. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 20 wt.% of the stabilizer. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 10 wt.% of the stabilizer. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 5 wt.% of the stabilizer. In some embodiments, the aqueous formulation comprises from about 0.1 wt.% to about 2 wt.% of the stabilizer.

[0158] In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1 : 1 to about 1 : 15, from about 1 : 1 to about 1 : 10, or from about 1 : 1 to about 1 :5 (perforin inhibitor : stabilizer). In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1 : 1 to about 1 : 15 (perforin inhibitor : stabilizer). In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1 : 1 to about 1 : 10 (perforin inhibitor : stabilizer). In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceuticallyacceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1 : 1 to about 1 :5 (perforin inhibitor : stabilizer). In some embodiments, the aqueous formulation comprises the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) and the stabilizer at a weight ratio of from about 1:2 to about 1 :5 (perforin inhibitor : stabilizer).

[0159] In some embodiments, the aqueous formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1 : 15, from about 1 : 1 to about 1 : 10, or from about 1 : 1 to about 1 :5 (stabilizer : albumin). In some embodiments, the aqueous formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1 : 15 (stabilizer : albumin). In some embodiments, the aqueous formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1: 10 (stabilizer : albumin). In some embodiments, the aqueous formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1:5 (stabilizer : albumin). In some embodiments, the aqueous formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 :2 to about 1 :5 (stabilizer : albumin).

[0160] In some embodiments, the aqueous formulation comprises a plurality of particles. The plurality of particles each comprise the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) encapsulated by the stabilizer and / or the albumin. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, from about 1 nm to about 150 nm, or from about 50 nm to about 150 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 300 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 200 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 150 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 50 nm to about 150 nm.

[0161] In some embodiments, the plurality of particles are each independently a liposome or a micelle. In some embodiments, the plurality of particles are each independently a liposome. In some embodiments, the plurality of particles are each independently a micelle.

[0162] In some embodiments, the aqueous formulation further comprises a saccharide (e.g., dextrose, sucrose, mannitol, a polysaccharide (e.g., Dextran 40, Dextran 70, etc.), etc.). In some embodiments, the aqueous formulation further comprises mannitol.

[0163] In an aspect, the present disclosure provides a lyophilized formulation prepared from an aqueous formulation. The aqueous formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the stabilizer is a surfactant. In some embodiments, the surfactant is a lipid or a polymer conjugated lipid.

[0164] In another aspect, the present disclosure provides a lyophilized formulation prepared from an aqueous formulation. The aqueous formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is a compound as set forth in Table 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), or ovalbumin (OVA). In some embodiments, the stabilizer is a surfactant. In some embodiments, the surfactant is a lipid or a polymer conjugated lipid. In some embodiments, the surfactant is a polymer conjugated lipid. In some embodiments, the surfacant is l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), l,2-distearoyl-rac-glycerol-3-methoxypoly ethylene glycol-2000 (DSG-PEG), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG), or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the surfactant is DSPE-PEG. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0165] In another aspect, the present disclosure provides a lyophilized formulation prepared from an aqueous formulation. The aqueous formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), or ovalbumin (OVA). In some embodiments, the stabilizer is a surfactant. In some embodiments, the surfactant is a lipid or a polymer conjugated lipid. In some embodiments, the surfactant is a polymer conjugated lipid. In some embodiments, the stabilizer is 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE- PEG), l,2-distearoyl-rac-glycerol-3-methoxypoly ethylene glycol-2000 (DSG-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3 -methoxypoly ethylene gly col-2000 (DMG- PEG), or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0166] In another aspect, the present disclosure provides a lyophilized formulation prepared from an aqueous formulation. The aqueous formulation comprises a perforin inhibitor, an albumin, and a stabilizer. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The albumin is human serum albumin (HSA) or bovine serum albumin (BSA). In some embodiments, the albumin is BSA. The stabilizer is l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), l,2-distearoyl-rac-glycerol-3-methoxypoly ethylene glycol-2000 (DSG-PEG), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG), or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the stabilizer is DSPE-PEG.

[0167] In an embodiment, the present disclosure provides a lyophilized formulation prepared from an aqueous formulation. The aqueous formulation comprises from about 0.01 wt.% to about 20 wt.% of a perforin inhibitor, from about 0.01 wt.% to about 20 wt.% of an albumin, and from about 0.01 wt.% to about 20 wt.% a stabilizer. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The albumin is human serum albumin (HSA) or bovine serum albumin (BSA). In some embodiments, the albumin is BSA. The stabilizer is l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG), l,2-distearoyl-rac-glycerol-3-methoxypoly ethylene glycol-2000 (DSG-PEG), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG), or 2-[(polyethylene glycol)-2000]-N,N- ditetradecyl acetamide. In some embodiments, the stabilizer is DSPE-PEG.

[0168] In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 1 wt.% of the perforin inhibitor. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 5 wt.% of the albumin. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 5 wt.% of the stabilizer.

[0169] In another aspect, the present disclosure provides a lyophilized formulation prepared from an aqueous formulation. The aqueous formulation comprises a perforin inhibitorand an albumin. The aqueous formulation comprises a plurality of particles. The plurality of particles each comprise the perforin inhibitor encapsulated by the albumin. The plurality of particles each have a Z-average particle size of from about 1 nm to about 300 nm.

[0170] In some embodiments, the perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 20 wt.% of the perforin inhibitor.

[0171] In some embodiments, the albumin is human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RS A), or ovalbumin (OVA). In some embodiments, the albumin is human serum albumin (HSA) or bovine serum albumin (BSA). In some embodiments, the albumin is BSA. In some embodiments, the aqueous formulation comprises from about 0.01 wt.% to about 20 wt.% of the albumin.

[0172] In some embodiments, the aqueous formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 : 1 to about 1 :20, from about 1 :5 to about 1 : 15, or from about 1 : 10 to about 1 : 15 (perforin inhibitor : albumin). In some embodiments, the aqueous formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 : 1 to about 1 :20 (perforin inhibitor : albumin). In some embodiments, the aqueous formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 :5 to about 1 : 15 (perforin inhibitor : albumin). In some embodiments, the aqueous formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 : 10 to about 1 : 15 (perforin inhibitor : albumin). In some embodiments, the aqueous formulation comprises the perforin inhibitor and the albumin at a weight ratio of from about 1 : 11 to about 1 : 14 (perforin inhibitor : albumin).

[0173] In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 300 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 200 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 150 nm.In some embodiments, the plurality of particles each have a Z-average particle size of from about 50 nm to about 150 nm.

[0174] In some embodiments, the aqueous formulation is free of a stabilizer.Reconstituted formulations

[0175] In an aspect, the present disclosure provides a reconstituted formulation comprising a lyophilized formulation. The lyophilized formulation has been reconstituted in one or more pharmaceutically acceptable carriers or diluents.

[0176] In an embodiment, the lyophilized formulation is any lyophilized formulation described herein.

[0177] As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0178] In some embodiments, the reconstituted formulation has a pH of from about 4 to about 10, from about 6 to about 10, from about 6.5 to about 9.5, from about 7 to about 8, or from about 7.2 to about 7.8. In some embodiments, the pharmaceutical formulation has a pH of from about 4 to about 10. In some embodiments, the reconstituted formulation has a pH of from about 4 to about 9. In some embodiments, the reconstituted formulation has a pH of from about 6 to about 10. In some embodiments, the reconstituted formulation has a pH of from about 6.5 to about 9.5. In some embodiments, the reconstituted formulation has a pH of from about 7 to about 8. In some embodiments, the reconstituted formulation has a pH of from about 7.2 to about 7.8.

[0179] In some embodiments, the reconstituted formulation has an osmolality of from about 200 mOsm / L to about 900 mOsm / L, from about 200 mOsm / L to about 500 mOsm / L, from about 200 mOsm / L to about 400 mOsm / L, or from about 225 mOsm / L to about 375 mOsm / L. In some embodiments, the reconstituted formulation has an osmolality of from about 200 mOsm / L to about 900 mOsm / L. In some embodiments, the reconstituted formulation has an osmolality of from about 200 mOsm / L to about 500 mOsm / L. In some embodiments, the reconstituted formulation has an osmolality of from about 200 mOsm / L to about 400 mOsm / L. In some embodiments, the reconstituted formulation has an osmolality of from about 225 mOsm / L to about 375 mOsm / L.

[0180] In some embodiments, the reconstituted formulation comprises a plurality of particles. The plurality of particles each comprise the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) encapsulated by the stabilizer and / or the albumin. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, from about 1 nm to about 150 nm, from about 50 nm to about 150 nm, from about 100 nm to about 300 nm, from about 150 nm to about 300 nm, or from about 200 nm to about 300 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 300 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 200 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 1 nm to about 150 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 50 nm to about 150 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 30 nm to about 300 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 100 nm to about 300 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 150 nm to about 300 nm. In some embodiments, the plurality of particles each have a Z-average particle size of from about 200 nm to about 300 nm.

[0181] In some embodiments, the pharmaceutically acceptable diluent comprises a saccharide (e.g., dextrose, sucrose, mannitol, a polysaccharide (e.g., Dextran 40, Dextran 70, etc.), etc.), propylene glycol, glycerol, water, or any combination thereof. In some embodiments, the pharmaceutically acceptable diluent comprises propylene glycol. In some embodiments, the pharmaceutically acceptable diluent comprises glycerol. In some embodiments, the pharmaceutically acceptable diluent comprises a saccharide. In some embodiments, the pharmaceutically acceptable diluent comprises dextrose. In some embodiments, the pharmaceutically acceptable diluent comprises water. In some embodiments, the pharmaceutically acceptable diluent is 2.5% (w / v) dextrose. In some embodiments, the pharmaceutically acceptable diluent is 5% (w / v) dextrose. In some embodiments, the diluent is water.

[0182] In some embodiments, the reconstituted formulation further comprises one or more pharmaceutically acceptable excipients or adjuvants.

[0183] In some embodiments, the reconstituted formulation comprises an isotonicity agent. As used herein, the term "isotonicity agent" refers to an agent that modifies the osmotic pressure of the reconstituted formulation. In some embodiments, the isotonicity agent modifies the osmotic pressure of the reconstituted formulation to thereby render the reconstituted formulation isotonic. Suitable isotonicity agents would be known to persons skilled in the art, illustrative examples of which include mannitol , dextrose, lactose, sorbitol, sucrose, trehalose, raffinose, sodium chloride, calcium chloride, magnesium chloride, polyethylene glycol, hydroxyethyl starch, and glycine. In some embodiments, the isotonicty agent is mannitol.

[0184] In some embodiments, the reconstituted formulation comprises a chelating agent. Suitable chelating agents would be known to persons skilled in the art, illustrative examples of which include ethylenediaminetetraacetic acid (EDTA) or a pharmaceutically acceptable salt thereof (e.g., disodium EDTA, sodium calcium EDTA, tetrasodium EDTA, etc.), citric acid, and salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanilate). In some embodiments, the chelating agent is EDTA or a pharmaceutically acceptable salt thereof. In some embodiments, the chelating agent is sodium calcium EDTA.

[0185] In some embodiments, the reconstituted formulation comprises an antimicrobial preservative. Suitable antimicrobial preservatives would be known to persons skilled in the art, illustrative examples of which include benzalkonium chloride and chlorobutanol. In some embodiments, the antimicrobial preservative is benzalkonium chloride.

[0186] In some embodiments, the reconstituted formulation comprises a buffer agent. Suitable buffer agents would be known to persons skilled in the art, illustrative examples of which include sodium phosphates (e.g., monosodium phosphate, disodium phosphate, trisodium phosphate, disodium diphosphate, tetrasodium diphosphate, etc.) and sodium citrates (e.g., monosodium citrate, disodium citrate, trisodium citrate, etc.). In some embodiments, the buffer agent is a sodium phosphate.

[0187] In some embodiments, the reconstituted formulation comprises an isotonicity agent (e.g., mannitol), a chelating agent (e.g., EDTA or a pharmaceutically acceptable salt thereof (e.g., sodium calcium EDTA)), an antimicrobial preservative (e.g., benzalkonium chloride), and a buffer agent (e.g., a sodium phosphate).

[0188] In some embodiments, the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) is at a concentration of from about 1 mg / mL to about 20 mg / mL, from about 3 mg / mL to about 15 mg / mL, from about 5 mg / mLto about 12 mg / mL, from about 7 mg / mL to about 12 mg / mL, or from about 7.5 mg / mL to about 12 mg / mL in the reconstituted formulation. In some embodiments, the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) is at a concentration of from about 1 mg / mL to about 20 mg / mL in the reconstituted formulation. In some embodiments, the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) is at a concentration of from about 3 mg / mL to about 15 mg / mL in the reconstituted formulation. In some embodiments, the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) is at a concentration of from about 5 mg / mL to about 12 mg / mL in the reconstituted formulation. In some embodiments, the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) is at a concentration of from about 7 mg / mL to about 12 mg / mL in the reconstituted formulation. In some embodiments, the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) is at a concentration of from 7.5 mg / mL to about 12 mg / mL in the reconstituted formulation.

[0189] In some embodiments, the reconstituted formulation is configured for parenteral administration. In some embodiments, the reconstituted formulation is configured for intravenous, topical, or ophthalmic administration. In some embodiments, the reconstituted formulation is configured for intravenous administration. In some embodiments, the reconstituted formulation is configured for topical administration. In some embodiments, the reconstituted formulation is configured for ophthalmic administration.

[0190] The reconstituted formulation is generally formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0191] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions are also contemplated herein. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, or liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion or by the use of surfactants. Prevention of the action of microorganisms can be achieved by incorporation of various antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, or sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, e.g., aluminum monostearate or gelatin.

[0192] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0193] Oral compositions generally comprise an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated withexcipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.

[0194] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurised container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0195] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished with nasal sprays or suppositories. The compounds can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0196] For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0197] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0198] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50.Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0199] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i. e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0200] Another example of determination of effective dose for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum of the test subject. Such assays may utilize antibody mimics and / or "biosensors" that have been created through molecular imprinting techniques. The compound which is able to modulate perforin activity is used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix that contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. el al. (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2: 166-173. Such "imprinted" affinity matrices are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrices in this way can be seen in Vlatakis, G. etal. (1993) Nature 361 :645- 647. Through the use of isotope-labeling, the "free" concentration of compound which modulates the expression or activity of perforin can be readily monitored and used in calculations of IC50. Such "imprinted" affinity matrices can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local andselective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. A rudimentary example of such a "biosensor" is discussed in Kriz, D. et al. (1995) Analytical Chemistry 67:2142-2144.

[0201] Persons skilled in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, the degree of expression or activity to be modulated, the severity of the disease or disorder, previous treatments and other diseases present.Topical formulations

[0202] In another aspect, the present disclosure provides a pharmaceutical formulation comprising a perforin inhibitor, a solvent, and a gelling agent. The pharmaceutical formulation is a gel. The pharmaceutical formulation is configured for topical administration.

[0203] In an embodiment, the perforin inhibitor is any perforin inhibitor described herein. In some embodiments, the perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0204] In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 20 wt.%, from about 0.1 wt.% to about 15 wt.%, from about 0.1 wt.% to about 10 wt.%, or from about 1 wt.% to about 5 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 20 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 15 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the topical formulation comprises from about 0.1 wt.% toabout 10 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the topical formulation comprises from about 1 wt.% to about 5 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the topical formulation comprises from about 1 wt.% to about 3.5 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the topical formulation comprises about 3 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 5 wt. % of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof). In some embodiments, the topical formulation comprises about 0.3 wt.% of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof).

[0205] The solvent is not particularly limited and may be any solvent suitable for forming a gel that dissolves the perforin inhibitor. In some embodiments, the solvent is a protic solvent (e.g., water, alcohols, etc.). As used herein, the term "protic solvent" refers to solvents having at least one hydrogen atom that is bound to an oxygen atom or a nitrogen atom. In some embodiments, the solvent is an alcohol. In some embodiments, the solvent is 2-(2- ethoxyethoxy)ethan-l-ol (also referred to herein as transcutol). In some embodiments, the topical formulation comprises more than one solvent.

[0206] In some embodiments, the topical formulation comprises at least about 10 wt.%, at least about 20 wt.%, at least about 30 wt.%, at least about 40 wt.%, at least about 50 wt. %, at least about 60 wt. %, at least about 70 wt.%, at least about 80 wt.%, at least about 90 wt.%, or at least about 95 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 10 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 20 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 30 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 40 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 50 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 60 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 70 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 80 wt.% of the solvent. In some embodiments, the topical formulation comprisesat least about 90 wt.% of the solvent. In some embodiments, the topical formulation comprises at least about 95 wt.% of the solvent. In some embodiments, the topical formulation comprises from about 10 wt.% to about 95 wt.% of the solvent.

[0207] The gelling agent is not particularly limited and may be any gelling agent suitable for forming a gel that is compatible with the perforin inhibitor. Suitable gelling agents include cellulose and derivatives thereof (e.g., methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), hydroxyethyl cellulose (HEC), etc.), vinyl alcohols, vinyl pyrrolidones (e.g., polyvinylpyrrolidone (PVP), etc.), natural gums (e.g., karaya gum, locust bean gum, guar gum, gelan gum, xanthan gum, gum arabic, tragacanth gum, etc.), carrageenan, pectin, agar, alginic acid, sodium alginate, and methacrylates. In some embodiments, the gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxy ethyl cellulose (HEC). In some embodiments, the gelling agent is hydroxypropyl methyl cellulose (HPMC). In some embodiments, the pharmaceutical formulation comprises more than one gelling agent.

[0208] In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 55 wt.%, from about 1 wt.% to about 55 wt.%, from about 10 wt.% to about 50 wt.%, from about 15 wt.% to about 45 wt.%, from about 20 wt.% to about 40 wt.%, or from about 25 wt.% to about 35 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 1 wt.% to about 55 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 10 wt.% to about 50 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 15 wt.% to about 45 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 20 wt.% to about 40 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 25 wt.% to about 35 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 1 wt.% to about 40 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 1 wt.% to about 30 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 1 wt.%to about 20 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 1 wt.% to about 10 wt.% of the gelling agent.

[0209] In some embodiments, the topical formulation further comprises an ultraviolet (UV) absorption agent. The UV absorption agent may be any agent suitable for absorbing UV rays. Suitable UV absorption agents would be known to persons skilled in the art, illustrative examples of which include 2-ethylhexyl 2-hydroxybenzoate (also referred to as 2-ethylhexyl salicylate and octyl salicylate; Escalol® 587), 3-methylbutyl 4-(dimethylamino)benzoate (also referred to as padimate A and isoamyl dimethyl PABA; Escalol® 506), 2-ethylhexyl 4- (dimethylamino)benzoate (also referred to as padimate O and 2-ethylhexyl dimethyl PABA; Escalol® 507), 2,4-dihydroxybenzophenone (also referred to as benzophenone- 1), 2,2'- dihydroxy-4,4'-dimethoxybenzophenone (also referred to as benzophenone-6; Uvinul® D-49), 2-(2H-Benzotriazol-2-yl)-4,6-di-tert-pentylphenol (Uvinul® 3028), ethyl-2-cyano-3,3- diphenylacrylate (Uvinul® 3035), homomenthyl salicylate (also referred to as homosalate), 2,2'-[6-(4-Methoxyphenyl)-l,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol} (also referred to as bemotrizinol; Escalol® S), 2-(2H-benzotriazole-2-yl)-4-methylphenol (Uvinul® 3033P), bis(2-ethylhexyl) 4,4'-[(6-{[4-(tert-butylcarbamoyl)phenyl]amino}-l,3,5-triazine-2,4- diyl)bis(azanediyl)]dibenzoate (also referred to as iscotrizinol; Uvasorb® HEB), lisadimate (also referred to as glyceryl PABA), Poly(4-hydroxy-2,2,6,6-tetramethyl-l -piperidine ethanol - a / M,4-butanedioic acid) (also referred to as plastic additive 22; Uvinul® 5062H, Uvinul® 5062GR), 4-[[4,6-bis[[4-(2-ethylhexoxy-oxomethyl)phenyl]amino]-l,3,5-triazin-2- yl]amino]benzoic acid 2-ethylhexyl ester (also referred to as ethylhexyl triazone; Uvinul® T- 150), 2-Ethylhexyl 2-cyano-3,3-diphenylprop-2-enoate (also referred to as octocrylene; Escalol® 597), 3-Methylbutyl (2E)-3-(4-methoxyphenyl)prop-2-enoate (also referred to as isoamyl-p-methoxycinnamate and amiloxate; Neo Hcliopan® E1000), 2-(2J / -Benzotriazol-2- yl)-4-methyl-6-[2-methyl-3-[l,3,3,3-tetramethyl-l-[(trimethylsilyl)oxy]-l- disiloxanyl]propyl]phenol (also referred to as drometrizole trisiloxane), titanium dioxide, [2- Hydroxy-4-(octyloxy)phenyl](phenyl)methanone (also referred to as octabenzone and benzophenone- 12); 2,2',4,4'-tetrahydroxybenzophenone (also referred to as benzophenone-2), diisopropyl methylcinnamate, (lA,3A,45)- / ?-Menthan-3-yl 2-aminobenzoate (also referred to as menthyl anthranilate and meradimate), bis-(l,2,2,6,6-pcntamcthyl-4-piperidyl)-sebacate, 3- (4-tert-Butylphenyl)-l-(4-methoxyphenyl)propane-l,3-dione (also referred to as butylmethoxy dibenzoylmethane and avobenzone), 2-ethoxy ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate 2-ethoxyethyl (also referred to as cinnoxate), benzylidene camphor sulfonic acid, zinc oxide, 2-Hydroxy-7V,7V-bis(2-hydroxyethyl)ethan-l-aminium 2- hydroxybenzoate (also referred to as trolamine salicylate and triethanolamine salicylate), polysilicone-15 (Parsol® SLX); (3E)-l,7,7-Trimethyl-3-[(4-methylphenyl)methylene]-2- norbornanone (also referred to as 4-methylbenzylidene camphor and enzacamene; Eusolex® 6300, Parsol® 5000), 2,2'-methylenebis[6-(2J / -l,2,3-benzotriazol-2-yl)-4-(2,4,4- trimethylpentan-2-yl)phenol] (also referred to as bisoctrizole), 5-benzoyl-4-hydroxy-2- m ethoxybenzene- 1 -sulfonic acid (also referred to as sulisobenzone and benzophenone-4), polyacrylamide methylbenzylidene camphor, glyceryl ethylhexanoate dimethoxycinnamate, benzophenone-5 (sulisobenzone sodium); (2-Hydroxy-4-methoxyphenyl)(2- hydroxyphenyl)methanone (also referred to as dioxybenzone and benzophenone-8), sodium 2,2'-dihydroxy-4,4'-dimethoxybenzophenone-5,5'-disulfonate (also referred to as benzophenone-9), and (2-Hydroxy-4-methoxyphenyl)(phenyl)methanone (also referred to as oxybenzone and benzophenone-3).

[0210] In some embodiments, the UV absorption agent absorbs UVA rays (i.e., UV rays having a wavelength of from about 315 nm to about 400 nm). In some embodiments, the UV absorption agent is avobenzone. In some embodiments, the UV absorption agent absorbs UVB rays (i.e., UV rays having a wavelength of from about 280 nm to about 315 nm). In some embodiments, the UV absorption agent is oxybenzone.

[0211] In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 20 wt.%, from about 0.1 wt.% to about 15 wt. %, from about 0.1 wt.% to about 10 wt.%, or from about 1 wt.% to about 5 wt.% of the UV absorption agent. In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 20 wt.% of the UV absorption agent. In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 15 wt. % of the UV absorption agent. In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 10 wt.%. of the UV absorption agent. In some embodiments, the topical formulation comprises from about 1 wt.% to about 5 wt.% of the UV absorption agent. In some embodiments, the topical formulation comprises about 3 wt.% of the UV absorption agent.

[0212] In some embodiments, the UV absorption agent is further defined as a first UV absorption agent, and the pharmaceutical formulation further comprises a second ultraviolet (UV) absorption agent. The second UV absorption agent and the first UV absorption agent aredifferent. In some embodiments, one of the first and second UV absorption agents absorbs UVA rays and the other of the first and second UV absorption agents absorbs UVB rays.

[0213] The second UV absorption agent may be any UV absorption agent described herein. In some embodiments, the second UV absorption agent absorbs UVB rays. In some embodiments, the second UV absorption agent is oxybenzone.

[0214] In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 20 wt.%, from about 0.1 wt.% to about 15 wt. %, from about 0.1 wt.% to about 10 wt.%, or from about 1 wt.% to about 5 wt.% of the second UV absorption agent. In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 20 wt.% of the second UV absorption agent. In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 15 wt. % of the second UV absorption agent. In some embodiments, the topical formulation comprises from about 0.1 wt.% to about 10 wt.% of the second UV absorption agent. In some embodiments, the topical formulation comprises from about 1 wt.% to about 5 wt.% of the second UV absorption agent. In some embodiments, the topical formulation comprises about 3 wt.% of the second UV absorption agent.

[0215] In some embodiments, the topical formulation comprises the first UV absorption agent and second UV absorption agent at a weight ratio of from about 5: 1 to about 1 :5, from about 3: 1 to about 1 :3, from about 2: 1 to about 1 :2, or about 1 : 1 (first UV absorption agent : second UV absorption agent). In some embodiments, the topical formulation comprises the first UV absorption agent and second UV absorption agent at a weight ratio of from about 5: 1 to about 1 :5 (first UV absorption agent : second UV absorption agent). In some embodiments, the topical formulation comprises the first UV absorption agent and second UV absorption agent at a weight ratio of from about 3: 1 to about 1 :3 (first UV absorption agent : second UV absorption agent). In some embodiments, the topical formulation comprises the first UV absorption agent and second UV absorption agent at a weight ratio of from about 2: 1 to about 1 :2 (first UV absorption agent : second UV absorption agent). In some embodiments, the topical formulation comprises the first UV absorption agent and second UV absorption agent at a weight ratio of about 1 : 1 (first UV absorption agent : second UV absorption agent).

[0216] In some embodiments, the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) degrades less than about 20% under UV light irradiation at a wavelength of about 350 nM for 7 hours.

[0217] Without wishing to be bound by theory or mode of operation, it is believed that UV absorption agents prevent or reduce degradation of the perforin inhibitor (e.g., Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof) when the topical formulation is exposed to UV rays (e.g., UVA rays and / or UVB rays).

[0218] In some embodiments, a container comprises the topical formulation. In some embodiments, the container absorbs UV rays (e.g., UVA rays and / or UVB rays). For example, when the container is a glass container, the glass container may be an amber glass container.

[0219] In an aspect, the present disclosure provides a pharmaceutical formulation comprising a perforin inhibitor, a solvent, and a gelling agent. The pharmaceutical formulation is a gel. The pharmaceutical formulation is configured for topical administration. The perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The solvent is 2-(2-ethoxyethoxy)ethan-l-ol. The gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxyethyl cellulose (HEC). In some embodiments, the gelling agent is HPMC.

[0220] In another aspect, the present disclosure provides a pharmaceutical formulation comprising from about 0.1 wt.% to about 20 wt.% of a perforin inhibitor, at least about 40 wt.% of a solvent, and from about 1 wt.% to about 55 wt.% of a gelling agent. The pharmaceutical formulation is a gel. The pharmaceutical formulation is configured for topical administration. The perforin inhibitor is a compound of Formula (A) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The solvent is 2-(2-ethoxyethoxy)ethan-l-ol. The gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxy ethyl cellulose (HEC). In some embodiments, the gelling agent is HPMC. In some embodiments, the topical formulation comprises from about 1 wt.% to about 10 wt.% of the gelling agent.

[0221] In another aspect, the present disclosure provides a pharmaceutical formulation comprising a perforin inhibitor, a solvent, and a gelling agent. The pharmaceutical formulationis a gel. The pharmaceutical formulation is configured for topical administration. The perforin inhibitor is a compound as set forth in Table 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The solvent is 2-(2-ethoxyethoxy)ethan-l-ol. The gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxyethyl cellulose (HEC). In some embodiments, the gelling agent is HPMC. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0222] In another aspect, the present disclosure provides a pharmaceutical formulation comprising from about 0.1 wt.% to about 20 wt.% of a perforin inhibitor, at least about 40 wt.% of a solvent, and from about 1 wt.% to about 55 wt.% of a gelling agent. The pharmaceutical formulation is a gel. The pharmaceutical formulation is configured for topical administration. The perforin inhibitor is a compound as set forth in Table 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The solvent is 2-(2-ethoxyethoxy)ethan-l-ol. The gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxy ethyl cellulose (HEC). In some embodiments, the gelling agent is HPMC. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the topical formulation comprises from about 1 wt.% to about 10 wt.% of the gelling agent. In some embodiments, the topical formulation comprises from about 1 wt.% to about 10 wt.% of the gelling agent.

[0223] In some embodiments, the pharmaceutical formulation further comprises a UV absorption agent. In some embodiments, the UV absorption agent absorbs UVA rays. In some embodiments, the UV absorption agent is avobenzone. In some embodiments, the pharmaceutical formulation comprises from about 0.1 wt.% to about 20 wt.% of the UV absorption agent.

[0224] In some embodiments, the UV absorption agent is further defined as a first UV absorption agent, and the pharmaceutical formulation further comprises a second UV absorption agent. The second UV absorption agent and the first UV absorption agent are different. In some embodiments, the second UV absorption agent absorbs UVB rays. In some embodiments, the UV absorption agent is oxybenzone. In some embodiments, the pharmaceutical formulation comprises from about 0.1 wt.% to about 20 wt.% of the second UV absorption agent.

[0225] In an aspect, the present disclosure provides a pharmaceutical formulation comprising a perforin inhibitor, a solvent, a gelling agent, and a UV absorption agent. The pharmaceutical formulation is a gel. The pharmaceutical formulation is configured for topical administration. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The solvent is 2-(2-ethoxyethoxy)ethan-l-ol. The gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxy ethyl cellulose (HEC). In some embodiments, the gelling agent is HPMC. The UV absorption agent absorbs UVA rays. In some embodiments, the UV absorption agent is avobenzone.

[0226] In another aspect, the present disclosure provides a pharmaceutical formulation comprising from about 0.1 wt.% to about 20 wt.% of a perforin inhibitor, at least about 40 wt.% of a solvent, from about 1 wt.% to about 55 wt.% of a gelling agent, and from about 0.1 wt.% to about 20 wt.% of a UV absorption agent. The pharmaceutical formulation is a gel. The pharmaceutical formulation is configured for topical administration. The perforin inhibitor is a compound as set forth in Table 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The solvent is 2-(2-ethoxyethoxy)ethan-l-ol. The gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxyethyl cellulose (HEC). In some embodiments, the gelling agent is HPMC. The UV absorption agent absorbs UVA rays. In some embodiments, the UV absorption agent isavobenzone. In some embodiments, the perforin inhibitor is a compound as set forth in Table 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0227] In another aspect, the present disclosure provides a pharmaceutical formulation comprising a perforin inhibitor, a solvent, a gelling agent, a first UV absorption agent, and a second UV absorption agent. The pharmaceutical formulation is a gel. The pharmaceutical formulation is configured for topical administration. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The solvent is 2-(2- ethoxyethoxy)ethan-l-ol. The gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxyethyl cellulose (HEC). In some embodiments, the gelling agent is HPMC. The first UV absorption agent absorbs UVA rays. In some embodiments, the first UV absorption agent is avobenzone. The second UV absorption agent absorbs UVB rays. In some embodiments, the second UV absorption agent is oxybenzone.

[0228] In another aspect, the present disclosure provides a pharmaceutical formulation comprising from about 0.1 wt.% to about 20 wt.% of a perforin inhibitor, at least about 40 wt.% of a solvent, from about 1 wt.% to about 55 wt.% of a gelling agent, from about 0.1 wt.% to about 20 wt.% of a first UV absorption agent, and from about 0.1 wt.% to about 20 wt.% of a second UV absorption agent. The pharmaceutical formulation is a gel. The pharmaceutical formulation is configured for topical administration. The perforin inhibitor is Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof. The solvent is 2-(2- ethoxyethoxy)ethan-l-ol. The gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate. In some embodiments, the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxyethyl cellulose (HEC). In some embodiments, the gelling agent is HPMC. The first UV absorption agent absorbs UVA rays. In some embodiments, the first UV absorption agent is avobenzone. The second UV absorption agent absorbs UVB rays. In some embodiments, the second UV absorption agent is oxybenzone.

[0229] In some embodiments, the topical formulation comprises the first UV absorption agent and second UV absorption agent at a weight ratio of from about 5: 1 to about 1 :5 (first UV absorption agent : second UV absorption agent).Therapeutic methods and uses

[0230] In another aspect of the present disclosure, it is provided a method of inhibiting activity of a perforin molecule, or a fragment or variant thereof, on a cell, the method comprising exposing the cell to a compound or a pharmaceutically acceptable salt, solvate, or hydrate thereof, as described herein. The cell may be a target cell (as described herein), or alternatively, it may be a CTL and / or NK cell that expresses perforin. The exposing of the cell to the compound, or a pharmaceutically acceptable salt, solvate, or hydrate thereof (as herein described), may occur in vitro, ex vivo or in vivo.

[0231] Where the exposing of a cell to the compound occurs in vitro or ex vivo, for example, the method of the present invention may be used as a diagnostic tool to determine the efficacy of certain compounds (alone or in combination) for inhibiting perforin activity in a patient. For example, a CTL and / or NK cell that expresses perforin may be removed from a patient and exposed to one or more compounds of the present invention (or a pharmaceutically acceptable salt, solvate, or hydrate thereof) in the presence of a suitable target cell (as described herein). The target cell may, though need not be, from the same patient. In another example, a target cell may be removed from a patient and exposed to one or more compounds of the present invention (or a pharmaceutically acceptable salt, solvate, or hydrate thereof) in the presence of perforin. The ability of the compound (or compounds) to inhibit the activity of perforin can be assessed by measuring the degree of target cell lysis by any method known to one skilled in the art. Thus, one may be able to ascertain whether a certain compound is more efficacious than another and tailor a specific treatment regime to that patient.

[0232] In some embodiments, the exposing of the cell to the compound, or a pharmaceutically acceptable salt or a derivative thereof, as herein described is in vivo.

[0233] Accordingly, in an aspect, the present disclosure provides the use of a pharmaceutical formulation (e.g., a lyophilized formulation, a reconstituted formulation, and / or a topical formulation) as described herein, for treating or preventing a disease or disorder associated with undesirable perforin activity.

[0234] In another aspect disclosed herein, the present disclosure provides the use of a pharmaceutical formulation (e.g., a lyophilized formulation, a reconstituted formulation, and / or a topical formulation) as described herein, in the manufacture of a medicament for treating or preventing a disease or disorder associated with undesirable perforin activity.

[0235] In another aspect of the present disclosure there is provided a prophylactic or therapeutic method of treating a subject at risk of or susceptible to a disease or disorder, or having a disease or disorder, associated with aberrant perforin expression and / or activity. Such disease or disorder will generally be associated with either an increase in levels of perforin molecules, an increase in perforin activity as compared to a healthy population, or a pathological attack of the subj ect' s tissues or by CTL, NK cells or other lymphocytes that utilise the perforin pathway.

[0236] In one embodiment, the prophylactic or therapeutic method comprises the steps of administering a pharmaceutical formulation (e.g., a lyophilized formulation, a reconstituted formulation, and / or a topical formulation) as described herein, to a subject who has a disease or disorder, a symptom of disease or disorder, or predisposition toward a disease or disorder associated with undesired perforin activity as herein described, for the purpose to cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition towards the disease or disorder. Pharmaceutical formulations (e.g., a lyophilized formulation, a reconstituted formulation, and / or a topical formulation) of the present invention will be administered in a therapeutically effective amount.

[0237] As used herein, the term "effective amount" refers to an amount of the pharmaceutical formulation or compound described herein which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur once, or at intervals of minutes or hours, or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. A typical dosage is in the range of 1 pg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.

[0238] It would also be appreciated by persons skilled in the art that the prophylactic or therapeutic methods as herein described could be used in any number of combinations with other treatment modalities currently employed in the art.

[0239] Conditions in which perforin expression and / or activity is increased, and where it is desirable to reduce said activity, may be identified by those skilled in the art by any or a combination of diagnostic or prognostic assays known in the art. For example, a biological sample obtained from a subject (e.g. blood, serum, plasma, urine, saliva, and / or cells derived therefrom) may be analysed for perforin expression and / or activity or the presence of CTL, NK cells or other lymphocytes capable of using perforin to induce tissue damage, as hereinbefore described. Such conditions include, but are not limited to, juvenile diabetes mellitus (type 1 or insulin dependent), graft-versus-host disease, chronic or acute allograft rejection, malaria and any other conditions associated with cytotoxic T lymphocyte- or natural killer cell-mediated immune pathology.

[0240] Thus, in one embodiment of the present invention, the prophylactic and therapeutic methods of treatment are applicable to the treatment and / or prevention of immune mediated conditions or inflammatory diseases and disorders such as, but not limited to, autoimmune or inflammatory diseases and disorders including juvenile diabetes mellitus (type 1 or insulin dependent), multiple sclerosis and variants thereof (e.g., Susac Syndrome), Crohn's disease, colitis, inflammatory bowel disease, fibrosis, fibrotic disorders (e.g., scleroderma), Guillain- Barre syndrome, lupus erythematosus, psoriasis, pancreatitis, rheumatoid arthritis, sepsis, vasculitis, Wegener's granulomatosis, proliferative retinopathies, as well as other conditions including but not limited to graft-versus-host disease, chronic or acute allograft rejection, infectious diseases and associated immune pathological complications (e.g., fulminant virus infections (e.g., fulminant liver failure following acute hepatitis B infection), mosquito-borne diseases of the Plasmodium genus, such as malaria, in particular cerebral malaria, sand-fly- bome diseases of the Leishmania genus, in particular cutaneous Leishmaniasis and other and conditions associated with cytotoxic T lymphocyte- or natural killer cell-mediated immune pathology.

[0241] With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expressionanalysis to drugs in clinical development and on the market. The term also refers to the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype"). Thus, another aspect of the present invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the pharmaceutical formulations of the present invention or agents that modulate perforin expression and / or activity (such as those identified by screening assays as herein described), according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0242] It is considered that the methods described herein are suitable for the prophylactic and therapeutic treatment of any species, including, but not limited to, all mammals including humans, canines, felines, cattle, horses, rats and mice, as well as birds, reptiles and lower organisms such as bacteria. In some embodiments, the methods described herein are suitable for the prophylactic and therapeutic treatment of humans.

[0243] For the above mentioned indications, the appropriate dosage will vary depending on, e.g. the compound employed, the age, sex, weight and general physical condition of the subject, the mode of administration, the nature and / or severity of the condition or the desired effect. By balancing these features it is well within the general skill of a medical practitioner to determine appropriate dosages.

[0244] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0245] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0246] All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entireties.

[0247] The various embodiments enabled herein are further described by the following non-limiting examples.ExamplesExample 1: Pharmaceutical Formulation Comprising a Plurality of LiposomesStability of liposomes at high pH

[0248] In a vial, water was adjusted to a pH of 10 to 12 with trisodium phosphate (NasPCU).A mixture of hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) was added to the vial (57:38:05 molar ratio (HPSC:cholesterol:DSPE-PEG2000)) and the vial was incubated at 65 °C for 1 hour (hr). Minimal changes were observed in the mixture after incubation. The absence of precipitation suggested that the liposomes were stable at high pH.Active Loading Trial 1

[0249] To form the blank liposome, HPSC, cholesterol, and DSPE-PEG2000 were extruded in 250 mM ammonium sulfate ((NEL^SCh) followed by dialysis in 5% glucose over 3 days to remove external (NEL^SCh.

[0250] Separately, in two vials, 8 mg of Compound (I) was suspended in a phosphate buffer (pH 11.2) with and without 10% dimethyl sulfoxide (DMSO). To the resultant solutions of Compound (I) was added the blank liposome at a ratio of 15: 1 w / w (blank liposome : Compound (I)), followed by incubation at 65 °C for 1 hr. After incubation, the pH was neutralized with hydrochloric acid (HC1). It was observed that the sample containing 10% DMSO appeared more transparent, whereas the sample without 10% DMSO appeared more turbid.

[0251] The samples were stored in a refrigerator overnight and then centrifuged at 8,000 revolutions per minute (RPM) for 10 minutes (min). High-performance liquid chromatography (HPLC) analysis of the supernatant of each sample indicated that more of Compound (I) was encapsulated in the sample without 10% DMSO (49.6% of Compound (I) encapsulated) as compared to the sample with 10 % DMSO (3.5% of Compound (I) encapsulated). These results suggest that, although DMSO appears to enhance solubility of Compound (I), the DMSO may disrupt the pH gradients required for active loading of the liposome.Active Loading Trial 2

[0252] To form the blank liposome, HPSC, cholesterol, and DSPE-PEG2000 were extruded in 250 mM ammonium sulfate ((NEU^SCU) followed by dialysis in 5% glucose over 3 days to remove external (NEU^SC .

[0253] Separately, in three vials, 8.4 mg of Compound (I) was suspended in a phosphate buffer (pH 11.2). To the resultant solutions of Compound (I) was added the blank liposome at a ratio of 15: 1 w / w (blank liposome : Compound (I)) , followed by incubation at 70 °C for 0 hrs, 1 hr, or 2 hrs. After incubation, the pH was neutralized with HC1 to a pH of 7.4 and the sample was stored in a refrigerator for 24 hrs. Each sample was then centrifuged at 8,000 RPM for 10 minutes (min). For the sample that was not incubated (i.e., 0 hrs), Compound (I) appeared as precipitation. For the sample that was incubated for 1 hr, there was less precipitation of Compound (I). For the sample that was incubated for 2 hrs, there was minimal precipitation of Compound (I).

[0254] However, after 3 days of storage in a refrigerator, the sample that was incubated for 2 hrs exhibited precipitate that was indicative of both Compound (I) and the liposome. This precipitation suggested that Compound (I) potentially destabilizes the liposomes.Example 2: Pharmaceutical Formulation Comprising a StabilizerPluronic® F-127

[0255] Pluronic® F-127 (CAS No. 9003-11-6) and Compound (I) were dissolved in acetone at a ratio of 1 :5 w / w. The acetone was removed under reduced procure to afford an orange plastic film. The resultant film was reconstituted in 0.4M sodium hydroxide (NaOH), with Compound (I) having a concentration of 10 mg / mL in the resultant reconstituted solution. The reconstituted solution was diluted with water (1 : 1) or HC1 (1 : 1). The average suspension size was 2,000 nm for the reconstituted solution diluted with water. The aggregate was too large for accurate sizing for the reconstituted solution diluted with HC1.Solutol® HS 15

[0256] Compound (I) (15 mg) and Solutol® HS 15 (250 mg; CAS No. 70142-34-6) were dissolved in 100 pL of DMSO to form a clear mixture. Water was added to form a 2 mLmixture, and the mixture was vortexed to afford a solution having 7.5 mg / mL of Compound (I). The solution was stored in a refrigerator for 3 days, after which minimal precipitation was observed.DSPE-PEG2000

[0257] Compound (I) (30 mg) and DSPE-PEG2000 (100 mg) were dissolved in 200 pL of DMSO. Phosphate buffered saline (PBS) was added to form a 4 mL mixture, and the mixture was vortexed to afford a solution having 7.5 mg / mL of Compound (I). Extended storage in a refrigerator resulted in observable precipitation.Example 3: Pharmaceutical Formulation Comprising an Albumin and a StabilizerSolutol® HS 15

[0258] Compound (I), DMSO, Solutol® HS 15, bovine serum albumin (BSA), and solvent were mixed in different amounts to form two samples, as set forth in Table 3 below. Sample 1 achieved a concentration of Compound (I) of 7.1 mg / mL before filtration and 6.9 mg / mL after filtration. Sample 2 achieved a concentration of Compound (I) of 12.5 mg / mL before filtration and 12.3 mg / mL after filtration. Throughout this disclosure, Compound (I) may be abbreviated as "Cmpd (I)".DSPE-PEG2000

[0259] Compound (I), DMSO, DSPE-PEG2000, and BSA were mixed in different amounts to form Samples 3-5, as set forth in Table 4 below. Sample 3 achieved a concentration of Compound (I) of 6.9 mg / mL before filtration and 6.4 mg / mL after 0.22 pm filtration. Sample 1 was slightly turbid and filtration proceeded without issue. Sample 4 exhibited a poor dispersion due to the DSPE-PEG2000 and DMSO becoming too stiff. The concentration of Compound (I) was not measured for Sample 4. Sample 5 exhibited large aggregate precipitation. Sample 5 achieved a concentration of Compound (I) of 6.1 mg / mL before filtration and 4.84 mg / mL after 0.22 pm filtration.

[0260] Sample 3 was subsequently lyophilized to remove the water and DMSO to produce a fluffy powder with some aggregation at the center of the sample. Sample 3 was reconstituted to form a slightly turbid solution that was readily filtered. The concentration of Compound (I)in reconstituted Sample 3 was 10.9 mg / L before filtration and 8.9 mg / mL after 0.22 pm filtration.Example 4: Lyophilized Pharmaceutical Formulation Comprising an Albumin and a Stabilizer

[0261] Compound (I), DMSO, DSPE-PEG2000, BSA, and water (Milli-Q® water) were mixed in different amounts to form Samples 6 and 7, as set forth in Table 5 below. Specifically, Compound (I) and DSPE-PEG2000 were dissolved in DMSO to form a mixture. Separately, BSA was dissolved in water to form a BSA solution. The BSA solution was vortex mixed with the mixture to achieve the final concentrations provided in Table 5. Sample 6 achieved a concentration of Compound (I) of 5.1 mg / mL before filtration and 4.5 mg / mL after 0.22 pm filtration. Sample 7 achieved a concentration of Compound (I) of 4.6 mg / mL before filtration and 4.6 mg / mL after 0.22 pm filtration.

[0262] Sample 7, having a higher DSPE-PEG2000 concentration, was clearer than Sample 6, indicating less aggregation. Minimal loss of Compound (I) during the filtration step was also observed for Sample 7.

[0263] Samples 6 and 7 were subsequently separated into smaller vials (Samples 8-10) and lyophilized. The lyophilized samples were then reconstituted in PBS at various concentrations, as set forth in Table 6. Samples 9 and 10 achieved concentrations of Compound (I) of greater than 7.5 mg / mL after 0.22 pm filtration.Example 5: Scale-up of Pharmaceutical Formulation Comprising an Albumin and a StabilizerMaterials

[0264] Compound (I) was synthesized as previously described in PCT Publication No. WO 2014 / 028968 Al and / or in Spicer, J. A. (2017, supra), including via methods used to synthesize compounds other than Compound (I)). l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) and bovine serum albumin (BSA) was purchased commercial suppliers. All solvents used were of HPLC grade.

[0265] Quantification of Compound (I) using HPLC Analysis: For HPLC analysis, the parenteral formulation was prepared by first, a 20* dilution in MilliQ water to reduce the matrix effect of BSA, followed by a 1 : 10 dilution in acetonitrile and finally, a 0.22 pm filtration to remove potential aggregates.

[0266] Compound (I) was assayed using a Shimadzu Nexera UHPLC system connected to a Nexera X2 UV detector system (Shimadzu Corp., Kyoto, Japan). Chromatographic separation was achieved using a RESTEK Ultra C18 column (150 * 3.2 mm, 5 pm, Pennsylvania, USA) maintained at 35 °C. The mobile phase comprising A (0.1% formic acid in water) and B (methanol) was delivered at 1 mL / min using a linear gradient at 5 to 95% B from 0-7 min, 95-5% from 7-13 min and then re-equilibrated at 5% B from 13-15 min. The Compound (I) peak eluted at 4.9 mins and detected a UV absorption wavelength of 270 nm. Data acquisition and processing were performed using LabSolutions software (Shimadzu Corporation).Batch 1

[0267] Method of Manufacture: Using a 500 mL Schott bottle, 0.50 g of Compound (I) and 1.81 g of DSPE-PEG2000 was dissolved in 4.02 g of DMSO at 60 °C. The resultant DMSO solution was allowed to cool to room temperature. BSA at 50 mg / mL was dissolved in purified water and 121.1 g of the solution was added to the DMSO solution and mixed with a magnetic stir bar at 500 RPM to form a yellow, cloudy dispersion. Mannitol at 1.0 g was pre-dissolved in purified water and added to the suspension. Purified water was added to the suspension to a final volume of 400 mL. Using 0.2-micron regenerated cellulose syringe filters, the formulation was extruded to form a slightly translucent solution.

[0268] The formulation was stored at -20 °C for 72 hrs and then under dry ice for 6 hrs before lyophilizing at 0.15 mBar for ten days. The resulting lyophilized mass remained near the bottom of the container, indicating collapsing of the scaffold. The dried mass was hard and brittle. A total of 10.5 g of the dried mass was decanted from the 500 mL container and stored in a 40 mL amber glass vial.

[0269] Reconstituted Formulation with Compound (I) at 10 mg / mL: Over 1 g of the dried mass was used to analyse the formulation to ensure consistency and homogeneity. 202.4 mg of the dried mass was reconstituted with 861 uL of 0.1 * PBS + 5 mg / mL benzyl alcohol. The unfiltered formulation had a Compound (I) concentration of 10.3 mg / mL according to HPLCanalysis. The formulation was filtered using a 0.2 gm regenerated cellulose filter and resulted in only 26.2% of Compound (I) remaining, whereas filtration with a 0.22 gm polyvinylidene fluoride (PVDF) filter resulted in 69% of Compound (I) remaining. These results suggested that the concentration of Compound (I) was too high for sterile filtration.

[0270] Reconstituted Formulation with Compound (I) at 5 mg / mL: 406.0 mg of the dried mass was reconstituted with 1676 uL with O.lx PBS + 5 mg / mL benzyl alcohol. The reconstituted formulation was diluted with an equal volume of 0.1 * PBS + 5 mg / mL benzyl alcohol solution. According to HPLC analysis, the unfiltered formulation had a concentration of 4.6 mg / mL. The formulation was filtered using a 0.2 pm regenerated cellulose filter, resulting in 63% of Compound (I) remaining after filtration. In contrast, using a 0.22 pm PVDF filter resulted in 80% of Compound (I) remaining. The high loss of Compound (I) from the regenerated cellulose filters suggested a potential interaction between the filter material and formulation.

[0271] Figure 1 shows Z-average particle size from Zetasizer analysis. The Z-average particle size was -287 nm with PDI of 1.0 indicating a polydisperse colloidal system.

[0272] The pH and osmolality of the 4.6 mg / mL unfiltered formulation was 7.6 and 322 mOsm / kg, respectively.Model Batch

[0273] Model Compound: A model compound was used in place of Compound (I). Like Compound (I), the model compound has a relatively large molecular weight (-800 Da), < Imcg / mL solubility in water, and -10 mg / mL solubility in DMSO. DSPE-PEG2000 was shown to significantly increase the solubility of the model compound to above 4 mg / mL in water and, as such, the model compound was deemed an acceptable substitute for Compound (I) to further optimize the manufacturing process.

[0274] DMSO Removal: The model compound with DSPE-PEG2000 was dissolved in DMSO and then dispersed in BSA solution using magnetic stirring, similar to Batch 1. The solution was then added into a semi-permeable membrane (10,000 MWCO Snakeskin™ dialysis tubing) and immersed in excess purified water for 24 hrs. The water for immersion was replaced three times during the 24 hrs, and the final osmolarity of the external media was - 9 mOsm / L, estimated to be less than 0.07% of DMSO. The external media remained clearwith minimal foam formation when shaken, indicating minimal loss of DSPE-PEG2000 or BSA through the semi-permeable membrane.

[0275] Microfluidization: After dialysis (i.e., removal of the DMSO), the formulation was microfluidized under 15,000 PSI using an LM20 Microfluidizer® Processor with a diamond interaction chamber. The formulation passed through the microfluidizer five times. The clearer solution after microfluidization suggested significant particle reduction, which was confirmed using Zetasizer analysis (Figure 2).

[0276] Lyophilization and Reconstitution: Mannitol was di ssolved in water and then added to the microfluidized formulation. The formulation was stored at -20 °C for 72 hrs, under dry ice for 6 hrs and then lyophilized at 0.15 mBar for ten days.

[0277] The lyophilized formulation retained a scaffold and was fluffy, which suggested minimal collapse during lyophilization. The lyophilized formulation was reconstituted with purified water to form a translucent dispersion having a Z-av erage particle size of 110 nm according to Zetasizer analysis. Over 95% of the model compound was recovered after sterile filtration with a regenerated cellulose filter. The results of this study on the model compound suggested that removal of DMSO and microfluidization may result in beneficial characteristics for pharmaceutical formulations comprising Compound (I).Batch 2

[0278] Method of Manufacture: Compound (I) at 0.50 g and DSPE-PEG200 at 1.84 g were dissolved in 6 g of DMSO at 60 °C. The DMSO solution was mixed with 120 g of a BSA solution (50 mg / mL) using a magnetic stirrer to form a cloudy yellow suspension.

[0279] The solution was then added to a semi-permeable membrane (10K MWCO dialysis bag) and immersed in 1 L of water for 4 hrs. The external water had an osmolality of 57 mOsm / L, indicating that most of the DMSO was removed. The external media was replaced, and the formulation was immersed overnight. The osmolality of external media was 29 mOsm / L after overnight dialysis, indicating that almost all of the DMSO had been removed from the dispersion.

[0280] The dialysed formulation was then microfluidized at 15,000 PSI for five passthroughs resulting in a transparent formulation. Mannitol at 1 g was dissolved in 40 mL of water and transferred into the bulk formulation. As shown in Figure 3, the undiluted formulation had an average particle size of - 135.7 nm and PDI of 0.12. The formulation wasthen stored in the freezer for two days, under dry ice overnight, and lyophilized at 0.15 mBar for ten days.

[0281] After ten days, the bulk of the formulation was collected, but the internal core remained frozen. Thus, the frozen core was extracted and lyophilized at 0.15 mBar for another seven days.

[0282] Approximately 8.9 g of lyophilized mass was collected. The dried mass from the periphery of the bulk formulation was extremely fluffy. Approximately 1 g of the dried mass occupied the 40 mL vial. The core of the bulk formulation was slightly less fully; approximately 2 g of the dried material occupied the 40 mL vial.

[0283] Reconstituted Formulation: The dried masses collected from the core and periphery (180 mg) of the bulk formulation were reconstituted with 0.1 * PBS+ 5 mg / mL benzyl alcohol (860 mg). HPLC analysis indicated a Compound (I) concentration of 8.9 mg / mL. After filtration with a 0.22 pm PVDF filter, 82- 86% of Compound (I) was recovered. Zetasizer analysis of the reconstituted formulations showed that the Z-average particle size (-200 nm) was similar between the materials from the core and the periphery of the bulk formulation, as shown in Figure 4.Batch 3

[0284] Method of Manufacture: BSA (6.28 g) was mixed with 122.13 g of water, resulting in a cloudy dispersion after 2 hrs of mixing with a magnetic stirrer. Compound (I) at 0.50 g and DSPE-PEG200 at 1.81 g were dissolved in 6.53 g ofDMSO at 60 °C. The DMSO solution was mixed with the BSA solution using a magnetic stirrer to form a cloudy yellow solution. The solution was then added to a semi-preamble membrane (10K MWCO dialysis bag) and immersed in 2 L of water for 4 hrs. The external water had an osmolality of 34 mOsm / L, suggesting that about 90% of the DMSO was removed. The external water was replaced, and the formulation was immersed overnight at 2 - 8 °C. The osmolality of external media after overnight dialysis was 5 mOsm / L, indicating that almost all of the DMSO was removed from the dispersion.

[0285] The dialysed formulation was then microfluidized at 15,000 PSI for two passthroughs resulting in a translucent formulation. The formulation was further microfluidized at 25,000 PSI for three pass-throughs. The resulting formulation showed improved clarity. Mannitol at 1 g was dissolved in 40 mL of water and transferred into the bulk formulation.

[0286] The formulation had an average particle size of ~ 91 nm and PDI of 0.2 according to dynamic light scattering. The formulation was then stored in the freezer for two days, under dry ice overnight, then lyophilized at 0.15 mBar.

[0287] On day 10 of lyophilizing, the lyophilization instrument failed due to prolonged power disruption. The partially lyophilized material melted and collapsed. The addition of water and repeated microfluidization failed to redisperse the particles, suggesting significant albumin denaturation during the collapse. Accordingly, the manufacturing of Batch 3 was abandoned.Batch 4

[0288] Method of Manufacture: Similar to Batch 3, 6.47 g of BSA was mixed with 155.9 g of water, resulting in a cloudy dispersion after 2 hrs of mixing with a magnetic stirrer. Compound (I) at 0.505 g and DSPE-PEG200 at 1.80 g were dissolved in 7.01 g of DMSO at 60 °C. The DMSO solution was mixed with the BSA solution using a magnetic stirrer to form a cloudy yellow solution. The solution was then added to a semi-permeable membrane (10K MWCO dialysis bag) and immersed in 2 L of water for 16 hrs. The external water had an osmolality of 43 mOsm / L, suggesting about 96% of the DMSO was removed. Mannitol at 1 g was dissolved in 40 mL of water and transferred into the bulk formulation.

[0289] The formulation was then microfluidized at 25,000 PSI for five pass-throughs resulting in a translucent formulation with a Z-ave particle size of 104.0 nm and PDI of 0.15, as shown in Figure 5. The formulation was diluted with MilliQ water due to rinsing to ensure a complete transfer of materials, and the final volume was ~ 500 mL. The formulation was stored under dry ice overnight before lyophilizing at 0.4 mBar for 2.5 weeks.

[0290] Approximately 9.51 g of lyophilized formulation was collected and filled in two 40 mL vials.

[0291] The lyophilized formulation (212.4 mg) was reconstituted with O.lx PBS + 5 mg / mL benzyl alcohol (1.005 g). Zetasizer analysis of the reconstituted formulation showed a Z-average particle size of 126 nm and PDI of 0.14 (Figure 6). HPLC analysis indicated a Compound (I) concentration of 8.0 mg / mL. After filtration with a 0.22 pm PVDF filter, ~ 94.5% of Compound (I) was recovered in the filtrate. The osmolality of the highly concentrated formulation was 318 mOsm / L. These results indicate that the lyophilized formulation can be reconstituted at a high concentration and remain suitable for injection.Batch 5

[0292] Method of Manufacture: Similar to Batch 4, 6.44 g of BSA was mixed with 159.0 g with water, resulting in a cloudy dispersion after 2 hrs of mixing with a magnetic stirrer. Compound (I) at 0.51 g and DSPE- PEG200 at 1.81 g were dissolved in 7.10 g of DMSO at 60 °C. The DMSO solution was mixed with the BSA solution using a magnetic stirrer to form a cloudy suspension. The solution was then added to a semi-permeable membrane (10K MWCO dialysis bag) and immersed in 2 L of water for 4 hrs. The external water was replaced twice over 16 hrs, and the osmolarity measurement indicated that over ~ 99% of the DMSO was removed from the formulation. Mannitol at 1.0 g was dissolved in 20 mL of water and transferred into the bulk formulation.

[0293] The formulation was then microfluidized at 25,000 PSI. The particle size was tracked during microfluidization. After three pass-throughs, the Z-average particle size was ~ 119 nm and a PDI of 0.14. The sample was diluted from 400 mL to ~ 600 mL using MilliQ water and microfluidized for two pass-throughs. The Z-ave particle size was reduced to 93 nm and PDI 0.13 (Figure 7). The formulation was then stored under dry ice overnight and lyophilized at 0.4 mBar for 2.5 weeks.

[0294] The lyophilized formulation showed minimal collapse and was fluffy. A total of 9.7 g of lyophilized powder was successfully transferred to 13 40 mL glass vials, with each 40 mL vial containing 650 - 800 mg of the lyophilized formulation.

[0295] The lyophilized formulation (212.4 mg) was reconstituted with O.lx PBS + 5 mg / mL benzyl alcohol (1.007 g). Zetasizer analysis of the reconstituted formulations showed a Z-average particle size of 118.9 nm and PDI of 0.14 (Figure 8). HPLC analysis indicated a Compound (I) concentration of 7.7 mg / mL. After filtration with a 0.22 pm PVDF filter, ~ 95.4% of Compound (I) was recovered in the filtrate. The osmolality of the concentrated formulation was 259 mOsm / L. These results indicate that the lyophilized formulation can be reconstituted at a high concentration and remain suitable for injection.

[0296] Reconstitution and Dilution System for Ophthalmic Administration: Because benzyl alcohol is not suitable as a preservative for ophthalmic applications, a reconstitution fluid was prepared that was free of benzyl alcohol. The reconstitution fluid is set forth in Table 7.

[0297] 215.3 mg of the lyophilized formulation from Batch 5 was reconstituted with 1.00 mL of the reconstitution fluid of Table 7 to achieve a Compound (I) concentration of ~ 7.7 mg / mL and an osmolality of 340 mOsm / L.

[0298] The reconstituted formulation was diluted *20 with either water, 5% mannitol, or 4.5% mannitol + 0.1 * PBS. The Z-average particle sizes in the formulation diluted with water (-110 nm) or 5% mannitol (-122 nm) were significantly smaller than when diluted with 4.5% mannitol + O.lx PBS (-235 nm). The diluted samples were stored in a refrigerator for 24 hrs and reanalysed using Zetasizer. The concentrate formulation was also stored for 24 hrs and then diluted with water to examine the stability of the concentrate. The minimal particle size change indicated that the concentrated and diluted formulations were thermodynamically stable.

[0299] Intravenous Administration System: The lyophilized formulation of Batch 2 was first reconstituted with O. l x PBS + 5mg / mL benzyl alcohol or 0.5% dextrose + 5mg / mL benzyl alcohol to - 9 mg / mL of Compound (I). The reconstituted formulations were then diluted 20- fold with different diluents, and the colloidal stability was tracked over time. The results are summarised in Table 8.

[0300] The formulations diluted with 5% (w / v) dextrose remained colloidally stable over 72 hours when stored in a refrigerator. Significant particle growth was observed after 1 hour of dilution with the reconstitution solution (0.1 x PBS, 5 mg / mL benzyl alcohol). This suggests that 5% (w / v) dextrose is a particularly suitable diluent.

[0301] Microfluidization and Particle Size: Figure 9 shows the correlation between the Z- average particle size and the particle concentration as the derived count during the microfluidization of Batches 3-5. The more diluted sample and, therefore, lower derived count, resulted in smaller particles. Further dilution did not appear to significantly reduce the size while decreasing lyophilization efficacy due to the larger aqueous volume.Example 6: Ultraviolet Light Catalyzed Degradation of Compound (I)

[0302] Compound (I) was added to methanol at a concentration of 0.1 mg / mL. The resultant samples were stored in clear and amber glass vials (Agilent, California, United States). The samples were placed in a Rayonet RPR-200 photochemical reactor (350 nm ultraviolet (UV) wavelength; 575 nm visible light wavelength) at operating temperature andanalysed after 1 and 3 days. HPLC and liquid chromatography-mass spectrometry (LC-MS) analysis indicated a decrease in intensity of the peak corresponding to Compound (I) and an increase in the intensity of other peaks for all samples (i.e., both clear and amber vials), suggesting degradation. Figure 10 shows the change in HPLC plots of the amber vial samples at 0 days (Figure 10A), 1 day (Figure 10B), and 3 days (Figure 10C).Example 7: Compound (I) Solubility in the Presence of UV Absorption Agents

[0303] Two pharmaceutical formulations, one comprising UV absorption agents (Sample 14) and one free of UV absorption agents (Sample 15), were prepared to determine whether the presence of UV Absorption Agents impacted the solubility of Compound (I). The formulations are set forth in Table 9.

[0304] Compound (I) 3.2 wt.% was mixed into Samples 14 and 15 in a roller mixer overnight, affording a final Compound (I) concentration of 2.43 wt.% for Sample 14 and 2.52 wt.% for Sample 15. These results suggest that UV absorption agents have minimal impact on the solubility of Compound (I).Example 8: Compound (I) Degradation in the Presence of UV Absorption Agents

[0305] Amber Vials: Samples 16-19, as set forth in Table 10, were prepared in transcutol. Specifically, Compound (I) was added to a mixture of the other components via overnight mixing in a roller mixer, followed by centrifugation at 12,000 RPM for 20 mins. The supernatant was collected and placed in amber vials. The resultant vials were exposed to UV radiation (350 nm; Rayonet RPR-200 photochemical reactor ) for 3 days. Minimal degradation was observed for all samples, suggesting that the amber vials protected against degradation.

[0306] Clear Vials: To accelerate degradation, Samples 16-19 were transferred to clear vials, and diluted with methanol such that the concentrations of avobenzone and oxybenzone in Samples 17 and 18 were each 0.1 wt.%. The clear vials were then exposed to UV radiation (350 nm; Rayonet RPR-200 photochemical reactor) for 18 hrs. As shown in Figure 11, samples containing the UV absorption agents (Samples 17 and 18) were lighter in color after UV radiation exposure, suggesting that the UV absorption agents provided protection against UV degradation. Sample 17, which included only the UV absorption agents, exhibited lessdegradation of Compound (I) based on HPLC analysis than Sample 16, 18, and 19, as shown in Figure 12. Specifically, Sample 17 exhibited less degradation than Sample 18, which included the UV absorption agents in addition to tocopherol, ascorbic acid, triethanolamine, and citric acid.Example 9: Concentration of UV Absorption Agents

[0307] First Study: Various concentrations of avobenzone and oxybenzone were dissolved in transcutol. Compound (I) (3-4 mg) was added to each mixture with mixing for 4 days. The resultant samples (i.e., Samples 20-25 as set forth in Table 11) were centrifuged, and the supernatant was isolated and diluted with methanol for HPLC analysis. Avobenzone and oxybenzone minimally impacted the solubility of Compound (I). The samples were transferred to glass vials and exposed to UV radiation (350 nm; Rayonet RPR-200 photochemical reactor) for 24 hrs. The amount of Compound (I) remaining in each sample was measured at 4 and 24 hours, as provided in Table 11. Sample 25, having 0.5 wt.% of avobenzone and 0.5 wt.% oxybenzone exhibited greater stability than the other samples.

[0308] Second Study: After the First Study, additional samples were prepared having avobenzone and oxybenzone concentrations up to 3.0 wt.% (Samples 26-28 as set forth in Table 12). UV degradation was carried out in a manner substantially identical to the First Study except that the amount of Compound (I) remaining in each sample was measured at 7 and 12 hours [Team: Please confirm that this statement is true of the study presented on Slide 27 of the PowerPoint presentation]. Sample 28, having 3 wt.% of avobenzone and 3 wt.% oxybenzone, exhibited greater stability than the other samples.Example 10: Stable Topical Formulation

[0309] Compound (I) was dissolved in either transcutol (Sample 29) or a mixture of 3 wt.% avobenzone and 3 wt.% oxybenzone in transcutol (Sample 30) to achieve a concentration of ~28 mg / mL of Compound (I). Hydroxypropyl methylcellulose (HPMC; ~10 wt.%) was then added to each sample to induce gel formation. The resultant gels were separated into amber glass vials and stored for 60 days in a cardboard box at 2 °C to 8 °C (in a refrigerator), at 25 °C, or at 40 °C. As shown in Table 13, degradation of Compound (I) was greater for thegel that was free of the UV absorption agents (i.e., avobenzone and oxybenzone) as compared to the gel that included the UV absorption agents.Example 11: Topical Treatment of Leishmania Braziliensis Infection with Compound (I)

[0310] NOD scid gamma mice (NSG™ mice; The Jackson Laboratory, Maine, United States of America) were inoculated subcutaneously into the right ear pinna at time = 0 with live Leishmania braziliensis parasites. By 3 weeks post-infection (wpi), minor ear swelling was noted, and this was regularly quantified with calipers that measure ear thickness. At 3 wpi, mice were randomized into two groups. With reference to Figure 13, one group of mice (N = 6) was treated twice daily with Compound (I) (1 mM, final concentration in diluent) applied topically; the other group (N = 6) received only the diluent ("not treated"). Inflammation was also scored using a pathology score on mice culled at the various time points, as shown in Figure 14. Treatment continued for 14 consecutive days between 3 and 5 wpi. All mice underwent euthanasia at 5 wpi. Figures 13-16 demonstrate that ulceration on the ears of mice infected with Leishmania parasites was greatly ameliorated by topical application of Compound (I), even when treatment was delayed until well after skin changes were observed.

[0311] Moreover, Compound (I) supressed immunopathology but had no effect on parasite burden. Ear pinna tissues harvested at 5 wpi were cultured for Leishmania parasites and viable parasites were enumerated. No difference in parasite burden was observed for mice treated with Compound (I) as compared to those treated with diluent alone, as shown in Figure 17. This data indicates that Compound (I) blocks parasite-induced inflammation, rather than reducing parasite viability and / or replication.Table 1: Example compounds of Formula ATable 2: Example compounds of Formula ATable 3: Samples 1 and 2 of Example 3Table 4: Samples 3-5 of Example 3Table 5: Samples 6 and 7 of Example 4Table 6: Samples 8-10 of Example 4Table 7: Exemplary Reconstitution Fluid for Ophthalmic ApplicationsTable 8: Reconstituted Samples 11-13 of Example 5Table 9: Samples 14 and 15 of Example 7Table 10: Samples 16-19 of Example 8aAll samples in transcutol.bAmount of Compound (I) added for mixing in the roller mixer overnight. No analysis was performed to determine the wt. % of Compound (I) in the supernatant after roller mixing.Table 11: Samples 20-25 of Example 9Table 12: Samples 26-28 of Example 9Table 13: Samples 29 and 30 of Example 10

Claims

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A lyophilized formulation comprising:Compound (I)or a pharmaceutically acceptable salt, solvate, or hydrate thereof; an albumin; and a stabilizer, wherein the stabilizer is a surfactant.

2. The lyophilized formulation of claim 1, wherein the stabilizer is a lipid or a polymer conjugated lipid.

3. The lyophilized formulation of claim 1 or claim 2, wherein the stabilizer is a polymer conjugated lipid, and wherein the polymer conjugated lipid is l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), 1,2-distearoyl- rac-glycerol-3 -methoxypoly ethylene glycol-2000 (DSG-PEG), l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), or 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

4. The lyophilized formulation of any one of claims 1-3, wherein the lyophilized formulation comprises a plurality of particles, and wherein the plurality of particles each comprise Compound (I), or a pharmaceutically acceptable salt thereof, encapsulated by the stabilizer and / or the albumin.

5. The lyophilized formulation of claim 4, wherein the plurality of particles each have a Z-average particle size of from about 1 nm to about 200 nm.

6. The lyophilized formulation of any one of claims 1-5, wherein the lyophilized formulation is prepared from an aqueous formulation.

7. The lyophilized formulation of claim 6, wherein the aqueous formulation comprises Compound (I), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and the albumin at a weight ratio of from about 1 : 1 to about 1 :20 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : albumin).

8. The lyophilized formulation of claim 6 or claim 7, wherein the aqueous formulation comprises Compound (I), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and the stabilizer at a weight ratio of from about 10: 1 to about 1 :40 (Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof : stabilizer).

9. The lyophilized formulation of any one of claims 6-8, wherein the aqueous formulation comprises the stabilizer and the albumin in a weight ratio of from about 1 : 1 to about 1 : 15 (stabilizer : albumin).

10. A pharmaceutical formulation comprising the lyophilized formulation of any one of claims 1-9, wherein the lyophilized formulation has been reconstituted in one or more pharmaceutically acceptable carriers or diluents.

11. The pharmaceutical formulation of claim 10, which has a pH of from about 4 to about 10.

12. The pharmaceutical formulation of claim 10 or claim 11, which has an osmolality of from about 200 mOsm / L to about 900 mOsm / L.

13. The pharmaceutical formulation of any one of claims 10-12, further comprising one or more pharmaceutically acceptable excipients or adjuvants.

14. The pharmaceutical formulation of any one of claims 10-13, which is configured for parenteral administration.

15. The pharmaceutical formulation of claim 14, which is configured for intravenous, topical, or ophthalmic administration.

16. The pharmaceutical formulation of any one of claims 10-15, wherein the pharmaceutical formulation comprises plurality of particles, wherein the plurality of particles each comprise Compound (I), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, encapsulated by the stabilizer and / or the albumin, and wherein the plurality of particles each have a Z-average particle size of from about 30 nm to about 300 nm following reconstitution.

17. The pharmaceutical formulation of any one of claims 10-16, wherein the pharmaceutically acceptable diluent comprises a saccharide, dextrose, propylene glycol, glycerol, water, or any combination of the foregoing.

18. A pharmaceutical formulation comprising: Compound (I)or a pharmaceutically acceptable salt, solvate, or hydrate thereof; a solvent, wherein the solvent is 2-(2-ethoxyethoxy)ethan-l-ol; and a gelling agent, wherein the pharmaceutical formulation is a gel, and wherein the pharmaceutical formulation is configured for topical administration.

19. The pharmaceutical formulation of claim 18, wherein the gelling agent is cellulose or a derivative thereof, a vinyl alcohol, a vinyl pyrrolidone, a natural gum, carrageenan, pectin, agar, alginic acid, sodium alginate, or a methacrylate.

20. The pharmaceutical formulation of claim 18 or claim 19, wherein the gelling agent is cellulose, methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose (HPMC), or hydroxyethyl cellulose (HEC).

21. The pharmaceutical formulation of claim 20, wherein the gelling agent is hydroxyproprly methylcellulose (HPMC).

22. The pharmaceutical formulation of any one of claims 18-21 , comprising from about 0.1 wt.% to about 20 wt.% of Compound (I) or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

23. The pharmaceutical formulation of any one of claims 18-22, comprising at least about 40 wt.% of the solvent.

24. The pharmaceutical formulation of any one of claims 18-23, comprising from about 0.1 wt.% to about 55 wt.% of the gelling agent.

25. The pharmaceutical formulation of any one of claims 18-24, further comprising an ultraviolet (UV) absorption agent.

26. The pharmaceutical formulation of claim 25, comprising from about 0.1 wt.% to about 20 wt.% of the UV absorption agent.

27. The pharmaceutical formulation of claim 25 or claim 26, wherein the UV absorption agent absorbs UVA rays.

28. The pharmaceutical formulation of any one of claims 25-27, wherein the UV absorption agent is avobenzone.

29. The pharmaceutical formulation of any one of claims 25-28, wherein the UV absorption agent is further defined as a first UV absorption agent, and wherein the pharmaceuticalformulation further comprises a second ultraviolet (UV) absorption agent, wherein the second UV absorption agent and the first UV absorption agent are different.

30. The pharmaceutical formulation of claim 29, comprising from about 0.1 wt.% to about 20 wt.% of the second UV absorption agent.

31. The pharmaceutical formulation of claim 29 or claim 30, wherein the second UV absorption agent absorbs UVB rays.

32. The pharmaceutical formulation of any one of claims 29-31, wherein the second UV absorption agent is oxybenzone.

33. The pharmaceutical formulation of any one of claims 29-32, comprising the first UV absorption agent and second UV absorption agent at a weight ratio of from about 5: 1 to about 1 :5 (first UV absorption agent : second UV absorption agent).

34. The pharmaceutical formulation of any one of claims 10-33 for use in a method for treating a disease or disorder associated with undesirable perforin activity.

35. A method for treating a disease or disorder associated with undesirable perforin activity in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical formulation of any one of claims 10-33.

36. Use of the lyophilized formulation of any one of claims 1-9, or the pharmaceutical formulation of any one of claims 10-33 in the manufacture of a medicament for treating a disease or disorder associated with undesirable perforin activity.

37. The pharmaceutical formulation for use of claim 34, the method of claim 35, or the use of claim 36, wherein the disease or disorder associated with undesirable perforin activity is selected from the group consisting of diabetes mellitus, Crohn's disease, colitis, inflammatory bowel disease, fibrosis and fibrotic disorders, Guillain-Barre syndrome, lupus erythematosus, psoriasis, pancreatitis, rheumatoid arthritis, sepsis, vasculitis and Wegener's granulmatosis,graft-versus-host disease, chronic or acute allograft rejection, infectious diseases, cancer and conditions associated with cytotoxic T lymphocyte- or natural killer cell-mediated immune pathology.

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