WO2025006988A2 - Galectin-3-targeted and p-selectin-targeted nanotherapies - Google Patents

Abstract

The present technology relates generally to lipid nanoparticles including a therapeutic agent and a sulfolipid targeting P-selectin or a ganglioside targeting Galectin-3 for treating or preventing a disease in a subject.

Description

GALECTIN-3-TARGETED AND P-SELECTIN-TARGETED NANOTHERAPIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/511,568, filed June 30, 2023, and U.S. Provisional Application No. 63/595,955, filed November 3, 2023, the contents of each of which are incorporated herein by reference in their entirety for any and all purposes.

U.S. GOVERNMENT SUPPORT

[002] This invention was made with government support under R01CA215719 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[003] The present technology relates generally to lipid nanoparticles including a therapeutic agent and a sulfolipid targeting P-selectin or a ganglioside targeting Galectin-3.

SUMMARY

[004] In an aspect, the present disclosure provides a composition comprising a plurality of lipid nanoparticles, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises about 5 wt.% to about 20 wt.% of a therapeutic agent, and a sulfolipid targeting P-selectin, a ganglioside targeting Galectin-3, or a PEG lipid; wherein the plurality of lipid nanoparticles is characterized by a light-intensity average diameter of about 10 nm to about 250 nm, as determined by dynamic light scattering, and a zeta potential of about -45 mV to about -30 mV, as determined by electrophoresis; and wherein the therapeutic agent is selected from 4-aminothiophenol, afatinib, alpelisib, amphotericin, artemisinin, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, budesonide, carbamazepine, celecoxib, clozapine, curcumin, cyclosporin, darapladib, DBetl, docetaxel, emricasan, erythromycin, etomidate, fulvestrant, ibrutinib, ibudilast, (-)JQ-l, ketoconazole, lamotrigine, mirdametinib, MS4078, naloxone, navitoclax, NVP-2, paclitaxel, pexidartinib, phenytoin, pimozide, rapamycin, regorafenib, resveratrol, rifampicin, rimonabant, risperidone, ropivacaine, ruxolitinib, SB203580, SC-43, SH-4-54, sorafenib, STM2457, sunitinib, terbinafine, THAL-SNS, TPCA-1, tofacitinib, topiramate, valrubicin, vemurafenib, venetoclax, vincristine, vismodegib, and WP1066. [005] In a related aspect, a method of treating or preventing kidney injury in a subject resulting from allogeneic hematopoietic cell transplantation is provided that includes administering an effective amount of the composition of any embodiment disclosed herein.

[006] In a related aspect, a method of treating or preventing graft-vs-host disease in a subject resulting from allogeneic hematopoietic cell transplantation is provided that includes administering an effective amount of the composition of any embodiment disclosed herein.

[007] In a related aspect, a method of reducing T-cell infiltration in a kidney of a subject is provided that includes administering an effective amount of the composition of any embodiment disclosed herein.

[008] In a related aspect, a method of reducing pSTAT3 phosphorylation in a kidney of a subject is provided that includes administering an effective amount of the composition of any embodiment disclosed herein.

[009] In a related aspect, a method of treating pancreatic ductal adenocarcinoma in a subject is provided that includes administering an effective amount of the composition of any embodiment disclosed herein that includes lipid nanoparticles (LNPs) comprising a ganglioside targeting Galectin-3 and a therapeutic agent comprising ARV-825.

[0010] In a related aspect, a method of reducing a tumor growth rate of a pancreatic ductal adenocarcinoma tumor in a subject is provided that includes administering an effective amount of the composition of any embodiment disclosed herein that includes lipid nanoparticles (LNPs) comprising a ganglioside targeting Galectin-3 and a therapeutic agent comprising ARV-825.

[0011] Further aspects and embodiments of the present technology are described herein.

BRIEF DESCRIPTION OF THE DRAWING

[0012] FIG. 1 is an illustration of a lipid nanoparticle with targeting lipids and hydrophobic drug.

[0013] FIGS. 2A-2C: Histology of allo-HCT murine model of graft-versus-host disease (GVHD) exhibited pathological and molecular features of AKI. FIG. 2A provides T-cell infiltration (CD3), apoptosis (TUNEL), kidney injury (NGAL, KIM-1) and renal morphology (PAS) on day 7 after bone marrow transplantation. (Scale bar: 50 pm.) FIG. 2B provides quantification of CD3, TUNEL, KIM-1 and NGAL positive cells over time. FIG. 2C provides levels of serum and urine markers of renal damage on day 7 after bone marrow transplantation. (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001, t-test.)

[0014] FIGS. 3A-3C: Multiplexed immunofluorescence and computer-aided quantification in whole tissue determined the immune landscape of the GVHD kidney. FIG. 3A provides a merged immunofluorescence image (top), and higher magnification image (bottom) of kidney tissue stained for CD8, Tbet, CD4, FoxP3, CD19, Ly6G, and DAPI. FIG. 3B provides immune cell subsets in each treatment condition. The sizes of the pie charts correspond to the relative number of immune cells per mouse group. FIG. 3C provides the ratio of Tbet+ or FoxP3+ populations within CD8+ or CD4+ cells. (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001, t-test.)

[0015] FIGS. 4A-4B: Transcriptome analysis of kidney lysates from GVHD mice revealed highly dysregulated immune-related pathways. FIG. 4A provides volcano plots of genes differentially expressed between GVHD and healthy mice on days 7, 10 and 14 post BMT. FIG. 4B provides a Hallmark pathway analysis of top dysregulated pathways between GVHD, BM only and healthy mice on days 7, 10 and 14 post transplantation.

[0016] FIGS. 5A-5G: Galectin-3 and P-selectin-targeted LNPs preferentially localize to GVHD-AKI kidneys. FIG. 5A shows Galectin-3 expression in healthy VS GVHD kidney tissues (Galectin-3 and DAPI indicated). FIG. 5B provides P-selectin expression in healthy VS GVHD kidney tissues (P-selectin and DAPI indicated). FIGS. 5C and 5D show hydrodynamic diameters of Galectin-3 and P-selectin-targeted LNPs. FIG. 5E shows a representative fluorescent image of the Galectin-3-Cy5 -LNPs biodistribution in the organs of GVHD and healthy mice (out of 5 per group) (left), and quantification of fluorescence intensity normalized per tissue area in kidney and liver (right). FIG. 5F shows a representative fluorescent image of the P-selectin-Cy5-LNPs biodistribution in the organs of GVHD and healthy mice (out of 5 per group) (left), and quantification of fluorescence intensity normalized per tissue area in kidney and liver (right). FIG. 5G provides merged fluorescence images of kidney tissue of mice injected with Galectin-3-Cy5-LNPs.

(*p<0.05, **p<0.01,***p<0.001, ****p<0.0001, t-test.) [0017] FIGS. 6A-6E: Pharmacological inhibition of Jak/Stat and NFkB pathways via LNPs on day 7 after BMT. FIG. 6A illustrates a schematic of model establishment and treatment regime for two efficacy studies. Gal: Galectin-3 targeting, pSel: P-selectin targeting. FIG. 6B provides p-STAT3 levels in mice treated with free or LNP -encapsulated Ruxolitinib. FIG. 6C is a graph of T-cell infiltration with LNP-encapsulated Ruxolitinib (left) or Tofacitinib (right) as compard to a healthy model (H). White blood cell count (FIG. 6D) and platelet count (FIG. 6E) on day 7 post BMT show that LNPs can mitigate Ruxolitinib ’s hematotoxicity. R-FD: Ruxolitinib free drug, R-LNP: Ruxolitinib LNP, T- FD: Tofacitinib free drug, T-LNP: Tofacitinib LNP. n=6 mice per group. (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001, One-way ANOVA.)

[0018] FIGS. 7A-7D: Galectin-3 -targeted Jak/Stat inhibition exhibited superior performance to free drug in attenuating severe GVHD. FIG. 7A illustrates a schematic of severe GVHD (BMT+2M T-cells) establishment and treatment regime for the third efficacy study measured on days 7 and 14 post-BMT. FIG. 7B shows Kaplan -Mei er survival curves of different groups (comparison with Mantel-Cox log rank test for survival), including LNPs without an encapsulated drug or targeting lipid (“vehicle”), ruxolitinib free drug (“RUX FD”), and LNPs encapsulating ruxolitinib (“RUX LNPs”). FIG. 7C provides graphs of body weight loss per group for the groups vehicle (“V”), ruxolitinib free drug (“R-FD”), and ruxolitinib encapsulated in LNPs (“R-LNP”). FIG. 7D provides graphs of GVHD clinical scoring, mice per group: nV =4, other groups: n=5 (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001, One-way ANOVA).

[0019] FIGS. 8A-8H: Galectin-3 -targeted Jak/Stat inhibition exhibited superior performance to free drug in the reduction of renal injury, restoration of kidney function in severe GVHD. FIG. 8A provides graphs of T-cell infiltration. Mice per group: nV=4, other groups: n=5. FIG. 8B provides graphs of pStat3 levels (IF quantification). Mice per group nV=4, other groups: n=5. FIG. 8C provides graphs of NGAL levels (IHC quantification). Mice per group: nV=4, other groups: n=5. FIG. 8D provides graphs of urine KIM-1. Mice per group: nV =4, other groups: n=5. FIG. 8E provides graphs of serum BUN levels on days 7, 10 and 14 post-BMT. Mice per group: nH=6, nV7=5, nRFD7= 11, nRLNP7 =11, nV10=4, nRFD10=6, nRLNP10=6 group. FIG. 8F is a graph of FITC-sinistrin normalized intensity curves for a healthy and a GVHD mouse measured with the Medibea-con transdermal monitor on day 7 post-BMT. FIG. 8G is a graph of half-time of FITC-sinistrin in the blood on day 7 post-BMT. FIG. 8H is a graph of glomerular filtration rate on day 7. H: healthy, V: vehicle, R-FD: Ruxolitinib Free Drug, R-LNP: Ruxolitinib LNP (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001, One-way ANOVA).

[0020] FIGS. 9A-9D: LNP size and structure for different drugs. Hydrodynamic diameter measurements (left) and cryogenic electron micrograph images (right) of Ruxolitinib Galectin-3 LNPs (FIG. 9A), WP1066 Galectin-3 LNPs (FIG. 9B), Tofacitinib P-selectin LNPs (FIG. 9C), and TPCA-1 P-selectin LNPs (FIG. 9D). (Scale bar: 50 nm).

[0021] FIGS. 10A-10D: LNP characterization with lipid ratio variation. FIG. 10A is a graph of hydrodynamic diameter of LNPs with different lipid ratios. FIG. 10B is a graph of zeta potential of LNPs with different lipid ratios. FIG. 10C is a graph of drug encapsulation of LNPs with different lipid ratios. FIG. 10D is a graph of poly dispersity index (PDI) of LNPs with different lipid ratios.

[0022] FIG. 11 is a graph of cell viability of LNPs with different lipid ratios and different particle concentrations.

[0023] FIG. 12A-12D: Efficacy of LNP-encapsulated ARV-825 in senescent pancreatic ductal adenocarcinoma (PDAC). FIG. 12A illustrates a schematic of model establishment and treatment of PDAC. FIG. 12B is a graph of Kaplan-Meier survival for PDAC BL/6 mice under different treatment conditions. FIG. 12C is a graph of tumor volume growth from day 14 to day 31 for PDAC BL/6 mice under different treatment conditions. FIG. 12D is a graph of body weight change over time for PDAC BL/6 mice under different treatment conditions.

[0024] FIGS. 13A-13F: Efficacy of Galectin-3 targeting LNP-encapsulated ARV-825 in senescent PDAC. FIG. 13A illustrates a schematic of model establishment and treatment of PDAC. FIGS. 13B and 13C are graphs of Kaplan-Meier survival for PDAC BL/6 mice comparing treatment with 1 mg/kg trametinib, 100 mg/kg Palbociclib, and LNPs as compared to treatment with LNPs alone, respectively. Kaplan-Meier survival, ** p < 0.01, * p < 0.05 by Log-rank (Mantel-Cox) test, n=5. FIGS. 13D and 13E are graphs of tumor volume change for PDAC BL/6 mice comparing treatment with 1 mg/kg trametinib, 100 mg/kg Palbociclib, and LNPs as compared to treatment with LNPs alone, respectively. ** p < 0.01, * p > 0.05 by ordinary one-way ANOVA analysis. FIG. 13F shows BRD4 immunohistochemistry for PDAC BL/6 mice under different treatment conditions. DETAILED DESCRIPTION

[0025] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

Definitions

[0026] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0027] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments, and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0028] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term - for example, “about 10 wt.%” would be understood to mean “9 wt.% to 11 wt.%.” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “ about” the term as well as the term without modification by “about” — for example, “about 10 wt.%” discloses “9 wt.% to 11 wt.%” as well as disclosing “10 wt.%.”

[0029] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another.

[0030] The phrase “and/or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof - for example, “A, B, and/or C” would mean “A, B, C, A and B, A and C, B and C, or the combination of A, B, and C.”

[0031] As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease or condition, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0032] As used herein, “prevention,” “prevent,” or “preventing” of a disease or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disease or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disease or condition relative to the untreated control sample. As used herein, prevention includes preventing or delaying the initiation of symptoms of the disease or condition. As used herein, prevention also includes preventing a recurrence of one or more signs or symptoms of a disease or condition.

[0033] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0034] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0035] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0036] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, ¹⁴C, ³²P, and ³⁵S are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.

[0037] In general, “substituted” refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group is substituted with one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents.

Examples of substituent groups include: halogens (z.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (z.e., SFs), sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (z.e., CN); and the like.

[0038] Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

[0039] As used herein, C_x-C_y, such as C1-C12, Ci-Cs, or Ci-Ce when used before a group refers to that group containing x toy carbon atoms

[0040] Alkyl groups include straight chain and branched chain alkyl groups having from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, and include without limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

[0041] Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups having from 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Exemplary monocyclic cycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 3 to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Substituted cycloalkyl groups may be substituted one or more times with non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups, which may be substituted with substituents such as those listed above. [0042] Cycloalkylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups may be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups may be substituted at the alkyl, the cycloalkyl or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.

[0043] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carboncarbon double bonds. Examples include, but are not limited to vinyl, allyl, -CH=CH(CH₃), -CH=C(CH₃)₂, -C(CH₃)=CH₂, -C(CH₃)=CH(CH₃), -C(CH₂CH₃)=CH₂ , among others. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.

[0044] Cycloalkenyl groups include cycloalkyl groups as defined above, having at least one double bond between two carbon atoms. Cycloalkenyl groups may be substituted or unsubstituted. In some embodiments the cycloalkenyl group may have one, two or three double bonds but does not include aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbon atoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.

[0045] Cycloalkenylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. Cycloalkenylalkyl groups may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted at the alkyl, the cycloalkenyl or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above. [0046] Alkynyl groups include straight and branched chain alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Alkynyl groups may be substituted or unsubstituted. Alkynyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group has one, two, or three carboncarbon triple bonds. Examples include, but are not limited to -C=CH, -C=CCH₃, -CH2OCCH3, -C=CCH2CH(CH₂CH3)₂, among others. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.

[0047] Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic, and tricyclic ring systems. Aryl groups may be substituted or unsubstituted. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. In some embodiments, the aryl groups are phenyl or naphthyl. The phrase “aryl groups” includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like). Representative substituted aryl groups may be mono-substituted (e.g., tolyl) or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.

[0048] Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Aralkyl groups may be substituted or unsubstituted. In some embodiments, aralkyl groups contain 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Substituted aralkyl groups may be substituted at the alkyl, the aryl or both the alkyl and aryl portions of the group. Representative aralkyl groups include but are not limited to benzyl and phenethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-indanylethyl. Representative substituted aralkyl groups may be substituted one or more times with substituents such as those listed above. [0049] Heterocyclyl groups include aromatic (also referred to as heteroaryl) and nonaromatic ring compounds containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Heterocyclyl groups may be substituted or unsubstituted. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4 heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi- and tricyclic rings having 3 to 16 ring members, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl groups encompass aromatic, partially unsaturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups, such as, for example, benzotri azolyl, 2,3-dihydrobenzo[l,4]dioxinyl, and benzo[l,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. The phrase includes heterocyclyl groups that have other groups, such as alkyl, oxo or halo groups, bonded to one of the ring members, referred to as “substituted heterocyclyl groups.” Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl,azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotri azolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadi azolyl, benzo [1,3] dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), tri azol opyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrol opy ri dy 1 , tetrahy dropy razol opy ri dy 1 , tetrahy droimi dazopy ri dy 1 , tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups. Representative substituted heterocyclyl groups may be mono-substituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed above.

[0050] Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotri azolyl, benzoxazolyl, benzothiazolyl, benzothiadi azolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fused ring compounds in which all rings are aromatic such as indolyl groups and include fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydro indolyl groups. Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above.

[0051] Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heterocyclyl group as defined above. Heterocyclylalkyl groups may be substituted or unsubstituted. Substituted heterocyclylalkyl groups may be substituted at the alkyl, the heterocyclyl or both the alkyl and heterocyclyl portions of the group. Representative heterocyclyl alkyl groups include, but are not limited to, morpholin-4-yl-ethyl, furan-2-yl-methyl, imidazol-4-yl-methyl, pyri din-3 -yl-m ethyl, tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those listed above.

[0052] Heteroaralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above. Heteroaralkyl groups may be substituted or unsubstituted. Substituted heteroaralkyl groups may be substituted at the alkyl, the heteroaryl or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those listed above. [0053] Groups described herein having two or more points of attachment (i.e., divalent, trivalent, or polyvalent) within the compound of the present technology are designated by use of the suffix, “ene.” For example, divalent alkyl groups are alkylene groups, divalent aryl groups are arylene groups, divalent heteroaryl groups are divalent heteroarylene groups, and so forth. Substituted groups having a single point of attachment to the compound of the present technology are not referred to using the “ene” designation. Thus, e.g., chloroethyl is not referred to herein as chloroethylene.

[0054] Alkoxy groups are hydroxyl groups (-OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Alkoxy groups may be substituted or unsubstituted. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.

[0055] The terms “alkanoyl” and “alkanoyloxy” as used herein can refer, respectively, to -C(O)-alkyl groups and -O-C(O)-alkyl groups, each containing 2-5 carbon atoms.

Similarly, “aryloyl” and “aryloyloxy” refer to -C(O)-aryl groups and -O-C(O)-aryl groups.

[0056] The terms "aryloxy" and “arylalkoxy” refer to, respectively, a substituted or unsubstituted aryl group bonded to an oxygen atom and a substituted or unsubstituted aralkyl group bonded to the oxygen atom at the alkyl. Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above.

[0057] The term “carboxylate” as used herein refers to a -COOH group.

[0058] The term “ester” as used herein refers to -COOR⁷⁰ and -C(O)O-G groups. R⁷⁰ is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. G is a carboxylate protecting group. Carboxylate protecting groups are well known to one of ordinary skill in the art. An extensive list of protecting groups for the carboxylate group functionality may be found in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999) which can be added or removed using the procedures set forth therein and which is hereby incorporated by reference in its entirety and for any and all purposes as if fully set forth herein.

[0059] The term “amide” (or “amido”) includes C- and N-amide groups, i.e., -C(O)NR⁷¹R⁷², and -NR⁷¹C(O)R⁷² groups, respectively. R⁷¹ and R⁷² are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Amido groups therefore include but are not limited to carbamoyl groups (-C(O)NH2) and formamide groups (-NHC(O)H).

In some embodiments, the amide is -NR⁷¹C(O)-(CI-5 alkyl) and the group is termed "carbonylamino," and in others the amide is -NHC(O)-alkyl and the group is termed "alkanoylamino."

[0060] The term “nitrile” or “cyano” as used herein refers to the -CN group.

[0061] Urethane groups include N- and O-urethane groups, i.e., -NR⁷³C(O)OR⁷⁴ and -OC(O)NR⁷³R⁷⁴ groups, respectively. R⁷³ and R⁷⁴ are independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R⁷³ may also be H.

[0062] The term “amine” (or “amino”) as used herein refers to -NR⁷⁵R⁷⁶ groups, wherein R⁷⁵ and R⁷⁶ are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. In some embodiments, the amine is alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.

[0063] The term “sulfonamido” includes S- and N-sulfonamide groups, i.e., -SO2NR⁷⁸R⁷⁹ and -NR⁷⁸SO2R⁷⁹ groups, respectively. R⁷⁸ and R⁷⁹ are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Sulfonamido groups therefore include but are not limited to sulfamoyl groups (-SO2NH2). In some embodiments herein, the sulfonamido is -NHSCh-alkyl and is referred to as the "alkylsulfonylamino" group. [0064] The term “thiol” refers to -SH groups, while “sulfides” include -SR⁸⁰ groups, “sulfoxides” include -S(O)R⁸¹ groups, “sulfones” include -SO2R⁸² groups, and “sulfonyls” include -SO2OR⁸³. R⁸⁰, R⁸¹, R⁸², and R⁸³ are each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. In some embodiments the sulfide is an alkylthio group, -S-alkyl.

[0065] The term “urea” refers to -NR⁸⁴-C(O)-NR⁸⁵R⁸⁶ groups. R⁸⁴, R⁸⁵, and R⁸⁶ groups are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0066] The term “amidine” refers to -C(NR⁸⁷)NR⁸⁸R⁸⁹ and -NR⁸⁷C(NR⁸⁸)R⁸⁹, wherein R⁸⁷, R⁸⁸, and R⁸⁹ are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0067] The term “guanidine” refers to -NR⁹⁰C(NR⁹¹)NR⁹²R⁹³, wherein R⁹⁰, R⁹¹, R⁹² and R⁹³ are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0068] The term “enamine” refers to -C(R⁹⁴)=C(R⁹⁵)NR⁹⁶R⁹⁷ and -NR⁹⁴C(R⁹⁵)=C(R⁹⁶)R⁹⁷, wherein R⁹⁴, R⁹⁵, R⁹⁶ and R⁹⁷ are each independently hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0069] The term “halogen” or “halo” as used herein refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

[0070] The term “hydroxyl” as used herein can refer to -OH or its ionized form, -O . A “hydroxyalkyl” group is a hydroxyl -substituted alkyl group, such as HO-CH2-.

[0071] The term “imide” refers to -C(O)NR⁹⁸C(O)R⁹⁹, wherein R⁹⁸ and R⁹⁹ are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. [0072] The term “imine” refers to -CR¹⁰⁰(NR¹⁰¹) and -N(CR¹⁰⁰R¹⁰¹) groups, wherein R¹⁰⁰ and R¹⁰¹ are each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein, with the proviso that R¹⁰⁰ and R¹⁰¹ are not both simultaneously hydrogen.

[0073] The term “nitro” as used herein refers to an -NO2 group.

[0074] The term “trifluoromethyl” as used herein refers to -CF3.

[0075] The term “trifluoromethoxy” as used herein refers to -OCF3.

[0076] The term “azido” refers to -N3.

[0077] The term “trialkyl ammonium” refers to a -N(alkyl)s group. A trialkylammonium group is positively charged and thus typically has an associated anion, such as halogen anion.

[0078] The term “isocyano” refers to -NC.

[0079] The term “isothiocyano” refers to -NCS.

[0080] The term “pentafluorosulfanyl” refers to -SF5.

[0081] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth. [0082] Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g., Na⁺, Li⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺), ammonia or organic amines (e.g., di cyclohexylamine, trimethylamine, tri ethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.

[0083] Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism and/or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and/or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms.

[0084] “ Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, which are referred to as tautomers of each other:

As another example, guanidines may exhibit the following isomeric forms in protic organic solution, also referred to as tautomers of each other:

Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology.

[0085] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.

[0086] The compounds of the present technology may exist as solvates, especially hydrates. Hydrates may form during manufacture of the compounds or compositions comprising the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. Compounds of the present technology may exist as organic solvates as well, including DMF, ether, and alcohol solvates, among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry. [0087] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. Also within this disclosure are Arabic numerals referring to referenced citations, the full bibliographic details of which are provided immediately preceding the claims. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

Graft-Versus-Host Disease Associated Kidney Injury

[0088] Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a highly effective treatment for a variety of hematologic malignancies, but it often leads to graft-versus host disease (GVHD), a condition where graft stem cells attack the recipient’s tissues. Approximately 50,000 allo-HCT procedures are performed annually worldwide, and the number continues to increase by 10-20% each year. GVHD occurs in up to 50% of patients receiving HCT from an (HLA)-matched sibling and up to 80% in unmatched donors, and carries approximately a 40% mortality rate. The clinical management of GVHD is challenging. Immuno-suppression with corticosteroids constitutes the basis of first-line therapy in both acute (aGVHD) and chronic GVHD (cGVHD), resulting in sustained responses in less than 50% of patients with aGVHD and 40-50% of patients with cGVHD, depending on initial disease severity. The JAK-STAT pathway inhibitor ruxolitinib is the only FDA-approved therapy for acute refractory GVHD. However, its use is severely limited by significant hematologic toxicity, including thrombocytopenia and anemia. Therefore, there is a substantial need to develop new therapies for the treatment of GVHD which reduces non-relapse mortality in these patients.

[0089] Acute kidney injury (AKI) is a relatively common complication that can develop after HCT, with a reported incidence rate of up to 80%, and it is increasingly recognized as a significant cause of morbidity and mortality in HCT recipients. A recent clinical study found that the incidence of acute kidney injury (AKI) was 64%, and chronic kidney disease (CKD) developed in 21% of these patients, while AKI was linked to a 2.77 HR of nonrelapse mortality. AKI post-HCT is also common within the pediatric population. Among patients with kidney injury who require dialysis, mortality approaches 100%. Thus, new methods are needed to improve patient outcomes after HCT therapy. Kidney injury directly associated with stem cell transplantation includes a wide range of structural and functional abnormalities, which may be vascular (hypertension, thrombotic microangiopathy), glomerular (albuminuria, nephrotic glomerulopathies), and tubulointerstitial. Although the etiology of AKI in HCT is multifactorial, and the kidney is not one of the traditional organtargets of GVHD, which are the gastrointestinal track, skin, and liver, there is growing evidence suggesting that immune mechanisms like those associated with GVHD play an important role in the development of kidney injury in these patients. Studies have described some of the features of renal injury in HCT in animal models; however, the progression of renal injury, the infiltration of immune cell subpopulations, and changes in gene expression are not well-characterized. Drug delivery strategies to target therapies to the kidneys constitute a clear unmet need. There are no therapeutics to treat AKI and few drugs to treat any kidney disease available, in part because of the poor pharmacokinetics of drugs with respect to the kidneys.

[0090] Therefore, in an aspect, the present technology provides Galectin-3 -targeting and P-selectin targeting lipid nanoparticles (LNPs) that target kidneys and are encapsulated with a therapeutic agent to treat AKI, as described herein.

Senescent pancreatic ductal adenocarcinoma (PDAC)

[0091] PDAC is the most common form of pancreatic cancer and one of the most aggressive solid tumor malignancies. The 5-year survival rate for patients with PDAC is less than 5%. To date, there is no effective treatment developed.

[0092] Bromodomain-containing protein 4 (BRD4) overexpression in pancreatic ductal adenocarcinoma (PDAC) promotes cancer proliferation. BRD4 is a member of the bromodomain and extraterminal domain (BET) family of proteins. BRD4 is overexpressed in PDAC tumors. BRD4 overexpression is linked to cancer progression. BRD4 binds to promoters of oncogenes like c-Myc and induces their overexpression.

[0093] ARV-825 is a senolytic BRD4 inhibitor. ARV-825 is a proteolysis-targeting chimera (PROTAC) having the formula

ARV-825, like other PROTACs, is a large molecule with lipophilic moieties at each end, which leads to poor solubility in aqueous conditions, poor permeability, and poor pharmacokinetics.

[0094] ARV-825 has been shown to provoke senolysis by targeting NHEJ and autophagy in senescent cells. Therapy-induced senescence acts as an initial anti-tumor mechanism to halt cancer cell proliferation in PDAC. Inducing senescence results in irreversible cell cycle arrest, offering a defense mechanism against cancer. Senescent cells develop a senescence- associated secretory phenotype (SASP), which includes cytokines, growth factors, and proteases. The SASP plays a dual role: reinforcing senescence to prevent cell proliferation and promoting immune surveillance to clear senescent cells. Additionally, SASP components can suppress vascularization (angiogenesis) at the tumor site, further inhibiting tumor growth and metastasis. However, persistence of therapy-induced senescence has been shown to be detrimental in the long term. Persistence of senescence can create an immunosuppressive microenvironment and promote tumor growth. Therefore, effective treatments may include therapy -induced senescence with selective killing of senescence cells by senolytic drugs.

[0095] Therefore, in an aspect, the present technology provides Galectin-3 -targeting and P-selectin targeting lipid nanoparticles (LNPs) that target PDAC tumors and are encapsulated with a therapeutic agent to treat PDAC.

Lipid Nanoparticles (LNPs)

[0096] In an aspect, the present technology provides LNPs encapsulating a therapeutic agent for delivery. The LNPs may include a targeting lipid for effectively targeting a predetermined biological tissue following the injection of the LNP formulation via localized and/or systemic routes of administration. LNPs can be administered by various means including, but not limited to, intravenous, intramuscular, intraperitoneal, or subcutaneous routes.

[0097] In an embodiment, the LNP formulation includes a targeting ligand for systemic and/or localized intravenous administration to target a predetermined tissue. The target may include a protein associated with inflammatory processes that can contribute to disease pathogenesis when dysregulated. The protein may be selected from Galectin-3 and P- selectin.

[0098] Galectin-3 is a P-galactoside-binding lectin that may be found extracellularly, making it a preferred therapeutic target. Galectin-3 plays roles in inflammation, immune response, cell adhesion, proliferation, and apoptosis. Extracellular Galectin-3 can interact with glycoproteins and glycolipids on cell surfaces, influencing cell-cell and cell-matrix interactions. Galectin-3 is implicated in various pathological conditions, including cancer (e.g., PDAC), fibrosis, cardiovascular diseases, and inflammatory disorders. It modulates immune responses and tissue remodeling processes.

[0099] P-selectin is a cell adhesion molecule (CAM) expressed on the surface of activated endothelial cells (lining blood vessels) and activated platelets, making it a preferred therapeutic target. P-selectin plays a role in the initial recruitment and adhesion of leukocytes to the site of inflammation or injury. Specifically, P-selectin interacts with specific ligands on leukocytes, such as P-selectin glycoprotein ligand- 1 (PSGL-1), facilitating leukocyte rolling along the endothelium and eventual transmigration into the tissues. P-selectin-mediated adhesion is a step in the inflammatory response, facilitating immune cell migration to sites of infection or tissue damage.

[00100] In another embodiment, the LNP formulation includes a polyethylene glycol (PEG) lipid for systemic and/or localized intravenous administration. Incorporation of the PEG lipid may impart several beneficial properties to the LNPs, including increasing LNP circulation half-life by reducing recognition and clearance by the immune system. The PEG lipid may facilitate passive targeting of LNPs to specific tissues or tumors through the enhanced permeability and retention (EPR) effect. [00101] FIG. 1 is an illustration of a non-limiting example of an LNP 100 of the present technology. The LNP 100 includes targeting lipids 110, filler lipids 120, and a therapeutic agent 130. The LNP 100 has a core-shell structure with lipid molecules (the targeting lipids 110 and filler lipids 120) encapsulating a core containing the therapeutic agent 130. The LNP 100 may protect the therapeutic agent 130 from degradation and promote controlled release of the therapeutic agent 130 at the target site. The LNP 100 may be stable in aqueous solutions, biocompatible, and well-tolerated in biological systems.

[00102] The targeting lipid 110 may include a targeting lipid targeting Galectin-3, P- selectin, or may be a passive targeting lipid. The LNP 100 may include the targeting lipid 110 in an amount of about 1% to about 80 wt.%, about 1 wt.% to about 70 wt.%, about 1 wt.% to about 60 wt.%, about 1 wt.% to about 50 wt.%, about 1 wt.% to about 40 wt.%, about 1 wt.% to about 30 wt.%, about 1 wt.% to about 20 wt.%, or about 1 wt.% to about 10 wt.%.

[00103] Galectin-3 -targeting lipids may include gangliosides. The ganglioside may include GM 1 -Ganglioside. LNPs targeting Galectin-3 may further include sphingolipid to promote targeting and/or to promote formation of the LNPs.

[00104] P-selectin targeting lipids may include sulfolipids. The sulfolipid may include sulfatide. LNPs targeting P-selectin may further include sulfated glycolipid to promote targeting and/or to promote formation of the LNPs.

[00105] The passive targeting lipid may include a PEG lipid. The PEG lipid includes a lipid molecule covalently attached to one or more PEG chains. The PEG lipid may include a PEGylated phospholipid. Examples of PEG lipids include l,2-dimyristoyl-rac-glycero-3- methoxy conjugated to a PEG chain. The PEG chain may have a chain length of about 10 carbons to about 128 carbons, including about 32 carbons to about 96 carbons, or about 64 carbons. The PEG chain may have a molecular weight of about 500 daltons to about 4000 daltons, or about 1000 daltons to about 3000 daltons, or about 2000 daltons.

[00106] The filler lipids 120 may include mixtures of phospholipids. The phospholipids may include L-a-phosphatidylglycerol and phosphatidylcholine. For example, the filler lipids 120 may include L-a-phosphatidylglycerol and phosphatidylcholine in a ratio of L-a- phosphatidylglycerol to phosphatidylcholine of about 1 to 10 to about 3.5 to 7.5, including about 1 to 10 to about 1.5:9.5, about 1 to 10 to about 2:9, about 1 to 10 to about 2.5:8.5, about 2:9 to about 2.5:8.5, about 2:9 to about 3:8, about 2.5:8.5 to and about 3:8, about 1 : 10, about 1.5:9.5, about 2:9, about 2.5:8.5, or about 3:8.

[00107] The amount of L-a-phosphatidylglycerol in the LNP 100 may be selected for biocompatibility of the LNP. The amount of L-a-phosphatidylglycerol in the LNP 100 may be about 1 wt.% to about 33 wt.% of the total lipids, including about 1 wt.% to about 3 wt.%, about 1 wt.% to about 6 wt.%, about 1 wt.% to about 9 wt.%, about 1 wt.% to about 12 wt.%, about 1 wt.% to about 18 wt.%, about 1 wt.% to about 21 wt.%, about 1 wt.% to about 24 wt.%, about 1 wt.% to about 27 wt.%, 1 wt.% to about 30 wt.%, 9 wt.% to about 18 wt.%, about 6 wt.% to about 9 wt.%, about 6 wt.% to about 12 wt.%, about 6 wt.% to about 18 wt.%, about 6 wt.% to about 21 wt.%, about 6 wt.% to about 24 wt.%, about 6 wt.% to about 27 wt.%, about 6 wt.% to about 30 wt.%, about 6 wt.% to about 33 wt.%, about 12 wt.% to about 18 wt.%, about 12 wt.% to about 21 wt.%, about 12 wt.% to about 24 wt.%, about 12 wt.% to about 27 wt.%, about 12 wt.% to about 30 wt.%, about 12 wt.% to about 33 wt.%, about 21 wt.% to about 24 wt.%, about 21 wt.% to about 27 wt.%, about 21 wt.% to about 30 wt.%, about 21 wt.% to about 33 wt.%, about 27 wt.% to about 30 wt.%, about 27 wt.% to about 33 wt.%, about 9 wt.%, about 16 wt.%, or about 33 wt.%.

[00108] The LNP 100 may further include ethyl acetate in an amount of about 2 wt.% to about 95 wt.%, including about 10 wt.% to about 90 wt.%, about 20 wt.% to about 90 wt.%, about 30 wt.% to about 90 wt.%, about 40 wt.% to about 90 wt.%, about 50 wt.% to about 90 wt.%, about 60 wt.% to about 90 wt.%, about 70 wt.% to about 90 wt.%, or about 80 wt.% to about 90 wt.%.

[00109] The LNPs may include the therapeutic agent in an amount of about 1 wt.% to about 20 wt.%, including 2 wt.% to about 18 wt.%, about 4 wt.% to about 16 wt.%, about 6 wt.% to about 14 wt.%, about 5 wt.% to about 15 wt.%, about 8 wt.% to about 12 wt.%, or about 10 wt.%.

[00110] The LNPs may have an intensity -weighted average diameter, as determined by dynamic light scattering (DLS), of about 10 to about 250 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm.

[00111] The LNPs may include a ganglioside targeting Galectin-3 and the intensity- weighted average diameter, as determined by dynamic light scattering (DLS), may be about 10 nm to about 120 nm, including about 20 nm to about 110 nm, about 30 nm to about 100 nm, or about 40 nm to about 90 nm.

[00112] The LNPs may include a sulfolipid targeting P-selectin and the intensity -weighted average diameter, as determined by dynamic light scattering (DLS), may be about 20 nm to about 250 nm, including about 30 nm to about 200 nm, 40 nm to about 180 nm, 50 nm to about 160 nm, 60 nm to about 140 nm, or about 80 nm to about 120 nm.

[00113] The LNPs may have a diameter from about 10 to 500 nm, about 20 to 250 nm, or about 80 nm to 120 nm.

[00114] The LNPs may have a poly dispersity index (PDI), as measured by DLS, of about of about 0.1 to about 0.5, such as, but not limited to, about 0.1 to 0.15, about 0.1 to 0.2, about 0.1 to 0.3, about 0.1 to 0.35, or about 0.1 to 0.4.

[00115] The LNPs may have a zeta potential, as characterized by electrophoresis, of about -100 mV to -5 mV, about -90 mV to -10 mV, about -80 mV to about -20 mV, about -70 mV to about -20 mV, about -60 mV to about -20 mV, about -50 mV to about -230 mV, about -45 mV to -30 mV, or about -40 mV to -30 mV.

[00116] In any embodiment, such LNPs are synthesized using methods comprising microfluidic mixers. For example, microfluidic mixers may include, but are not limited to a slit interdigitial micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing. Langmuir. 2012. 28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 2012. I :e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51 ; each of which is herein incorporated by reference in its entirety). In some embodiments, methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure- induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Application Publication Nos. 2004/0262223 and 2012/0276209, each of which is expressly incorporated herein by reference in their entirety. The SHM may be, for example, a NanoAssemblr™ Benchtop (Precision Nanosystems, Canada).

Therapeutic Agent

[00117] In an aspect, the present disclosure provides a LNP composition comprising LNPs encapsulating a therapeutic agent. The LNP composition may be adjusted to better encapsulate different therapeutic agents. For example, the ratio of different filler lipids may be adjusted, and the type of targeting lipid may be changed to successfully form LNPs. The size and surface properties of the LNP may vary depending on the therapeutic agent encapsulated.

[00118] The therapeutic agent may be selected from 4-aminothiophenol, afatinib, alpelisib, amphotericin, artemisinin, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, budesonide, carbamazepine, celecoxib, clozapine, curcumin, cyclosporin, darapladib, DBetl, docetaxel, emricasan, erythromycin, etomidate, fulvestrant, ibrutinib, ibudilast, (- )JQ- 1 , ketoconazole, lamotrigine, mirdametinib, MS4078, naloxone, navitoclax, NVP-2, paclitaxel, pexidartinib, phenytoin, pimozide, rapamycin, regorafenib, resveratrol, rifampicin, rimonabant, risperidone, ropivacaine, ruxolitinib, SB203580, SC-43, SH-4-54, sorafenib, STM2457, sunitinib, terbinafine, THAL-SNS, TPCA-1, tofacitinib, topiramate, valrubicin, vemurafenib, venetoclax, vincristine, vismodegib, and WP1066.

[00119] Where the LNP composition includes LNPs including a ganglioside for targeting Galectin-3, the therapeutic agent may be selected from aminothiophenol, afatinib, amphotericin, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, celecoxib, curcumin, darapladib, emricasan, fulvestrant, ibrutinib, ibudilast, (-)JQ-l, ketoconazole, lamotrigine, mirdametinib, naloxone, NVP-2, pimozide, rapamycin, regorafenib, resveratrol, rimonabant, risperidone, ropivacaine, ruxolitinib, SB203580, SH-4-54, sorafenib, sunitinib, THAL-SNS, topiramate, valrubicin, venetoclax, vincristine, vismodegib, and WP1066.

[00120] Where the LNP composition includes LNPs including a sulfolipid for targeting P- selectin, the therapeutic agent may be selected from 4-aminothiophenol, afatinib, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, celecoxib, clozapine, curcumin, erythromycin, fulvestrant, ibudilast, (-)JQ-l, mirdametinib, naloxone, navitoclax, pexidartinib, pimozide, rapamycin, regorafenib, resveratrol, rifampicin, rimonabant, risperidone, ropivacaine, ruxolitinib, SC-43, SH-4-54, STM2457, sunitinib, terbinafine, tofacitinib, topiramate, valrubicin, venetoclax, vincristine, and WP1066.

[00121] Where the LNP composition includes LNPs including a PEG lipid, the therapeutic agent may be selected from 4-aminothiophenol, afatinib, alpelisib, artemisinin, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, budesonide, carbamazepine, celecoxib, clozapine, curcumin, cyclosporin, DBetl, docetaxel, erythromycin, fulvestrant, ibrutinib, ibudilast, (-)JQ-l, ketoconazole, mirdametinib, MS4078, NVP-2, paclitaxel, phenytoin, pimozide, rapamycin, regorafenib, resveratrol, rifampicin, rimonabant, risperidone, ruxolitinib, SB203580, SC-43, SH-4-54, sorafenib, terbinafine, THAL-SNS, TPCA-1, tofacitinib, topiramate, vemurafenib, venetoclax, vincristine, vismodegib, and WP1066.

Pharmaceutical Compositions and Preparations of the Present Technology

[00122] In any and all embodiments of the LNP composition disclosed herein, the composition may further comprise a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment. Disclosed herein are pharmaceutical compositions comprising LNPs of the present disclosure that may contain a carrier or diluent, which can be a solvent or dispersion medium containing, for example, water, saline, Tris buffer, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. 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 dispersion and by the use of surfactants. Prevention of the action of microorganisms can be affected by various antibacterial and antifungal agents and preservatives, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars (e.g., sucrose) or sodium chloride, and buffering agents are included. Isotonic agents may be present in an amount of about 1% (w/v) to about 40% (w/v), about 5 % (w/v) to about 30% (w/v), about 5% (w/v) to about 20% w/v), or about 10% (w/v).

[00123] Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin or carrier molecules. Other excipients may include wetting or emulsifying agents. In general, excipients suitable for injectable preparations can be included as apparent to those skilled in the art.

[00124] Pharmaceutical compositions and preparations comprising LNPs may be manufactured by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries that facilitate formulating preparations suitable for in vitro, in vivo, or ex vivo use. The compositions can be combined with one or more additional biologically active agents and may be formulated with a pharmaceutically acceptable carrier, diluent, or excipient to generate pharmaceutical (including biologic) or veterinary compositions of the instant disclosure suitable for parenteral or intravenous administration.

[00125] Many types of formulation are possible as is appreciated by those skilled in the art. The particular type chosen is dependent upon the route of administration chosen, as is well- recognized in the art. For example, systemic formulations will generally be designed for administration by injection, e.g., intravenous, as well as those designed for intratumoral delivery. In some embodiments, the systemic or intratumoral formulation is sterile. [00126] Sterile injectable solutions are prepared by incorporating LNPs in the required amount of the appropriate solvent with various other ingredients enumerated herein, as required, followed by suitable sterilization means. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.

[00127] In some embodiments, the LNP compositions of the present disclosure may be formulated in aqueous solutions, or in physiologically compatible solutions or buffers such as Hanks’s solution, Ringer’s solution, mannitol solutions or physiological saline buffer. In certain embodiments, any of the LNP compositions may contain formulator agents, such as suspending, stabilizing, penetrating or dispersing agents, buffers, lyoprotectants or preservatives such as polyethylene glycol, polysorbate 80, l-dodecylhexahydro-2H-azepin- 2-one (laurocapran), oleic acid, sodium citrate, Tris HC1, dextrose, propylene glycol, mannitol, polysorbate polyethylene sorbitan monolaurate (Tween®-20), isopropyl myristate, benzyl alcohol, isopropyl alcohol, ethanol sucrose, trehalose and other such generally known in the art may be used in any of the compositions of the instant disclosure. (Pramanick et al., Pharma Times 45(3):65-76 (2013)).

Modes of Administration of LNP Compositions of the Present Technology

[0001] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Administration of LNP compositions of the present technology can be achieved using more than one route. Examples of routes of administration include, but are not limited to parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), intratumoral, intrathecal, intranasal, systemic, transdermal, iontophoretic, intradermal, intraocular, pleural, intranodal, intrapleural, or topical administration. In one embodiment, LNP compositions of the present technology are administered directly into a tumor, e.g., by intratumoral injection, where a direct local reaction is desired. Additionally, administration routes of LNP compositions of the present technology can vary, e.g., first administration using an intratumoral injection, and subsequent administration via an intravenous injection, or any combination thereof. A therapeutically effective amount of LNP compositions of the present can be administered by injection for a prescribed period of time and at a prescribed frequency of administration. In certain embodiments, LNP compositions of the present technology can be used in conjunction with other therapeutic treatments. [0002] In some embodiments, the therapeutically effective amount of LNP compositions is administered to a subject with graft-versus-host (GVH) disease with kidney injury. The therapeutically effective amount of LNP compositions may be administered to a subject with kidney injury resulting from allogeneic hematopoietic cell transplantation. The therapeutically effective amount of LNP compositions may be administered to a subject to reduce T-cell infiltration in the subject’s kidney. The therapeutically effective amount of LNP compositions may be administered to a subject to reduce pSTAT3 phosphorylation in the subject’s kidney.

[0003] In some embodiments, the LNP compositions of the present are administered to a subject with pancreatic ductal adenocarcinoma (PDAC). The therapeutically effective amount of LNP compositions may be administered to a subject to reduce the tumor growth rate of the PDAC tumor.

Effective Amount and Dosage of LNP Compositions of the Present Technology

[00128] A therapeutically effective amount of LNP compositions of the present technology can be administered in one or more divided doses for a prescribed period of time and at a prescribed frequency of administration.

[00129] For example, as is apparent to those skilled in the art, a therapeutically effective amount of LNP compositions of the present technology in accordance with the present disclosure may vary according to factors such as the disease state, age, sex, weight, and general condition of the subject, and the ability of LNP compositions of the present technology to elicit a desired therapeutic response in the particular subject (the subject’s response to therapy). In delivering LNP compositions of the present technology to a subject, the dosage will also vary depending upon such factors as the general medical condition, previous medical history, disease type and progression, tumor burden, the presence or absence of tumor infiltrating immune cells in the tumor, and the like.

[00130] In general, the subject is administered a dosage of the LNP composition of the present technology in the range of between about 1 mg/kg and about 100 mg /kg, including about 10 mg/kg to about 40 mg/kg, about 5 mg/kg to about 20 mg/kg, about 10 mg/kg, or about 20 mg/kg.

[00131] In some embodiments, it may be advantageous to formulate compositions of the present disclosure 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 mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically or veterinary acceptable carrier.

Therapeutic Methods of the Present Technology

[00132] In an aspect, the present disclosure provides a method for treating or preventing kidney injury in a subject resulting from allogeneic hematopoietic cell transplantation. The method may include administering an effective amount of an LNP composition of the present technology to the subject.

[00133] In an aspect, the present disclosure provides a method of treating or preventing graft-vs-host disease in a subject resulting from allogeneic hematopoietic cell transplantation. The method may include administering an effective amount of an LNP composition of the present technology to the subject.

[00134] In an aspect, the present disclosure provides a method of reducing T-cell infiltration in a kidney of a subject. The method may include administering an effective amount of an LNP composition of the present technology to the subject.

[00135] In an aspect, the present disclosure provides a method of reducing pSTAT3 phosphorylation in a kidney of a subject. The method may include administering an effective amount of an LNP composition of the present technology to the subject.

[00136] In any of the above methods in this section, the LNPs may include a ganglioside targeting Galectin-3 or a sulfolipid targeting P-selectin. The therapeutic agent may include ruxolitinib or WP 1066

[00137] In an aspect, the present disclosure provides a method of treating pancreatic ductal adenocarcinoma in a subject. The method may include administering an effective amount of an LNP composition of the present technology to the subject, wherein the LNPs may include the ganglioside targeting Galectin-3 and the therapeutic agent may be ARV-825. This method may further include administering an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the LNP composition to the subject to induce senescence. [00138] In an aspect, the present disclosure provides a method of reducing a tumor growth rate of a pancreatic ductal adenocarcinoma tumor in a subject. The method may include administering an effective amount of an LNP composition of the present technology to the subject, wherein the LNPs may include the ganglioside targeting Galectin-3 and the therapeutic agent may be ARV-825. This method may further include administering an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the LNP composition to the subject to induce senescence.

EXAMPLES

[00139] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compositions and systems of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects, or embodiments of the present technology. The following Examples demonstrate the preparation, characterization, and use of illustrative compositions of the present technology treat or prevent kidney injury associated with graft-versus-host disease.

EXAMPLE 1: Graft-Versus-Host Disease Promotes Inflammatory Kidney Injury Reversed by Galectin-3-Targeted Nanodelivery of Jak/Stat Therapy

Materials and Methods

[00140] Mice and bone marrow transplantation and assessment of GVHD. All mice in this study were maintained under protocols approved by the Institutional Animal Care and Use Committee at Weill Cornell Medicine and MSKCC. Eight- to ten-week-old female C57BL/6 and BALB/c recipient mice were purchased from Jackson Laboratories. Mouse allogeneic hematopoietic stem cell transplantation (allo-HSCT) experiments were performed with BALB/c mice receiving 900 centiGray (cGy) of fractionated lethal radiation followed by tail vein injection of 5 * 10⁶ T-cells depleted (with anti-Thy-1.2 and Low-Tox- M rabbit complement (CEDARLANE Laboratories) bone marrow from C57BL/6 mice. A bone marrow only (BMO) group received only bone marrow, while the graft-versus-host disease (GVHD) group intravenously received splenic T-cells. Donor T-cells were prepared by harvesting donor splenocytes and enriching T-cells by Miltenyi MACS purification of CD5 (routinely >90% purity). Before the beginning of each experiment, each cage of mice was separated from the vendor to be divided as equally as possible among the various treatment groups. This helped to minimize the chances of incorrectly attributing differences observed in renal pathology to treatment effects, when in reality they are due to cage effects. Animals were housed under a 12/12 hour light/dark cycle and given access to food and water ad libitum. All groups were followed up a week after the bone marrow (BM) transplant for GVHD and scored according to five clinical parameters (weight, posture, fur, skin, activity).

[00141] Serum and urine biomarker quantification. For serum chemistry, blood was collected into tubes containing a serum separator. The tubes were then centrifuged, and the serum was obtained for analysis. Blood urea nitrogen concentration (BUN) and creatinine concentration were measured in a Beckman Coulter AU680 analyzer. Neutrophil Gelatinase- Associated Lipocalin (NGAL) and Kidney Injury Molecule-1 (KIM-1) were measured using Abeam ELISA kits (Mouse Lipocalin-2 ELISA Kit (NGAL) (ab 199083) and Mouse KIM-1 ELISA Kit (TIM1) (ab213477). Urine was centrifuged before ELISA measurements according to the kit’s instructions. Urine NGAL/KIM-1 values were normalized to urine creatinine, also measured in a Beckman Coulter AU680 analyzer.

[00142] Glomerular filtration rate (GFR) measurements. Transcutaneous glomerular filtration rate (tGFR) measurements, normalized for animal weight, were performed using a transdermal continuous renal function monitor (MediBeacon GMBH, Manheim, Germany). The mice involved in the experiment were weighed, anesthetized, and had their fur removed from the upper back region in the thoracic area by shaving and epilation cream. The devices were then secured on the hairless area using double-sided adhesive patches provided by MediBeacon and surgical tape (Micropore, 3M Health Care, St Paul, MN). A mixture of normal saline and FITC-sinistrin (Mannheim Pharma & Diagnostics, Mannheim, Germany) was injected intravenously via tail vein. Devices were left on for at least 65 minutes while mice were placed in individual cages. Measures were taken to ensure minimal stimulus including lower lighting and minimal sound. GFR was calculated from FITC-sinistrin plasma clearance using an established two-compartment model. [00143] RNA sequencing: RNA extraction. On the day of harvesting, mice were anesthetized with ketamine/xylazine and then subjected to transcardial perfusion with PBS until colorless fluid was observed coming from the right atrium. Kidneys were immediately harvested, and flash frozen in liquid nitrogen. 20-30 mg frozen tissue were homogenized in 1 mL TRIzol Reagent (ThermoFisher catalog # 15596018) and phase separation was induced with 200 pL chloroform. RNA was extracted from 350 pL of the aqueous phase using the miRNeasy Mini Kit (Qiagen catalog # 217004) on the QIAcube Connect (Qiagen) according to the manufacturer’s protocol. Samples were eluted in 35 pL RNase-free water.

[00144] Transcriptome sequencing. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 500 ng of total RNA with RIN values of 8.9-9.8 underwent polyA selection and TruSeq library preparation according to instructions provided by Illumina (TruSeq Stranded mRNA LT Kit, catalog # RS-122-2102), with 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PEI 00 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina). An average of 37 million paired reads was generated per sample. Ribosomal reads represented 0.62% of the total reads generated and the percent of mRNA bases averaged 87%.

[00145] Transcriptome sequencing analysis. Quality control (QC) was performed with FastQC (vO.11.9). Raw reads were trimmed using Trimmomatic (v0.38) with default parameters for paired-end reads and the cropping option specific to the TruSeq PE adapters (TruSeq3-PE-2.fa). Trimmed reads were then aligned with STAR aligner (v2.7.0e) against the mouse genome assembly (GRCm38.p5) and the aligned reads were in turn assigned to genes using featureCounts (vl .6.3). The resulting count tables were imported to R for further processing, analysis, and visualization. Lowly expressed genes were filtered out using edgeR’s filterByExpr function and normalization factors for library size scaling were calculated using edgeR’s cal cNormF actors function. Differential expression analysis was performed using the limma’s voom function and a threshold of FDR<0.05 was set to define genes that change with significance between the different datasets. The table of all differentially expressed genes and their fold changes was used as a pre-ranked list in GSEA (v4.2.2) against the mouse gene set hallmark resource (mh. all. vO.3. symbols. gmt) to predict signaling pathways that are enriched in any of the pairwise comparisons. Pathways were defined as enriched if they had a false discovery rate (FDR) value < 0.05. [00146] Staining and Image Analysis. Immunohistochemistry. Immunohistochemical staining of murine kidney tissues was performed at the Molecular Cytology Core Facility of Weill Cornell Medicine or MSKCC. Kidney tissues were fixed in 4% paraformaldehyde overnight. Fixed tissues were embedded in paraffin and sections prepared at a thickness of 5 pm. CD3, NGAL: IHC was performed using a rabbit polyclonal CD3 antibody

(Agilent, Santa Clara, CA) and a Rabbit monoclonal NGAL antibody [EPR21092] (Abeam, Cambridge, MA) on a Leica Bond system (Buffalo Grove, IL) following the manufacturer’s protocol. The sections were pre-treated using heat mediated antigen retrieval with Tris- EDTA buffer (pH = 9, epitope retrieval solution 2) for 20 minutes or sodium citrate buffer (pH = 6, epitope retrieval solution 1) for 30 minutes. Tissue sections were incubated with CD3 or NGAL antibody (1 : 100 dilution) for 15 minutes at room temperature. Target protein was detected using a horseradish peroxidase (HRP) conjugated compact polymer system and 3, 3 '-Diaminobenzidine (DAB) as the chromogen. Each section was counterstained with hematoxylin and mounted with Leica Micromount. All images were taken with either a bright-field and fluorescence microscope (Zeiss Axio Observer) or digital Panoramic Slide Scanner (3D Histech, Budapest Hungary).

[00147] Multiplex Immunofluorescence. Multiplex immunofluorescence followed by sequential immunohistochemistry (IHC) with automated external controls (AEC) chromogen was performed on Leica Bond staining processors with paraffin tissue sections. Slides were scanned using a Panoramic 250 scanner (3DHistech, Budapest, Hungary). A 20* 0.8 numerical aperture (NA) objective was used. The multiplexed fluorescence was imaged with a pco.edge 4.2 4 MP camera, while the AEC slides were imaged with a CIS VCC-FC60FR19CL camera.

[00148] Whole slide image analysis. Immunofluorescence image analysis. Cell segmentation was carried out using the open-source digital pathology software QuPath vO.4.3 (qupath.github.io). Nuclear detection was performed on the DAPI channel using an unsupervised watershed algorithm with parameters tuned on a validation set of 10 ROI. After nuclear detection, the cytoplasm around each nucleus was simulated by cell expansion of 5 pm and measurements generated for marker intensity in different compartments (mean, minimum, maximum, and standard deviation of intensity in cytoplasm, nucleus, or the whole cell). Positivity was determined by the intensity of each marker in the primary cell compartment where it is usually expressed. In this study, markers were cytoplasmic or membranous. Guided by our pathology core, a single threshold for each marker was selected as a cut-off to determine positivity across the entire dataset. The threshold was identified by its ability to separate positive from negative cells in a set of 10 ROIs from 10 different samples. In order to avoid bias introduced during ROI selection, each ROI included a whole kidney slice, which resulted in detection of 200,000-400,000 cells per kidney, with the associated intensity values for each channel.

[00149] Immunohistochemistry image analysis. Nuclear detection was performed on the hematoxylin channel and positivity was determined by the intensity of the DAB chromogen, with the thresholding validation performed as described above.

[00150] LNP Synthesis. Chicken egg L-a-phosphatidylglycerol (sodium salt) (PG) was purchased from Avanti Polar Lipids, Inc. (AL, USA) and soy Phosphatidylcholine (PC) was from Lipoid GmbH (Germany). Particles were prepared using the NanoAssemblr™ Benchtop (Precision Nanosystems, Canada), which is a staggered herringbone micromixer (SHM). The organic phase consisted of a lipid mixture and a small molecule drug in ethyl acetate. PG, PC, and ethyl acetate were mixed in a 1 : 10:90 mass ratio. The small molecule drug of choice was added to this lipid mixture at 5.6 mM final concentration. The aqueous phase consisted of GM 1 -Ganglioside sodium (Carbosynth Limited, UK) or sulfatides (Matreya Inc, PA, USA) dissolved in water, in a concentration of 2 mg/mL and 3 mg/mL, respectively. Before the formulation the cartridge was prewashed with water and ethyl acetate at a flow rate of 4 mL/min, total volume 2 mL, and water: ethyl acetate ratio of 1 : 1. Afterwards the aqueous and organic phases were loaded into separate syringes and pumped through the NanoAssemblr cartridge at a total flow of 8 mL/min and volume ratio of 8.5: 1.5. For fluorescent LNPs used in biodistribution studies, after the microfluidic mixing, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5) purchased from

Avanti Polar Lipids, Inc. (AL, USA) was dissolved in ethanol to a stock concentration of 5 mg/mL and added to the LNP mixture at a final concentration of 0.1 mg/mL. The resulting mixture was briefly vortexed and placed into a rotor evaporator to remove the organic solvent. For in vivo administration, D(+)-sucrose was added to the mixture to a final concentration of 10% (w/v) and the resulting mixture was filtered through a 0.2 pm filter.

[00151] LNP characterization. Size measurements. Size (including number, volume, and intensity mean) as well as PDI were determined by dynamic light-scattering (DLS) measurements acquired with a Malvern Zetasizer Nano ZS. Nanoparticles were prepared for DLS by diluting 1 : 100 in saline solution. Size and homogeneity of nanoparticles was further characterized by cryogenic electron microscopy (cryo-EM) using a Titan Krios G2 (ThermoFisher, MA, USA) with a K3 detector (Gatan, CA, USA).

[00152] Drug loading. Drug concentration in the LNPs was quantified using high- performance liquid chromatography (HPLC). Nanoparticles were prepared for HPLC analysis by first vortexing the LNP suspension with a 50/50 methanol/saline solution in a 1 :4 (LNP: solution) ratio to release the drug from the nanoparticles. This was further diluted in methanol for a final drug dilution of 1 :50. Samples were then assessed on an Agilent 1260 Infinity II HPLC system with an InfinityLab Poroshell 120 EC-C18, 4.6 x 75 mm², 2.7 pm analytical LC column. The mobile phase consisted of acetonitrile and/or deionized water, each containing 0.1% trifluoroacetic acid. Chromatographic separation was achieved by gradient elution with acetonitrile (0-95%) at a flow rate of 1 mL min^-1. For each small molecule drug used, a standard curve was also prepared and used for quantification. For each drug, a single peak was observed at their corresponding retention times and absorbances. Prior to HPLC analysis, The UV-visible absorbance spectrum for each drug tested was obtained using a UV/visible/near infrared spectrophotometer (Jasco V-670, Tokyo, Japan) to select the appropriate absorbance wavelength for HPLC measurements.

[00153] Drug Release. The drug release kinetics of LNPs was characterized by performing dialysis. LNPs were confined within a pre-wetted dialysis membrane compatible with hydrophobic drugs (Spectra/Por® 6 Standard RC tubing, MWCO 50 kD, 28 mm width, 1 m length) which allowed the release of free drug into the dialysate outside the membrane. The dialysate was comprised of equal parts PBS and adult bovine serum (ABS) (ThermoFisher, MA, USA) to simulate physiological conditions, with 1% DMSO (Alfa Aesar, MA, USA) and 1% Tween-80 (MP Biomedicals, France) added to increase drug solubility and thus ensure sink conditions were satisfied. LNPs within the dialysis membrane were in a solution of the same composition. Moreover, the volume of LNPs to dialysate was 1 : 100. The dialysis system was maintained at a constant 37 °C and mixed at 75 rpm by a stirring hot plate. Over a 48-hour period, 150 pL was sampled from within the dialysis bag at each timepoint.

[00154] Quantification of the drug remaining within the dialysis bag was conducted by HPLC. LNPs withdrawn from the dialysis bag were diluted 1 : 50 in methanol to precipitate serum proteins, which were then spun down at 30,000 ref for 6 minutes. The supernatant was subsequently filtered through 0.2 pm Whatman syringeless filter devices made of polytetrafluoroethylene (PTFE) (Cytiva, MA, USA). HPLC analysis was then conducted.

[00155] LNP biodistribution studies. GVHD (1 M T-cells) was induced as described above. The study groups were: GVHD- IM - 5 mice, Healthy - 5 mice, Uninjected control (for background fluorescence normalization) - 1 healthy and 1 GVHD mouse. On day 6 after bone marrow transplant (BMT), Cy5-conjugated LNPs were injected intraperitoneally (IP) into the mice, and mice were sacrificed 24 hours later. Liver, kidneys, heart, lung, and intestines were harvested and washed briefly with PBS. Tissues were placed on a petri dish (tissues from the same mouse were grouped together), marked with a permanent marker for identification. Tissues were imaged in an Invitrogen iBright FL1500 Imaging System, in fluorescence mode, choosing the Cy5 excitation and detection wavelengths. Various durations of exposure time were experimented, until 1 second was chosen for all samples as it produced no saturated pixels but enough intensity for quantification. Mouse intestines were highly auto-fluorescent, so they were excluded from subsequent images as they were saturating the receptor signal. All other mouse organs from uninjected mice were placed in the same imaging plane for autofluorescence normalization. Average fluorescence intensity normalized by the area was quantified by Fiji.

[00156] Efficacy Studies 1 and 2 (IM T-cells, 7 days). Efficacy study 1 (ruxolitinib, WP1066): GVHD (1 M T-cells) in mice was induced as described above. Mice were treated with vehicle (V groups), 20 mg/kg free drug (FD groups) or drug encapsulated in Galectin-3 LNPs (LNP groups), administered intraperitoneally on days 2, 4 and 6 after BMT. Mice were scored on day 7, then euthanized and blood and organs were harvested for analysis. Ruxolitinib was dissolved in 5% DMSO and 2% Tween80, and WP1066 was dissolved in 10% DMSO and 40% PEG300.

[00157] Efficacy study 2 (Tofacitinib, TPCA-1): GVHD (1 M T-cells) in mice was induced as described above. Mice were treated with vehicle (V groups), 20 mg/kg free drug or drug encapsulated in Galectin-3 LNPs, administered intraperitoneally daily on days 2 to 6 after BMT. The last dose of TPCA-1 was not administered because mice died on day 5 (see notes on on-target toxicity of NFKB inhibition above). Mice were scored on day 7, then euthanized and blood and organs were harvested for analysis. Tofacitinib was dissolved in 5% DMSO and WP1066 was dissolved in 5% DMSO and 30% PEG300. [00158] Efficacy Study 3 (2M T-cells, 14 days). GVHD (2 M T-cells) in mice was induced as described above. Mice were divided in day 7 groups and day 14 groups. Each day group consisted of vehicle (V), free drug (FD) and LNP (R-LNP) groups, that received free vehicle, free Ruxolitinib or Ruxolitinib Galectin 3 -binding LNPs, daily from day 2 after BMT until the day of sacrifice. Day 7 and day 14 groups were sacrificed on the respective days and blood and organs were collected for analysis.

Results and Discussion

[00159] GHVD induces kidney inflammation and injury. The pathology and molecular signatures of hematopoietic stem cell (HCT) mediated kidney injury were investigated to understand the disease time course and tissue/cell -types involved, to identify therapeutic targets, and to determine the degree to which the murine model recapitulates the immune modulation in the kidneys of patients after allogeneic hematopoietic stem cells (allo-HCT). HCT was conducted in mice by administering 900 cGy of fractionated lethal radiation to BALB/c mice followed by retroorbital injection of 5 x io⁶ T-cell depleted bone marrow from C57BL/6 mice. 1 million (M) splenic T-cells were then injected intravenously into one group (the GVHD group), and a second group was not given T-cells (bone marrow only (BMO) group). The number of injected T-cells, correlated with the severity of the induced GVHD.

[00160] Kidney histo-pathologic analysis was performed to determine the degree and nature of renal injury. PAS stain and immunohistochemistry (IHC) were used for Cluster of Differentiation 3 (CD3), Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL), and Neutrophil Gelatinase-Associated Lipocalin (NGAL) analysis. At day 7 (FIG. 2A), transplanted mice had significant tubular injury including tubular necrosis, loss of brush border, and vacuolization (blind analysis by a renal pathologist). In addition, extensive CD3⁺ cell infiltration was observed in both tubules and glomeruli, apoptosis, as well as increased Kidney Injury Molecule-1 (KIM-1) and NGAL, both sensitive markers of kidney injury. While NGAL damage decreased from day 7 to day 14, CD3 infiltration increased, indicating continuous activation of the immune system (FIG. 2B). A significant increase in NGAL and KIM-1 was also observed in serum and urine (FIG. 2C).

[00161] To obtain a better understanding of the immune landscape in GVHD kidneys, the immune cell subpopulations infiltrating the kidneys was characterized. Multiplexed immunofluorescence and computer-aided quantification was performed in whole tissue to determine how immune cell numbers differ from healthy tissue or the tissues of BMO controls (FIG. 3A). In GVHD kidneys, 50.6% of the infiltrated immune cells were CD8+ cytotoxic T-cells, while Tbet+ Thl cells were the second most prominent population (33.17%). The distribution of other populations was 8.77% CD4+ T-helper cells, 4.17% FoxP3+ Regulatory T-cells, 2.34% B cells and 0.95% Neutrophils. (FIG. 3B). Along with the total number of infiltrated immune cells, which was 10 times higher in GVHD kidneys compared to healthy and BMO groups, the ratios of specific subpopulations were also different. The fast recruitment of CD8+ cells and their higher percentage compared to CD4+ cells (10: 1) was consistent with findings from clinical studies in the most common target-organs of GVHD, namely liver, colon, and skin (FIG. 3C)

[00162] In order to better understand the gene expression profile of GVHD-AKI kidneys and identify therapeutic targets, bulk sequencing of RNA isolated from the kidneys 7, 10 and 14 days post transplantation, from healthy, BMO and GVHD (1 M) mice was performed. On day 7, 1838 genes were down-regulated and 1542 up-regulated (log2(fold change)>l) in GVHD mice compared to healthy control (FIG. 4A) while 1508 genes were down-regulated, and 1495 genes were upregulated in GVHD mice compared to mice receiving bone marrow only. There was strong evidence of persistent immune pathway activation, in accordance with data from human kidneys (FIG. 4A). Significant upregulation (more than 4-fold) was seen in genes associated with the IFN-y, IFN-a, Jak- Stat3 and NFKB pathways, as indicated by Hallmark Pathway Analysis (FIG. 4B). An increase in genes associated with renal injury (Ubd -ubiquitin d, Lcn2 -NGAL), and pro- fibrotic pathways (FGF family proteins) was observed. Hallmark pathway analysis indicated that the top upregulated pathways included IFN-y, IFN-a, Jak-Stat3 and NFKB. Jak-Stat signaling pathway was identified as a key player in the development and maintenance of GVHD throughout the disease progress and within T-cells, B-cells, macrophages, neutrophils, and natural killer cells. The JAK/STAT pathways were active in each step of acute GVHD: (a) tissue damage from disease and conditioning chemoradiotherapy, (b) donor T-cell activation, and (c) recruitment and activation of other immune cells.

[00163] To complement RNAseq results, biochemical assays were conducted.

Quantitative polymerase chain reaction (qPCR) was performed on effectors of the disregulated pathways, including STAT1, STAT3, TNFRSF1A, TLR2, MAP3K8, RELB, S0CS3 NFKB2, MAP3K8, TNF, PLIN2, S0CS1 and LIF. The expression of those genes was significantly elevated in the GVHD group compared to control. Elevated phospho- NFKB was observed in kidney lysates of GVHD mice via ELISA and histology, and elevation of STAT3, pSTAT3, NFKB, SOCS2 and RELB with Western Blot. Gene expression profiles were analyzed for evidence of modulation of known drug delivery targets. Elevated P-selectin (SELP, logfold change = 3.32, p.adj= 2.8E-10), a transmembrane protein normally overexpressed in activated endothelial cells, was observed. Substantially enhanced galectin-3/ Gal-3 binding protein (LGALS3, logfold change = 1.4, p.adj=6E-15, LGALSBP, logfold change = 3.29, p.adj= 1.7E-55), a multifunctional protein with a role in interstitial fibrosis and progression of chronic kidney disease, was also observed. Overexpression of the associated proteins in the kidneys by immunofluorescence staining was indicated in FIG. 5A.

[00164] To evaluate the expression enhancement in other target organs of GVHD, P- selectin and galectin-3 were measured in the liver, small and large intestine, and spleen. Results indicated relative levels of expression of Galectin-3 and endothelial P-selectin were higher in kidneys and intestines as compared to liver and spleen, both in baseline (healthy animals) and upon GVHD induction.

[00165] Galectin-3 and P-selectin-targeted nanodelivery in GVHD-AKI kidneys. The potential for targeted delivery of nanomedicines to GVHD kidney tissues was investigated. Lipid nanoparticles (LNPs) were synthesized consisting of a small molecule drug core and a monolayer of structural phospholipids using microfluidics. To facilitate binding of the LNPs to P-selectin (P-sel LNPs), a sulfated glycolipid, sulfatide, was incorporated into the lipid mix. Sulfatide was substituted with a sphingolipid, GM 1 -Ganglioside to develop LNPs that bind to Galectin-3 (Gal-3 LNPs). The hydrodynamic diameter of the particles was drug dependent and ranged from 40-90 nm (FIGS. 9A-9D), while the zeta potential was -40 ± 5 mV for all of the LNPs in this study. The average drug loading of the particle was 10 wt.%. For biodistribution studies, Gal-3 LNPs or P-sel LNPs decorated with a cy5- lipid and encapsulating betamethasone dipropionate as the cargo (Beta LNPs) were used (FIG. 5A).

[00166] The biodistribution of the targeted LNPs in the context of GVHD-AKI was assessed. On day 6 after BMT, Gal-3-Cy5-LNPs or P-sel-Cy5-LNPs were injected by tail intravenous injection in healthy or GVHD mice and euthanized after 24 hours. Organs were harvested, and fluorescence was measured with a gel reader. Both P-sel LNPs and Gal-3 LNPs accumulated in the kidneys of GVHD mice with higher selectivity than the kidneys of healthy mice. Gal-3 LNPs showed a preferential accumulation in kidneys of healthy mice, as compared to the liver (a common site of nanoparticle accumulation). Without being bound by any theory, these differences may be due to the significant levels of Galectin-3 in kidneys of healthy mice and the smaller hydrodynamic diameter of the Gal-3 LNPs. The in vivo therapeutic response of the nanoparticles incorporating inhibitors of the heretofore identified therapeutic targets and with binding affinity to the identified delivery targets in the GVHD model were investigated. Jak-Statl (IFN-gamma), Jak-Stat3, and NFKB were identified as the top candidates for inhibition and therapeutic intervention in the GVHD- AKI model.

[00167] A small library of LNPs were synthesized incorporating ruxolitinib (Jak/Statl inhibitor), WP1066 (Stat3 inhibitor), Tofacitinib (pan-Jak inhibitor), or TPCA-1 (NfkB inhibitor). Ruxolitinib is FDA-approved for the treatment of steroid-refractory acute GVHD (aGVHD), Tofacitinib is FDA-approved for the treatment of arthritis and colitis, while WP1066 and TPCA-1 are still in preclinical development. Mice received daily or bidaily intraperitoneal injections of either the free drug or the drug encapsulated in LNPs, starting from day 2 after transplantation. Ruxolitinib (20mg/kg) or WP1066 (20mg/kg) were tested in Gal-3 LNPs, while in the second study Tofacitinib (20 mg/kg) or TPCA-1 (20mg/kg) were investigated in P-sel LNPs (FIG. 6A). These combinations of encapsulated drugs with targeted lipid carriers were selected based on the stabilities of the resulting particles. Both Jak and Stat3 inhibitors (ruxolitinib, tofacitinib, and WP1066) were efficacious in reducing kidney damage biomarkers (NGAL and KIM-1) which were quantified in serum and urine as well as by immunohistochemistry in the kidney tissues. Strikingly, the white blood cell count and platelet count on day 7 post-BMT in mice treated with ruxolitinib-LNPs were higher as compared to free drug groups (FIGS. 6D and 6E). This result indicated that the nanoparticles mitigated the known hematologic toxicity of ruxolitinib, which is associated with thrombocytopenia and anemia in patients treated with this drug. A striking result was noted regarding NFKB inhibition in this model. The NFKB inhibitor (TPCA-1) resulted in 100% mortality in all drug-receiving groups (both free drug and encapsulated form) after only two doses in GVHD mice, while it did not cause any weight loss or death in healthy mice even at a higher dose (40 mg/kg, daily), indicating on- target toxicity of the drug especially in GVHD. This deleterious effect was consistent with reports from the use of bortezomid (proteasome and NFKB inhibitor) in GVHD, which paradoxically amplified GVHD mortality when administered late, due to amplification of inflammatory cytokine generation but can have a beneficial effect when administered very early after BMT. This narrow therapeutic window in NFKB inhibition prompted us to exclude this pathway from later studies.

[00168] Ruxolitinib LNPs restore kidney impairment in severe GVHD. The first efficacy study (7 days, 1 million T-cells) highlighted the superior performance of LNPs compared to free drug in terms of pharmacodynamics and T-cell infiltration reduction. Ruxolitinib and tofacitinib reduced T-cell infiltration more efficiently than other drug candidates, but ruxolitinib required less frequent administration, making it a better performer in this preliminary study.

[00169] A more severe, lethal GVHD model was used to investigate the systemic and kidney-specific efficacy of ruxolitinib-LNPs including glomerular filtration rate and general GVHD scoring. The GVHD-AKI model was modified by injecting 2 million T-cells instead of 1 M, and the mice were followed for 14 days instead of 7 days (FIG. 7A). Mice received daily IP injections of vehicle (DMSO/Tween 80) (14 mice), free ruxolitinib (11 mice), or LNP-ruxolitinib (11 mice) and were then euthanized 7 or 14 days after transplantation (FIG. 7A). Blood was drawn at 7, 10 and 14 days and urine was collected at 7 and 14 days (day 10 was omitted because mice were severely dehydrated).

[00170] LNP-ruxolitinib improved the overall health of the mice more efficiently than the free drug. As expected, increasing the amount of T-cells during transplantation dramatically increased the severity of GVHD: 40% of mice receiving the vehicle died before day 7. (FIG. 7B). Strikingly, the administration of LNP-ruxolitinib restored the weight loss and GVHD clinical scoring (blind evaluation) faster than the free drug (FIGS. 7A and 7B), suggesting potential benefits to other affected organs.

[00171] Consistent with the first study, LNPs reduced T-cell infiltration and pSTAT3 phosphorylation more than the free drug (FIGS. 8A and 8B). Kidney injury was assessed by quantifying NGAL by immunohistochemistry and urine KIM-1 by ELISA. NGAL expression was significantly increased until day 14 but was reduced more by treatment with LNPs as compared to free drug, with the effect of LNPs more evident at earlier timepoints (FIG. 8C). Urine KIM-1 was elevated in GVHD mice, but the difference between the performance of LNPs and free drug was not significant (FIG. 8D), possibly due to the small number of samples, as urine collection from these mice was not always possible. The severity of renal injury was reflected in the abnormal elevation of serum BUN, a rather insensitive biomarker in mice, which was significantly higher in GVHD mice (FIG. 8E). Treatment with ruxolitinib maintained serum BUN close to the physiological range, with LNPs performing better at the earlier timepoint (FIG. 8E). Importantly, the same trend was reflected when glomerular filtration rate was measured, which significantly reduced in GVHD mice and was improved by LNP -ruxolitinib administration (FIGS. 8F-8H). Transcutaneous glomerular filtration rate (tGFR) measurements, normalized for animal weight, were performed using a transdermal continuous renal function monitor (MediBeacon, Germany). Overall, those results indicated that severe GVHD has a significant impact in kidney function that can be restored more rapidly and efficiently with the administration of an LNP-encapsulated ruxolitinib as compared to free drug.

[00172] Discussion. Prevention and treatment of acute graft-versus-host disease following allogeneic bone marrow transplantation remains a fundamental aspect of modem hematology and oncology. Kidney injury in the setting of allo-HCT has an incidence as high as 80% and increases patient morbidity and mortality, however, its progression and mechanisms remain poorly characterized. Kidney biopsies are rarely performed early after HCT due to thrombocytopenia, which contributes to the complexity of the disease detection and understanding.

[00173] In this work, the contribution of GVHD alone was investigated in the development of kidney injury and kidney function impairment. An irradiation-conditioned, fully major histocompatibility complex (MHC)-mismatched model of GVHD was used, and evidence of renal injury was indicated as soon as day 7 after transplantation. The gene expression patterns in the kidneys of healthy versus irradiation-conditioned versus GVHD mice were analyzed to understand the underlying mechanisms of early T-cell trafficking. Extensive upregulation of genes related to inflammation and graft rejection pathways was indicated. Histological investigations of the kidney showed increased T-cell infiltration, tubulitis and glomerulitis. A comprehensive immune cell repertoire analysis was performed in the infiltrated T-cells and found that they consisted of CD8+ and Tbet+ cells. The animals had significantly elevated serum and urine markers of kidney injury as well as substantial renal damage adrenal function impairment proportional to the severity of GVHD.

[00174] Previous research demonstrated that AKI in the setting of allo-HCT is associated with an inflammatory processes characterized by the infiltration of CD3+ and CD4+ cells and the release of pro-inflammatory cytokines that have a profound effect on renal tubules, vasculature, renal interstitium, and glomeruli. Transcriptome analysis in kidney lysates from mice with acute GVHD revealed a similar expression profile as in usual target organs of GVHD, while multiplex analysis of infiltrated immune cells in the kidneys revealed an immune landscape typical of inflammation and allograft rejection.

[00175] The understanding of the T-cell repertoire involved in kidney infiltration is useful for understanding the disease and to determine if a limited number of clones may be involved in the disease and thus if a specific depletion of donor inoculum may be envisaged to prevent the development of GVHD. The severity of kidney injury was proportional to the severity of GVHD, with severe GVHD resulting not only to the elevation of renal injury biomarkers, but also of the otherwise insensitive kidney function biomarkers BUN and creatinine. In severe GVHD, mice displayed decreased glomerular filtration rate.

[00176] The analysis highlighted two (out of potentially more) overexpressed proteins in the GVHD kidneys which may be used as binding means for targeted lipid nanoparticles. Inhibiting a number of overexpressed pathways including Jak/Stat3, Jak/Statl and NFkB was investigated.

EXAMPLE 2: Lipid Ratio Variation for LNPs Encapsulating Active Therapeutics

[00177] The procedure described in Example 1 for preparing LNPs was used in Example 2.

[00178] The aim of this study was to determine the effect of the amount of PG in the mixture of lipid mixture used to form LNPs. The tested amounts of PG in the lipid mixture were 9 wt.%, 16 wt.%, 33 wt.%, 50 wt.% and 66 wt.% of total lipids, with the balance made up by PC. The lipid mixture included PC and ethyl acetate, with ethyl acetate being 90 wt.% in the mixture. The encapsulated drug was rapamycin (10 wt.%).

[00179] FIGS. 10A-10D provide graphs characterizing LNPs with lipid ratio variation.

FIG. 10A is a graph of hydrodynamic diameter of LNPs with different lipid ratios. FIG. 10B is a graph of zeta potential of LNPs with different lipid ratios. FIG. IOC is a graph of drug encapsulation of LNPs with different lipid ratios. FIG. 10D is a graph of poly dispersity index (PDI) of LNPs with different lipid ratios. These results indicated that increasing PG decreased the particle size and zeta potential, slightly increased the PDI, and had little effect on encapsulation.

[00180] FIG. 11 is a graph of cell viability of LNPs with different lipid ratios and different particle concentrations. Increasing the amount of PG above 33 wt.% in the lipid mixture dramatically increased LNP in vitro toxicity on Bend3 endothelial cells. Considering in vitro toxicity, the amount of PG in the lipid mixture may be less than or equal to about 33 ± 2 wt.% of total lipids, with the balance made up by PC. In lipid mixtures, formulations of PG:PC:ethyl acetate mass ratios can vary from 1 : 10:90 to 3.5 :7.5 :90, without exhibiting in vitro toxicity.

EXAMPLE 3: Efficacy of LNP-encapsulated proteolysis-targeting chimera (PROTAC) in senescent pancreatic ductal adenocarcinoma (PDAC)

[00181] The procedure described in Example 1 for preparing LNPs was used in Example 3.

[00182] The efficacy of LNP-encapsulated ARV-825 was evaluated in senescent pancreatic ductal adenocarcinoma (PDAC). FIG. 12A illustrates a schematic of model establishment and treatment of PDAC. C57BL/6 mice were subjected to orthotopic transplantation of PDAC cells. Fourteen days after transplantation, senescence was induced by administering 1 mg/kg trametinib and 100 mg/kg Palbociclib by oral gavage. Seven days after senescence induction, mice were split into 4 groups: administration of free drug ARV-825, administration of ARV-825 encapsulated in untargeted LNPs, administration of ARV-825 encapsulated in LNPs targeting p-selectin, and administration of ARV-825 encapsulated in LNPs targeting Galectin-3. LNP or free drug was administered via intraperitoneal injection every other day. Tumor size was measured by ultrasound weekly.

[00183] FIG. 12B is a graph of Kaplan-Meier survival for PDAC BL/6 mice under the treatment conditions described with respect to FIG. 12A. FIG. 12C is a graph of tumor volume growth from day 14 to day 31 for PDAC C57BL/6 mice under the treatment conditions described with respect to FIG. 12A. FIG. 12D is a graph of body weight change over time for PDAC BL/6 mice under the treatment conditions described with respect to FIG. 12A [00184] The efficacy of Gal ectin-3 targeting LNP-encapsulated ARV-825 was further evaluated for treating PDAC. FIG. 13A illustrates a schematic of model establishment and treatment of PDAC. C57BL/6 mice were subjected to orthotopic transplantation of KPC-1 cells. Fourteen days after transplantation, mice were split into two groups - with one group having senescence induced by administering 1 mg/kg trametinib and 100 mg/kg Palbociclib by oral gavage and the other group not having senescence induced. Seven days later, mice from each group were further split into three groups: administration of ARV-825 encapsulated in untargeted LNPs, administration of ARV-825 encapsulated in LNPs targeting Galectin-3, administration of free ARV-825 (“free drug”), and administration of LNPs targeting Galectin-3 without drug (“vehicle”).

[00185] FIGS. 13B and 13C are graphs of Kaplan-Meier survival for PDAC BL/6 mice comparing treatment with 1 mg/kg trametinib, 100 mg/kg Palbociclib, and LNPs as compared to treatment with LNPs alone, respectively, according to the study described in FIG. 13A. Kaplan-Meier survival, ** p < 0.01, * p < 0.05 by Log-rank (Mantel-Cox) test, n=5. FIGS. 13D and 13E are graphs of tumor volume change for PDAC BL/6 mice comparing treatment with 1 mg/kg trametinib, 100 mg/kg Palbociclib, and LNPs as compared to treatment with LNPs alone, respectively. ** p < 0.01, * p > 0.05 by ordinary one-way ANOVA analysis, , according to the study described in FIG. 13A. FIG. 13F shows BRD4 immunohistochemistry for PDAC BL/6 mice under different treatment conditions, according to the study described in FIG. 13A.

EXAMPLE 4: Formulations of PEG-Substituted LNP-Encapsulated Drugs

[00186] The procedure described in Example 1 for preparing LNPs was used in Example 4, except that instead of a targeting ligand, PEG lipid, l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene gly col-2000, was used. Formulations were prepared with different drugs to determine whether LNPs can be formed with these drugs.

EXAMPLE 5: Formulations of Galectin-3-Targeting LNP-Encapsulated Drugs

[00187] The procedure described in Example 1 for preparing LNPs was used in Example 5. To facilitate binding of the LNPs to Galectin-3 (Gal-3 LNPs), a sphingolipid, GM1- Ganglioside was used. Formulations were prepared with different drugs to determine whether LNPs targeting Galectin-3 can be formed with these drugs.

EXAMPLE 6: Formulations of P-Selectin-Targeting LNP-Encapsulated Drugs

The procedure described in Example 1 for preparing LNPs was used in Example 6. To facilitate binding of the LNPs to P-selectin (P-sel LNPs), a sulfated glycolipid, sulfatide, was incorporated into the lipid mix. Formulations were prepared with different drugs to determine whether LNPs targeting P-selectin can be formed with these drugs.

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EQUIVALENTS

[00188] While certain embodiments have been illustrated and described a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers, or racemic mixtures thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.

[00189] The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof.

[00190] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation, or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[00191] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00192] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[00193] All publications, patent applications, issued patents, and other documents (for example, journals, articles and/or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[00194] The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims:

A. A composition comprising a plurality of lipid nanoparticles, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises about 5 wt.% to about 20 wt.% of a therapeutic agent, and a sulfolipid targeting P-selectin, a ganglioside targeting Galectin- 3, or a PEG lipid; wherein the plurality of lipid nanoparticles is characterized by a lightintensity average diameter of about 10 nm to about 250 nm, as determined by dynamic light scattering, and a zeta potential of about -45 mV to about -30 mV, as determined by electrophoresis; and wherein the therapeutic agent is selected from 4-aminothiophenol, afatinib, alpelisib, amphotericin, artemisinin, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, budesonide, carbamazepine, celecoxib, clozapine, curcumin, cyclosporin, darapladib, DBetl, docetaxel, emricasan, erythromycin, etomidate, fulvestrant, ibrutinib, ibudilast, (-)JQ-l, ketoconazole, lamotrigine, mirdametinib, MS4078, naloxone, navitoclax, NVP-2, paclitaxel, pexidartinib, phenytoin, pimozide, rapamycin, regorafenib, resveratrol, rifampicin, rimonabant, risperidone, ropivacaine, ruxolitinib, SB203580, SC-43, SH-4-54, sorafenib, STM2457, sunitinib, terbinafine, THAL-SNS, TPCA-1, tofacitinib, topiramate, valrubicin, vemurafenib, venetoclax, vincristine, vismodegib, and WP1066.

B. The composition of paragraph A, wherein the plurality of lipid nanoparticles comprises L-a-phosphatidylglycerol and phosphatidylcholine.

C. The composition of paragraph B, wherein the plurality of lipid nanoparticles comprises a ratio of L-a-phosphatidylglycerol to phosphatidylcholine of about 1 to 10 to about 3.5 to 7.5.

D. The composition of paragraph C, wherein the plurality of lipid nanoparticles comprises a ratio of L-a-phosphatidylglycerol to phosphatidylcholine of about 1 to 10.

E. The composition according to any one of the preceding paragraphs, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside and the ganglioside comprises GM 1 -Ganglioside.

F. The composition of paragraph E, wherein the lipid nanoparticle of the plurality of lipid nanoparticles further comprises sphingolipid.

G. The composition of paragraph E or F, wherein the therapeutic agent comprises 4- aminothiophenol, afatinib, amphotericin, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, celecoxib, curcumin, darapladib, emricasan, fulvestrant, ibrutinib, ibudilast, (-)JQ-l, ketoconazole, lamotrigine, mirdametinib, naloxone, NVP-2, pimozide, rapamycin, regorafenib, resveratrol, rimonabant, risperidone, ropivacaine, ruxolitinib, SB203580, SH-4-54, sorafenib, sunitinib, THAL-SNS, topiramate, valrubicin, venetoclax, vincristine, vismodegib, and WP1066. H. The composition of any one of paragraphs E-G, wherein the plurality of lipid nanoparticles is characterized by an intensity -weighted average diameter of about 10 nm to about 120 nm, as determined by dynamic light scattering.

I. The composition of any one of paragraphs E-H, wherein the therapeutic agent comprises ruxolitinib or WP1066; and the plurality of lipid nanoparticles is characterized by an intensity -weighted average diameter of about 40 nm to about 90 nm, as determined by dynamic light scattering.

J. The composition of any one of paragraphs A to D, wherein the lipid nanoparticle of the plurality of lipid nanoparticles comprises the sulfolipid and the sulfolipid comprises sulfatide.

K. The composition of paragraph J, wherein the lipid nanoparticle of the plurality of lipid nanoparticles further comprises sulfated glycolipid.

L. The composition of paragraph J or K, wherein the therapeutic agent comprises 4- aminothiophenol, afatinib, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, celecoxib, clozapine, curcumin, erythromycin, fulvestrant, ibudilast, (-)JQ-l, mirdametinib, naloxone, navitoclax, pexidartinib, pimozide, rapamycin, regorafenib, resveratrol, rifampicin, rimonabant, risperidone, ropivacaine, ruxolitinib, SC-43, SH-4-54, STM2457, sunitinib, terbinafine, tofacitinib, topiramate, valrubicin, venetoclax, vincristine, and WP1066.

M. The composition of any one of paragraphs J to L, wherein the plurality of lipid nanoparticles is characterized by an intensity-weighted average diameter of about 20 nm to about 250 nm, as determined by dynamic light scattering.

N. The composition of any one of paragraphs J-M, wherein the therapeutic agent comprises tofacitinib; and the plurality of lipid nanoparticles is characterized by an intensity -weighted average diameter of about 80 nm to about 120 nm, as determined by dynamic light scattering.

O. The composition of any one of paragraphs J-N, wherein the lipid nanoparticle of the plurality of lipid nanoparticles comprises about 10 wt.% of the therapeutic agent. P. The composition of any one of the preceding paragraphs, wherein the plurality of lipid nanoparticles is characterized by a poly dispersity index of about 0.1 to about 0.35, as determined by dynamic light scattering.

Q. The composition of any one of the preceding paragraphs, wherein the composition comprises an aqueous solvent.

R. The composition of any one of the preceding paragraphs, further comprising about 5% (w/v) to about 20% (w/v) sucrose.

S. The composition of any one of the preceding paragraphs, wherein the composition is effective to reduce or prevent kidney injury in a subject resulting from allogeneic hematopoietic cell transplantation.

T. The composition of any one of the preceding paragraphs, wherein the composition is effective to reduce or prevent graft-vs-host disease in a subject resulting from allogeneic hematopoietic cell transplantation.

U. The composition of any one of the preceding paragraphs, wherein administration of the composition to a subject reduces T-cell infiltration, pSTAT3 phosphorylation, or both T-cell infiltration and pSTAT3 phosphorylation in a kidney of the subject after allogeneic hematopoietic cell transplantation as compared to administration of the therapeutic agent in free form.

V. The composition of any one of the preceding paragraphs, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside targeting Galectin-3; the therapeutic agent is ARV-825; and the composition is effective to treat pancreatic ductal adenocarcinoma in a subject.

W. The composition of any one of paragraphs A-F, H, I, O-Q, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside targeting Galectin-3; the therapeutic agent is ARV-825; and the composition is effective to reduce tumor growth rate of a pancreatic ductal adenocarcinoma tumor in a subject.

X. A method of treating or preventing kidney injury in a subject resulting from allogeneic hematopoietic cell transplantation, the method comprising administering an effective amount of the composition of any one of the preceding paragraphs to the subject. Y. The method of paragraph X, wherein the effective amount of the composition is about 10 mg/kg to about 40 mg/kg.

Z. The method of paragraph X or Y, wherein the effective amount of the composition is about 20 mg/kg.

AA. The method of any one of paragraphs X-Z, wherein administering comprises intravenous administration of the composition to the subject.

AB. A method of treating or preventing graft-vs-host disease in a subject resulting from allogeneic hematopoietic cell transplantation, the method comprising administering an effective amount of the composition of any one of the preceding paragraphs to the subject.

AC. The method of paragraph AB, wherein the effective amount of the composition is about 10 mg/kg to about 40 mg/kg.

AD. The method of paragraph AB or AC, wherein the effective amount of the composition is about 20 mg/kg.

AE. The method of any one of paragraphs AB-AD, wherein administering comprises intravenous administration of the composition to the subject.

AF. A method of reducing T-cell infiltration in a kidney of a subject, the method comprising administering an effective amount of the composition of any one of the preceding paragraphs to the subject.

AG. The method of paragraph AF, wherein the effective amount of the composition is about 10 mg/kg to about 40 mg/kg.

AH. The method of paragraph AF or AG, wherein the effective amount of the composition is about 20 mg/kg.

Al. The method of paragraphs AF-AH, wherein administering comprises intravenous administration of the composition to the subject.

AJ. A method of reducing pSTAT3 phosphorylation in a kidney of a subject, the method comprising administering an effective amount of the composition of any one of the preceding paragraphs to the subject. AK. The method of paragraph AJ, wherein the effective amount of the composition is about 10 mg/kg to about 40 mg/kg.

AL. The method of paragraph AJ or AK, wherein the effective amount of the composition is about 20 mg/kg.

AM. The method of paragraphs AJ-AL, wherein administering comprises intravenous administration of the composition to the subject.

AN. A method of treating pancreatic ductal adenocarcinoma in a subject comprising administering an effective amount of the composition of any one of paragraphs A-F, H, I, O-Q to the subject, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside targeting Gal ectin-3; the therapeutic agent is ARV-825.

AO. The method of paragraph AN, further comprising administering an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the composition of any one of paragraphs A-F, H, I, O-Q to the subject.

AP. The method of paragraph AO, wherein the effective amount of trametinib is 1 mg/kg and the effective amount of Palbociclib is 100 mg/kg.

AQ. The method of paragraph AO or AP, wherein administering the trametinib and Palbociclib comprises oral administration.

AR. The method of paragraph AO, wherein the subject has been administered an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the composition of any one of paragraphs A-F, H, I, O-Q to the subject.

AS. The method of any one of paragraphs AO- AR, wherein the effective amount of the composition is about 10 mg/kg.

AT. The method of any one of paragraphs AO-AS, wherein administering the composition comprises intraperitoneal administration of the composition to the subject.

AU. A method of reducing a tumor growth rate of a pancreatic ductal adenocarcinoma tumor in a subject comprising administering an effective amount of the composition of any one of paragraphs A-F, H, I, O-Q to the subject, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside targeting Galectin-3; and the therapeutic agent is ARV-825.

AV. The method of paragraph AU, further comprising administering an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the composition of any one of paragraphs A-F, H, I, O-Q to the subject.

AW. The method of paragraph AV, wherein the effective amount of trametinib is 1 mg/kg and the effective amount of Palbociclib is 100 mg/kg.

AX. The method of paragraph AV or AW, wherein administering the trametinib and Palbociclib comprises oral administration.

AY. The method of paragraph AV, wherein the subject has been administered an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the composition of any one of paragraph A-F, H, I, O-Q to the subject.

AZ. The method of any one of paragraphs AV- AY, wherein the effective amount of the composition is about 10 mg/kg.

BA. The method of any one of paragraph AV-AZ, wherein administering the composition comprises intraperitoneal administration of the composition to the subject.

Claims

1. A composition comprising a plurality of lipid nanoparticles, wherein: each lipid nanoparticle of the plurality of lipid nanoparticles comprises about 5 wt.% to about 20 wt.% of a therapeutic agent; and a sulfolipid targeting P-selectin, a ganglioside targeting Galectin-3, or a PEG lipid; the plurality of lipid nanoparticles is characterized by a light-intensity average diameter of about 10 nm to about 250 nm, as determined by dynamic light scattering, and a zeta potential of about -45 mV to about -30 mV, as determined by electrophoresis; the therapeutic agent is selected from 4-aminothiophenol, afatinib, alpelisib, amphotericin, artemisinin, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, budesonide, carbamazepine, celecoxib, clozapine, curcumin, cyclosporin, darapladib, DBetl, docetaxel, emricasan, erythromycin, etomidate, fulvestrant, ibrutinib, ibudilast, (-)JQ-l, ketoconazole, lamotrigine, mirdametinib, MS4078, naloxone, navitoclax, NVP-2, paclitaxel, pexidartinib, phenytoin, pimozide, rapamycin, regorafenib, resveratrol, rifampicin, rimonabant, risperidone, ropivacaine, ruxolitinib, SB203580, SC-43, SH-4-54, sorafenib, STM2457, sunitinib, terbinafine, THAL-SNS, TPCA-1, tofacitinib, topiramate, valrubicin, vemurafenib, venetoclax, vincristine, vismodegib, and WP1066.

2. The composition of Claim 1, wherein the plurality of lipid nanoparticles comprises L-a- phosphatidylglycerol and phosphatidylcholine.

3. The composition of Claim 2, wherein the plurality of lipid nanoparticles comprises a ratio of L-a-phosphatidylglycerol to phosphatidylcholine of about 1 to 10 to about 3.5 to 7.5.

4. The composition of Claim 3, wherein the plurality of lipid nanoparticles comprises a ratio of L-a-phosphatidylglycerol to phosphatidylcholine of about 1 to 10.

5. The composition of Claim 1, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside and the ganglioside comprises GM1- Ganglioside.

6. The composition of Claim 5, wherein the lipid nanoparticle of the plurality of lipid nanoparticles further comprises sphingolipid.

7. The composition of Claim 5 or 6, wherein the therapeutic agent comprises 4- aminothiophenol, afatinib, amphotericin, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, celecoxib, curcumin, darapladib, emricasan, fulvestrant, ibrutinib, ibudilast, (-)JQ-l, ketoconazole, lamotrigine, mirdametinib, naloxone, NVP-2, pimozide, rapamycin, regorafenib, resveratrol, rimonabant, risperidone, ropivacaine, ruxolitinib, SB203580, SH-4-54, sorafenib, sunitinib, THAL-SNS, topiramate, valrubicin, venetoclax, vincristine, vismodegib, and WP1066.

8. The composition of any one of Claims 5-7, wherein the plurality of lipid nanoparticles is characterized by an intensity -weighted average diameter of about 10 nm to about 120 nm, as determined by dynamic light scattering.

9. The composition of any one of Claims 5-8, wherein the therapeutic agent comprises ruxolitinib or WP1066; and the plurality of lipid nanoparticles is characterized by an intensity -weighted average diameter of about 40 nm to about 90 nm, as determined by dynamic light scattering.

10. The composition of Claim 1, wherein the lipid nanoparticle of the plurality of lipid nanoparticles comprises the sulfolipid and the sulfolipid comprises sulfatide.

11. The composition of Claim 10, wherein the lipid nanoparticle of the plurality of lipid nanoparticles further comprises sulfated glycolipid.

12. The composition of Claim 10 or 11, wherein the therapeutic agent comprises 4- aminothiophenol, afatinib, ARV-825, BI 2536, betamethasone, betamethasone dipropionate, celecoxib, clozapine, curcumin, erythromycin, fulvestrant, ibudilast, (- )JQ-1, mirdametinib, naloxone, navitoclax, pexidartinib, pimozide, rapamycin, regorafenib, resveratrol, rifampicin, rimonabant, risperidone, ropivacaine, ruxolitinib, SC-43, SH-4-54, STM2457, sunitinib, terbinafine, tofacitinib, topiramate, valrubicin, venetoclax, vincristine, and WP1066.

13. The composition of any one of Claims 10-12, wherein the plurality of lipid nanoparticles is characterized by an intensity-weighted average diameter of about 20 nm to about 250 nm, as determined by dynamic light scattering.

14. The composition of any one of Claims 10-13, wherein the therapeutic agent comprises tofacitinib; and the plurality of lipid nanoparticles is characterized by an intensity- weighted average diameter of about 80 nm to about 120 nm, as determined by dynamic light scattering.

15. The composition of any one of Claims 1-14, wherein the lipid nanoparticle of the plurality of lipid nanoparticles comprises about 10 wt.% of the therapeutic agent.

16. The composition of any one of Claims 1-15, wherein the plurality of lipid nanoparticles is characterized by a polydispersity index of about 0.1 to about 0.35, as determined by dynamic light scattering.

17. The composition of any one of Claim 1-16, wherein the composition comprises an aqueous solvent.

18. The composition of any one of Claims 1-17, further comprising about 5% (w/v) to about 20% (w/v) sucrose.

19. The composition of any one of Claims 1-18, wherein the composition is effective to reduce or prevent kidney injury in a subject resulting from allogeneic hematopoietic cell transplantation.

20. The composition of any one of Claims 1-19, wherein the composition is effective to reduce or prevent graft-vs-host disease in a subject resulting from allogeneic hematopoietic cell transplantation.

21. The composition of any one of Claims 1-20, wherein administration of the composition to a subject reduces T-cell infiltration, pSTAT3 phosphorylation, or both T-cell infiltration and pSTAT3 phosphorylation in a kidney of the subject after allogeneic hematopoietic cell transplantation as compared to administration of the therapeutic agent in free form.

22. The composition of any one of Claims 1-20, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside targeting Galectin-3; the therapeutic agent is ARV-825; and the composition is effective to treat pancreatic ductal adenocarcinoma in a subject.

23. The composition of any one of Claims 1-6, 8, 9, 15-18, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside targeting Galectin-3; the therapeutic agent is ARV-825; and the composition is effective to reduce tumor growth rate of a pancreatic ductal adenocarcinoma tumor in a subject.

24. A method of treating or preventing kidney injury in a subject resulting from allogeneic hematopoietic cell transplantation, the method comprising: administering an effective amount of the composition of any one of Claims 1-21 to the subject.

25. The method of Claim 24, wherein the effective amount of the composition is about 10 mg/kg to about 40 mg/kg.

26. The method of Claims 24 or 25, wherein the effective amount of the composition is about 20 mg/kg.

27. The method of any one of Claims 24-26, wherein administering comprises intravenous administration of the composition to the subject.

28. A method of treating or preventing graft-vs-host disease in a subject resulting from allogeneic hematopoietic cell transplantation, the method comprising: administering an effective amount of the composition of any one of Claims 1-21 to the subject.

29. The method of Claim 28, wherein the effective amount of the composition is about 10 mg/kg to about 40 mg/kg.

30. The method of Claims 28 or 29, wherein the effective amount of the composition is about 20 mg/kg.

31. The method of Claims 28-30, wherein administering comprises intravenous administration of the composition to the subject.

32. A method of reducing T-cell infiltration in a kidney of a subject, the method comprising: administering an effective amount of the composition of any one of Claims 1-21 to the subject.

33. The method of Claim 32, wherein the effective amount of the composition is about 10 mg/kg to about 40 mg/kg.

34. The method of Claims 32 or 33, wherein the effective amount of the composition is about 20 mg/kg.

35. The method of Claims 32-34, wherein administering comprises intravenous administration of the composition to the subject.

36. A method of reducing pSTAT3 phosphorylation in a kidney of a subject, the method comprising: administering an effective amount of the composition of any one of Claims 1-21 to the subject.

37. The method of Claim 36, wherein the effective amount of the composition is about 10 mg/kg to about 40 mg/kg.

38. The method of Claims 36 or 37, wherein the effective amount of the composition is about 20 mg/kg.

39. The method of Claims 36-38, wherein administering comprises intravenous administration of the composition to the subject.

40. A method of treating pancreatic ductal adenocarcinoma in a subject comprising: administering an effective amount of the composition of any one of Claims 1-6, 8, 9, 15-18 to the subject, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside targeting Galectin-3; the therapeutic agent is ARV-825.

41. The method of Claim 40, further comprising administering an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the composition of any one of Claims 1-6, 8, 9, 15-18 to the subject.

42. The method of Claim 41, wherein the effective amount of trametinib is 1 mg/kg and the effective amount of Palbociclib is 100 mg/kg.

43. The method of claim 41 or 42, wherein administering the trametinib and Palbociclib comprises oral administration.

44. The method of Claim 40, wherein the subject has been administered an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the composition of any one of Claims 1-6, 8, 9, 15-18 to the subject.

45. The method of any one of Claims 40-44, wherein the effective amount of the composition is about 10 mg/kg.

46. The method of any one of Claims 40-45, wherein administering the composition comprises intraperitoneal administration of the composition to the subject.

47. A method of reducing a tumor growth rate of a pancreatic ductal adenocarcinoma tumor in a subject comprising: administering an effective amount of the composition of any one of Claims 1-6, 8, 9, 15-18 to the subject, wherein each lipid nanoparticle of the plurality of lipid nanoparticles comprises the ganglioside targeting Galectin-3; the therapeutic agent is ARV-825.

48. The method of Claim 47, further comprising administering an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the composition of any one of Claims 1-6, 8, 9, 15-18 to the subject.

49. The method of Claim 48, wherein the effective amount of trametinib is 1 mg/kg and the effective amount of Palbociclib is 100 mg/kg.

50. The method of claim 48 or 49, wherein administering the trametinib and Palbociclib comprises oral administration.

51. The method of Claim 47, wherein the subject has been administered an effective amount of trametinib and Palbociclib to the subject prior to administering the effective amount of the composition of any one of Claims 1-6, 8, 9, 15-18 to the subject.

52. The method of any one of Claims 47-51, wherein the effective amount of the composition is about 10 mg/kg.

53. The method of any one of Claims 47-52, wherein administering the composition comprises intraperitoneal administration of the composition to the subject.