WO2025235487A1

WO2025235487A1 - Lpa receptor antagonist-derived lipid compounds and uses thereof

Info

Publication number: WO2025235487A1
Application number: PCT/US2025/027959
Authority: WO
Inventors: Yizhou Dong; Jingyue YAN; Diana KANG; Chang Wang
Original assignee: Icahn School of Medicine at Mount Sinai; Ohio State Innovation Foundation
Current assignee: Icahn School of Medicine at Mount Sinai; Ohio State Innovation Foundation
Priority date: 2024-05-06
Filing date: 2025-05-06
Publication date: 2025-11-13
Anticipated expiration: 2026-11-06

Abstract

Disclosed herein are lipid compounds and compositions comprising lipid compounds and methods of making and use thereof.

Description

LPA RECEPTOR ANTAGONIST-DERIVED LIPID COMPOUNDS AND

USES THEREOF

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government Support under Grant Nos. R35GM119679 and R35GM144117 awarded by the National Institutes of Health. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. Provisional Patent Application No. 63/643,200 filed May 6, 2024, which is hereby incorporated herein by reference in its entirety.

TECHNICAL FIELD

This application generally relates to lipid formulations that can be used in drug delivery and screening.

BACKGROUND

Efficient delivery of vaccines, gene therapeutics, mRNA, and drug delivery is a key step and challenge for the application of mRNA therapeutics. Despite promising data from ongoing clinical trials, there are currently problems restricting the widespread use of oligonucleotides in therapeutic and diagnostic contexts. First, free RNAs are susceptible to nuclease digestion in plasma, facilitating degradation of the therapeutic agent. Second, these oligonucleotides are often unable to access the intracellular compartment where the relevant translation machinery resides.

As a result, lipid nanoparticles formed from cationic lipids with other lipid components have been used to as a possible way to traverse these barriers in delivery and increase the cellular uptake of oligonucleotides. However, the efficacy of these delivery systems typically stems from the compositional structure of the base lipid molecule, and new compositions and methods are needed for delivering mRNA to cells for treating various disease states. Thus, there remains a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides.

SUMMARY

In accordance with the purposes of the disclosed compounds and methods as embodied and broadly described herein, the disclosed subject matter relates to compounds and methods of making and use thereof.

Disclosed herein are compounds defined by Formula I, or a pharmaceutically acceptable salt thereof: i wherein p is an integer from 0 to 5; n is an integer from 1 to 10; m, when present, is an integer from 1 to 10;

R¹ is an alkyl or ether linker, wherein the alkyl or ether linker is substituted with a lysophosphatidic acid (LPA) receptor antagonist; each R² is independently a substituted or unsubstituted Ci-Cis alkyl; each R³, when present, is independently hydrogen, OH, a substituted or unsubstituted Ci- Ci8 alkyl, a substituted or unsubstituted C1-C5 alkyl alcohol.

In some examples of Formula I, each R² is independently an unsubstituted linear Cs-Cis alkyl. In some examples of Formula I, each R² is independently an unsubstituted branched Cs- Ci8 alkyl. In some examples of Formula I, each R² is independently a linear Ci-Cis alkyl (e.g., C4-C6 alkyl) substituted with one or more substituents selected from the group consisting of acetal, amine, amide, ester, ether (e.g., acetal), and carbonate ester. In some examples of Formula I, each R² is independently a branched Ci-Cis alkyl (e.g., C4-C6 alkyl) substituted with one or more substituents selected from the group consisting of amine, amide, ester, ether (e.g., acetal), and carbonate ester. In some examples of Formula I, each R² is independently selected from the group consisting of: wherein G and I each independently represent integers from 1 to 8. In some examples of Formula I, each R² is independently selected from the group consisting of: wherein G and I each independently represent integers from 1 to 8. In some examples of Formula I, each R² is independently selected from the group consisting of:

In some examples of Formula I, each R² is the same. In some examples of Formula I, at least one R² is different. In some examples of Formula I, p is 0. In some examples of Formula I, p is 1. In some examples of Formula I, the LPA receptor antagonist comprises a lysophosphatidic acid receptor 1 (LPAi) antagonist. In some examples of Formula I, R¹ is a Ci-Ce alkyl linker substituted with the LPA receptor antagonist.

In some examples according to Formula I, the LPA receptor antagonist is represented by:

II wherein

X is N or C;

Z is N or O;

R⁴, R⁵, R⁶, R⁷, R⁸ are each independently H, OH, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or NR^xR^y, or wherein, as valence permits, R⁴, R⁵, R⁶, R⁷, R⁸, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; R^x and R^y, when present, are independently selected from substituted or unsubstituted Ci- C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, and substituted or unsubstituted C1-C20 acyl;

R⁹ is H or cyclopropyl;

R¹⁰, R¹¹, R¹² are each independently H or methyl; and

L is selected from:

III wherein

X is N or C;

Z is N or O;

R⁴, R⁵, R⁶, R⁷, R⁸ are each independently H, OH, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or NR^xR^y, or wherein, as valence permits, R⁴, R⁵, R⁶, R⁷, R⁸, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms;

R^x and R^y, when present, are independently selected from substituted or unsubstituted Ci- C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl; and

R⁹ is H or cyclopropyl.

IV wherein R⁴, R⁵, R⁶, R⁷, R⁸ are each independently H, OH, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or NR^xR^y, or wherein, as valence permits, R⁴, R⁵, R⁶, R⁷, R⁸, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; and

R^x and R^y are independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl. In some examples of Formula I, the LPA receptor antagonist is selected from the group consisting of AM966, AM095, RO-6842262, and BMS-986020.

In some examples, the compound is selected from the group consisting of: Also disclosed herein are compositions comprising any of the compounds disclosed herein.

Also disclosed herein are compositions comprising any of the compounds disclosed herein further comprising an agent. In some examples, the agent comprises a polynucleotide. In some examples, the agent comprises an RNA. In some examples, the agent comprises an mRNA. In some examples, the agent comprises a polynucleotide encoding a ubiquitin-modifying enzyme. In some examples, the agent comprises Tumor Necrosis Factor a-Induced Protein 3 (TNFAIP3), Suppressor of Cytokine Signaling 1 (SOCS1), Smad7, Krtippel-like Factor 2 (KLF2), Matrix metalloproteinases (MMPs), Thrombospondin- 1 (TSP-1), Interleukin- 10 (IL-10), and/or Decorin. In some examples, the agent comprises a polynucleotide encoding Tumor Necrosis Factor a- Induced Protein 3 (TNFAIP3).

Also disclosed herein are methods of making any of the compounds and compositions disclosed herein.

Also disclosed herein are lipid nanoparticles comprising any of the compounds disclosed herein a non-cationic lipid; a polyethylene glycol-lipid; and a sterol. In some examples, the noncationic lipid comprises l,2-dioleoyl- w-glycero-3-phosphoethanolamine (DOPE), l-palmitoyl-2- oleoyl- w-glycero-3-phosphoethanolamine (POPE), l,2-distearoyl-sw-glycero-3-phosphocholine (DSPC), l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE), DPPC (1,2- dipalmitoyl-sn-glycero-3- phosphocholine), l,2-dioleyl-sn-glycero-3-phosphotidylcholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn- glycero-3 -phosphoethanolamine (DMPE), l,2-dioleoyl-5/7-glycero-3- phospho-(l'-rac-glycerol) (DOPG), or combinations thereof.

In some examples, the lipid nanoparticle further includes an agent. In some examples, the agent comprises a polynucleotide. In some examples, the agent comprises an RNA. In some examples, the agent comprises an mRNA. In some examples, the agent comprises a polynucleotide encoding a ubiquitin-modifying enzyme. In some examples, the agent comprises Tumor Necrosis Factor a- Induced Protein 3 (TNFAIP3), Suppressor of Cytokine Signaling 1 (SOCS1), Smad7, Krtippel-like Factor 2 (KLF2), Matrix metalloproteinases (MMPs), Thrombospondin- 1 (TSP-1), Interleukin- 10 (IL- 10), and/or Decorin. In some examples, the agent comprises a polynucleotide encoding Tumor Necrosis Factor a-Induced Protein 3 (TNFAIP3). In some examples, the agent is encapsulated by the nanoparticle.

In some examples, the sterol comprises a cholesterol-based lipid. In some examples, a molar ratio of the non-cationic lipid is from 20% to 50%. In some examples, a molar ratio of the compound is from 5% to 60%. In some examples, a molar ratio of the sterol is from 20% to 50%. In some examples, a molar ratio of the PEG-lipid is from 0.1% to 2%. In some examples, a molar ratio of the compound is from 35% to 45%, a molar ratio of the non-cationic lipid is from 45% to 55%, a molar ratio of the sterol is from 35% to 45%, and a molar ratio of the polyethylene glycollipid is from 0.1% to 1%. In some examples, a weight fraction of the agent is from 5% to 20%.

Also disclosed herein are pharmaceutical compositions comprising a pharmaceutically ac ceptable carrier and an effective amount of any of the compounds and compositions disclosed he rein.

Also disclosed herein are methods for delivering an agent into a cell, comprising: introducing into the cell any of the compositions, nanoparticles or pharmaceutically acceptable compositions described herein.

Further disclosed herein are methods of treating a disease or disorder in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of any of the compositions, nanoparticles or pharmaceutically acceptable compositions described herein. In some examples, the disease or disorder comprises a connective tissue disorder. In some examples, the disease or disorder comprises fibrosis. In some examples, the fibrosis is lung fibrosis.

Additional advantages of the disclosed compounds, compositions and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed compounds, compositions and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed compounds, compositions and methods, as claimed.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure.

Figure 1. Shows an overview of an example approach for the delivery of mRNA to transiently elevate A20 levels in lung fibroblasts using LPAi antagonist-derived amino lipid nanoparticles (LA-LNPs). These LA A20 mRNA-LNPs exhibit strong antifibrotic activities by blocking the LPAi signaling pathway and restoring A20 enzymatic activities in fibroblasts, resulting in a lower rate of migration and collagen synthesis.

Figure 2. Synthesis of LPAi -antagonist derived lipids (LAs) and characterization of LA- LNPs. (Panel A) Synthetic routes to LA1-LA6. (Panel B) Size and PDI of LA-LNPs and MC3- LNPs encapsulated with FLuc mRNA. LA-LNPs were formulated at the same lipid molar ratio of LA:DOPE:Chol:PEG=20:30:40:0.75. (Panel C) Luminescence intensity in MLg cells. Data in B and C are presented as the mean ± S.D. (n=3). Statistical significance in C is analyzed by one-way ANOVA with Dunnett’s multiple comparison test. ****p < 0.0001.

Figure 3. Orthogonal optimization and characterization of LA5-LNPs. (Panel A) Orthogonal optimization table with 4 levels for each lipid. (Panel B) Luminescence intensity after the delivery of FLuc mRNA in 16 formulated LA5-LNPs with different lipid compositions. (Panels C-F) Impact trend of each lipid component in LA5-LNPs. The optimized LA5-LNP has a molar ratio of LA5:DOPE:Chol:PEG=40:50:30:0.5. (Panel G) Size and PDI of optimized LA5- LNPs carrying FLuc mRNAs. (Panel H) Encapsulation efficiency and zeta potential of LA5-LNPs carrying FLuc mRNAs. Data in B-H are presented as the mean ± S.D. (n=3).

Figure 4. LA5-LNPs mediated mRNA delivery to lung fibroblasts. (Panel A) Cryo-TEM characterization of LA5-LNPs (scale bar = 50 nm). (Panel B) MLg cell uptake of LA5-LNPs containing Alexa-Fluor 647 RNAs was investigated with endocytosis inhibitors, EIP A, CPZ, and MpCD. (Panel C) Confocal microscopy images of MLg cells incubated with calcein with or without LA5-LNPs (scale bar = 10 pm). (Panel D) Mean GFP intensity after the treatment with LA5-LNPs carrying GFP mRNA. (Panel E) LA5-LNPs mediated delivery of A20 mRNA in MLg cells. Data in Panels A, B, E are presented as the mean ± S.D. (n=3). Statistical significance in Panels B and D are analyzed by one-way ANOVA with Dunnett’s multiple comparison test. Statistical significance in Panel E is analyzed by student’s t test. ****p < 0.0001.

Figure 5. Delivery of A20 mRNA by LA5-LNPs ameliorates fibroblast activation by reducing collagen synthesis and abating fibroblast migration in primary mice lung fibroblasts. (Panel A) LA5-LNPs mediated A20 mRNA delivery in MLF cells. (Panels B and C) Tnfaip3 and Collal gene expression levels after the treatment with TGF-pi as determined by qRT-PCR. (Panel D and E) Representative images of wound-healing assay in MLFs treated with PBS, free LA5-LNPs, or LA5 A20-LNPs (N = 6). Data in Panels A-D are presented as the mean ± S.D. (n=3). Statistical significance in Panels A-D is analyzed by one-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05; ns, not significant. Figure 6. LA5 LNPs characterization. (Panel A) Luminescence intensity in MLg cells after treatment with LA5 or ALC-0315 LNPs with firefly luciferase mRNA. (Panel B) Relative cell viability of isolated primary mouse lung fibroblasts after treatment with LA5, MC3, or ALC-0315 LNPs with firefly luciferase mRNA. Relative cell viability measured with an MTT assay. Data in Panels A-B are presented as the mean ± S.D. (n=3).

DETAILED DESCRIPTION

The compounds, compositions and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.

Before the present compounds, compositions and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, 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.

Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

General Definitions

In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

As used herein, the article “a,” “an,” and “the” means “at least one,” unless the context in which the article is used clearly indicates otherwise.

The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides. The term “oligonucleotide” denotes single- or double-stranded nucleotide multimers of from about 2 to up to about 100 nucleotides in length. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22: 1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS™ technology. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded,” as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes.

The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers. In some embodiments, the polynucleotide is composed of nucleotide monomers of generally greater than 100 nucleotides in length and up to about 8,000 or more nucleotides in length.

The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

The term “complementary” refers to the topological compatibility or matching together of interacting surfaces of a probe molecule and its target. Thus, the target and its probe can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.

The term “hybridization” refers to a process of establishing a non-covalent, sequencespecific interaction between two or more complementary strands of nucleic acids into a single hybrid, which in the case of two strands is referred to as a duplex.

The term “anneal” refers to the process by which a single-stranded nucleic acid sequence pairs by hydrogen bonds to a complementary sequence, forming a double-stranded nucleic acid sequence, including the reformation (renaturation) of complementary strands that were separated by heat (thermally denatured). The term “melting” refers to the denaturation of a double-stranded nucleic acid sequence due to high temperatures, resulting in the separation of the double strand into two single strands by breaking the hydrogen bonds between the strands.

The term “target” refers to a molecule that has an affinity for a given probe. Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species.

The term “promoter” or “regulatory element” refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of bacterial origin, for example, promoters derived from viruses or from other organisms can be used in the compositions, systems, or methods described herein. The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41 :521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It is appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.

The term “recombinant” refers to a human manipulated nucleic acid (e.g. polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g. polynucleotide), or if in reference to a protein (i.e, a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g. polynucleotide). In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning — A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise nucleic acids (e.g. polynucleotides) combined in such a way that the nucleic acids (e.g. polynucleotides) are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g. polynucleotide). One of skill will recognize that nucleic acids (e.g. polynucleotides) can be manipulated in many ways and are not limited to the examples above.

The term “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. In embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning — A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid (e.g. polynucleotide) may include a terminator that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation. In some embodiments, the expression cassette comprises a promoter operably linked to a second nucleic acid (e.g. polynucleotide) and a terminator operably linked to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation. In some embodiments, the expression cassette comprises an endogenous promoter. In some embodiments, the expression cassette comprises an endogenous terminator. In some embodiments, the expression cassette comprises a synthetic (or non-natural) promoter. In some embodiments, the expression cassette comprises a synthetic (or non-natural) terminator.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

The phrase “codon optimized” as it refers to genes or coding regions of nucleic acid molecules for the transformation of various hosts, refers to the alteration of codons in the gene or coding regions of polynucleic acid molecules to reflect the typical codon usage of a selected organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that selected organism.

Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g. enhancers and coding sequences) do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked with a coding sequence when it is capable of affecting (e.g. modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

The term "nucleobase" refers to the part of a nucleotide that bears the Watson/Crick basepairing functionality. The most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.

As used throughout, by a "subject" (or a “host”) is meant an individual. Thus, the "subject" can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human. The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

A nucleic acid sequence is “heterologous” to a second nucleic acid sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a heterologous promoter (or heterologous 5’ untranslated region (5’UTR)) operably linked to a coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from naturally occurring allelic variants.

As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, and improvement or remediation of damage.

As used herein, the term “preventing” a disease, a disorder, or unwanted physiological event in a subject refers to the prevention of a disease, a disorder, or unwanted physiological event or prevention of a symptom of a disease, a disorder, or unwanted physiological event.

“Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. In certain embodiments, the effective amount per dose varies from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, and from about 10.0 mg/kg to about 150 mg/kg.

"Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

"Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

“Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “therapeutic agent” is used, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

As used herein, the term “controlled-release” or “controlled-release drug delivery” or “extended release” refers to release or administration of a drug from a given dosage form in a controlled fashion in order to achieve the desired pharmacokinetic profile in vivo. An aspect of “controlled” drug delivery is the ability to manipulate the formulation and/or dosage form in order to establish the desired kinetics of drug release.

The phrases "concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or immediately following one another.

The term “antibodies” is used herein in a broad sense and includes both polyclonal and m onoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or huma nized versions of immunoglobulin molecules or fragments thereof. The antibodies can be tested f or their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinic al testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG- 2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable cla sses for mouse. The heavy chain constant domains that correspond to the different classes of imm unoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a sub stantially homogeneous population of antibodies, i.e., the individual antibodies within the popula tion are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimer ic" antibodies in which a portion of the heavy and/or light chain is identical with or homologous t o corresponding sequences in antibodies derived from a particular species or belonging to a parti cular antibody class or subclass, while the remainder of the chain(s) is identical with or homolog ous to corresponding sequences in antibodies derived from another species or belonging to anoth er antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

The disclosed monoclonal antibodies can be made using any procedure which produces m onoclonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybri doma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hy bridoma method, a mouse or other appropriate host animal is typically immunized with an immu nizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will sp ecifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vit ro.

The monoclonal antibodies may also be made by recombinant DNA methods. DNA enco ding the disclosed monoclonal antibodies can be readily isolated and sequenced using convention al procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to ge nes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active a ntibody fragments can also be generated and screened using phage display techniques, e.g., as de scribed in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et a 1.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antib odies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routi ne techniques known in the art. For instance, digestion can be performed using papain. Example s of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4, 342,566. Papain digestion of antibodies typically produces two identical antigen binding firagme nts, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pep sin treatment yields a fragment that has two antigen combining sites and is still capable of cross-1 inking antigen.

As used herein, the term “antibody or antigen binding fragment thereof’ or “antibody or f ragments thereof’ encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, sFv, scFv and the 1 ike, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind t heir specific antigens are provided. For example, fragments of antibodies which maintain bindin g activity are included within the meaning of the term “antibody or antigen binding fragment ther eof.” Such antibodies and fragments can be made by techniques known in the art and can be sere ened for specificity and activity according to the methods set forth in the Examples and in genera 1 methods for producing antibodies and screening antibodies for specificity and activity (See Harl ow and Lane. Antibodies, A Laboratory Manual . Cold Spring Harbor Publications, New York, (1 988)).

Also included within the meaning of “antibody or antigen binding fragment thereof’ are c onjugates of antibody fragments and antigen binding proteins (single chain antibodies). Also incl uded within the meaning of “antibody or antigen binding fragment thereof’ are immunoglobulin single variable domains, such as for example a nanobody.

The fragments, whether attached to other sequences or not, can also include insertions, de letions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bon ding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antib ody or antibody fragment must possess a bioactive property, such as specific binding to its cogna te antigen. Functional or active regions of the antibody or antibody fragment may be identified b y mutagenesis of a specific region of the protein, followed by expression and testing of the expre ssed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can in elude site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to a human antibody an d/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or r abbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the meth ods serves to lessen the chance that an antibody administered to a human will evoke an undesirab le immune response.

Chemical Definitions

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

The organic moieties mentioned when defining variable positions within the general formulae described herein (e.g., the term “halogen”) are collective terms for the individual substituents encompassed by the organic moiety. The prefix C_n-C_m preceding a group or moiety indicates, in each case, the possible number of carbon atoms in the group or moiety that follows.

The term “ion,” as used herein, refers to any molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom that contains a charge (positive, negative, or both at the same time within one molecule, cluster of molecules, molecular complex, or moiety (e.g., zwitterions)) or that can be made to contain a charge. Methods for producing a charge in a molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom are disclosed herein and can be accomplished by methods known in the art, e.g., protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, de-esterification, hydrolysis, etc.

The term “anion” is a type of ion and is included within the meaning of the term “ion.” An “anion” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains a net negative charge or that can be made to contain a net negative charge. The term “anion precursor” is used herein to specifically refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation).

The term “cation” is a type of ion and is included within the meaning of the term “ion.” A “cation” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom, that contains a net positive charge or that can be made to contain a net positive charge. The term “cation precursor” is used herein to specifically refer to a molecule that can be converted to a cation via a chemical reaction (e.g., protonation or alkylation).

As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

“Z¹,” “Z²,” “Z³,” and “Z⁴” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

The term “aliphatic” as used herein refers to a non-aromatic hydrocarbon group and includes branched and unbranched, alkyl, alkenyl, or alkynyl groups. As used herein, the term “alkyl” refers to saturated, straight-chained or branched saturated hydrocarbon moieties. Unless otherwise specified, C1-C24 (e.g., C2-C22, C4-C22, C6-C22, C8-C22, C10-C22, C12-C22, C14-C22, C16-C22, C2-C20, C4-C20, C6-C20, C8-C20, C10-C20, C12-C20, C14-C20, Ci6- C20, C1-C20, C1-C18, C1-C16, C1-C14, C1-C12, C1-C10, Ci-C₈, Ci-C₆, or C1-C4) alkyl groups are intended. Examples of alkyl groups include methyl, ethyl, propyl, 1-methyl-ethyl, butyl, 1 -methylpropyl, 2-methyl-propyl, 1,1 -dimethyl -ethyl, pentyl, 1-methyl-butyl, 2-methyl-butyl, 3-methyl- butyl, 2, 2-dimethyl -propyl, 1 -ethyl -propyl, hexyl, 1,1-dimethyl-propyl, 1,2-dimethyl-propyl, 1- methyl-pentyl, 2-methyl-pentyl, 3-methyl-pentyl, 4-methyl-pentyl, 1,1-dimethyl-butyl, 1,2- dimethyl-butyl, 1,3-dimethyl-butyl, 2,2-dimethyl-butyl, 2,3-dimethyl-butyl, 3,3-dimethyl-butyl, 1 -ethyl -butyl, 2-ethyl-butyl, 1,1,2-trimethyl-propyl, 1,2,2-trimethyl-propyl, 1 -ethyl -1 -methyl - propyl, l-ethyl-2-m ethyl -propyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Alkyl substituents may be unsubstituted or substituted with one or more chemical moieties. The alkyl group can be substituted with one or more groups including, but not limited to, hydroxyl, halogen, acetal, acyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied.

Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halides (halogens; e.g., fluorine, chlorine, bromine, or iodine). The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.

This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

As used herein, the term “alkenyl” refers to unsaturated, straight-chained, or branched hydrocarbon moieties containing a double bond. Unless otherwise specified, C2-C24 (e.g., C2-C22, C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10, C₂-C₈, C₂-C₆, or C2-C4) alkenyl groups are intended. Alkenyl groups may contain more than one unsaturated bond. Examples include ethenyl,

1 -propenyl, 2-propenyl, 1 -methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1 -methyl- 1 -propenyl,

2-methyl-l-propenyl, 1 -methyl -2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3- pentenyl, 4-pentenyl, 1 -methyl- 1-butenyl, 2-methyl- 1-butenyl, 3-methyl-l-butenyl, l-methyl-2- butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, l-methyl-3-butenyl, 2-methyl-3-butenyl, 3- methyl-3-butenyl, l,l-dimethyl-2-propenyl, 1,2-dimethyl-l -propenyl, l,2-dimethyl-2-propenyl, 1-ethyl-l-propenyl, 1 -ethyl -2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl,

1-methyl-l-pentenyl, 2-methyl- 1-pentenyl, 3-methyl-l-pentenyl, 4-methyl- 1-pentenyl, 1-methyl-

2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, l-methyl-3- pentenyl, 2-methyl-3 -pentenyl, 3-methyl-3-pentenyl, 4-methyl-3 -pentenyl, l-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, l,l-dimethyl-2-butenyl, 1,1- dimethyl-3-butenyl, 1,2-dimethyl- 1-butenyl, l,2-dimethyl-2-butenyl, l,2-dimethyl-3-butenyl,

1.3-dimethyl- 1-butenyl, 1,3-dimethyl -2-butenyl, l,3-dimethyl-3-butenyl, 2, 2-dimethyl -3-butenyl,

2.3-dimethyl- 1-butenyl, 2, 3-dimethyl -2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-l-butenyl,

3.3-dimethyl-2-butenyl, 1-ethyl-l-butenyl, 1 -ethyl -2-butenyl, l-ethyl-3-butenyl, 2-ethyl-l- butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, l,l,2-trimethyl-2-propenyl, 1 -ethyl- l-methyl-2- propenyl, l-ethyl-2-m ethyl- 1 -propenyl, and 1 -ethyl -2-methyl -2-propenyl. The term “vinyl” refers to a group having the structure -CEUCH2; 1 -propenyl refers to a group with the structure - CEUCH-CH3; and 2-propenyl refers to a group with the structure -CH2-CEUCH2. Asymmetric structures such as (Z¹Z²)C=C(Z³Z⁴) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. Alkenyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied.

As used herein, the term “alkynyl” represents straight-chained or branched hydrocarbon moieties containing a triple bond. Unless otherwise specified, C2-C24 (e.g., C2-C24, C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4) alkynyl groups are intended. Alkynyl groups may contain more than one unsaturated bond. Examples include C2-Ce-alkynyl, such as ethynyl, 1-propynyl, 2-propynyl (or propargyl), 1-butynyl, 2-butynyl, 3-butynyl, l-methyl-2- propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 3 -methyl- 1-butynyl, l-methyl-2- butynyl, l-methyl-3-butynyl, 2-methyl-3-butynyl, 1,1 -dimethyl -2-propynyl, 1 -ethyl -2-propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 3-methyl-l -pentynyl, 4-methyl-l- pentynyl, 1 -methyl -2-pentynyl, 4-methyl-2-pentynyl, l-methyl-3 -pentynyl, 2-methyl-3-pentynyl, 1 -methyl -4-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, l,l-dimethyl-2-butynyl, 1,1- dimethyl-3-butynyl, l,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3, 3 -dimethyl- 1-butynyl, 1- ethyl-2-butynyl, 1 -ethyl -3-butynyl, 2-ethyl-3-butynyl, and 1 -ethyl- l-methyl-2-propynyl. Alkynyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

As used herein, the term “aryl,” as well as derivative terms such as aryloxy, refers to groups that include a monovalent aromatic carbocyclic group of from 3 to 50 carbon atoms. Aryl groups can include a single ring or multiple condensed rings. In some embodiments, aryl groups include Ce-Cio aryl groups. Examples of aryl groups include, but are not limited to, benzene, phenyl, biphenyl, naphthyl, tetrahydronaphthyl, phenyl cyclopropyl, phenoxybenzene, and indanyl. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bound, z.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

The term “cyclic group” is used herein to refer to either aryl groups, non-aryl groups (z.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems (e.g., monocyclic, bicyclic, tricyclic, polycyclic, etc.) that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more nonaryl groups, or one or more aryl groups and one or more non-aryl groups.

The term “acyl” as used herein is represented by the formula -C(O)Z¹ where Z¹ can be a hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. As used herein, the term “acyl” can be used interchangeably with “carbonyl.” Throughout this specification “C(O)” or “CO” is a shorthand notation for C=O.

The term “acetal” as used herein is represented by the formula (Z¹Z²)C(OZ³)(OZ⁴), where Z¹, Z², Z³, and Z⁴ can be, independently, a hydrogen, halogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “alkanol” as used herein is represented by the formula Z'OH, where Z¹ can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

As used herein, the term “alkoxy” as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group can be defined as to a group of the formula Z¹- O-, where Z¹ is unsubstituted or substituted alkyl as defined above. Unless otherwise specified, alkoxy groups wherein Z¹ is a C1-C24 (e.g., C2-C22, C4-C22, C6-C22, C8-C22, C10-C22, C12-C22, C14- C22, C16-C22, C2-C20, C4-C20, C6-C20, C8-C20, C10-C20, C12-C20, C14-C20, C16-C20, C1-C20, C1-C18, C1-C16, C1-C14, C1-C12, C1-C10, Ci-Cs, Ci-Ce, or C1-C4) alkyl group are intended. Examples include methoxy, ethoxy, propoxy, 1 -methyl -ethoxy, butoxy, 1-methyl-propoxy, 2-methyl-propoxy, 1,1- dimethyl-ethoxy, pentoxy, 1-methyl-butyloxy, 2-methyl -butoxy, 3-methyl-butoxy, 2,2-di-methyl- propoxy, 1-ethyl-propoxy, hexoxy, 1, 1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl- pentoxy, 2-methyl-pentoxy, 3-methyl-pentoxy, 4-methyl-penoxy, 1, 1 -dimethyl -butoxy, 1,2- dimethyl-butoxy, 1,3-dimethyl-butoxy, 2,2-dimethyl-butoxy, 2,3-dimethyl-butoxy, 3, 3 -dimethylbutoxy, 1-ethyl-butoxy, 2-ethylbutoxy, 1,1,2-trimethyl-propoxy, 1,2,2-trimethyl-propoxy, 1- ethyl- 1-methyl-propoxy, and 1 -ethyl -2-methyl -propoxy.

The term “aldehyde” as used herein is represented by the formula — C(O)H. Throughout this specification “C(O)” is a shorthand notation for C=O.

The terms “amine” or “amino” as used herein are represented by the formula — NZ^JZ²Z³, where Z¹, Z², and Z³ can each be substitution group as described herein, such as hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The terms “amide” or “amido” as used herein are represented by the formula — C(O)NZ¹Z², where Z¹ and Z² can each be substitution group as described herein, such as hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “anhydride” as used herein is represented by the formula Z¹C(O)OC(O)Z² where Z¹ and Z², independently, can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “cyclic anhydride” as used herein is represented by the formula:

O X where Z¹ can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “azide” as used herein is represented by the formula -N=N=N.

The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

A “carboxylate” or “carboxyl” group as used herein is represented by the formula — C(O)O’

A “carbonate ester” group as used herein is represented by the formula Z¹OC(O)OZ².

The term “cyano” as used herein is represented by the formula — CN.

The term “ester” as used herein is represented by the formula — OC(O)Z¹ or — C(O)OZ¹, where Z¹ can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “ether” as used herein is represented by the formula Z^XOZ², where Z¹ and Z² can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “epoxy” or “epoxide” as used herein refers to a cyclic ether with a three atom ring and can represented by the formula: where Z¹, Z², Z³, and Z⁴ can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above

The term “ketone” as used herein is represented by the formula Z¹C(O)Z², where Z¹ and Z² can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “halide” or “halogen” or “halo” as used herein refers to fluorine, chlorine, bromine, and iodine.

The term “hydroxyl” as used herein is represented by the formula — OH.

The term “nitro” as used herein is represented by the formula — NO2.

The term “phosphonyl” is used herein to refer to the phospho-oxo group represented by the formula — P(O)(OZ¹)2, where Z¹ can be hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “silyl” as used herein is represented by the formula — SiZ^JZ²Z³, where Z¹, Z², and Z³ can be, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “sulfonyl” or “sulfone” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2Z where Z¹ can be hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term “sulfide” as used herein is comprises the formula — S — .

The term “thiol” as used herein is represented by the formula — SH.

“R¹,” “R²,” “R³,” “Rⁿ,” etc., where n is some integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R¹ is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an amine group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible stereoisomer or mixture of stereoisomer (e.g., each enantiomer, each diastereomer, each meso compound, a racemic mixture, or scalemic mixture).

Compounds

Disclosed herein are compounds defined by Formula I, or a pharmaceutically acceptable salt thereof:

I wherein p is an integer from 0 to 5; n is an integer from 1 to 10; m, when present, is an integer from 1 to 10;

In some examples of Formula I, each R² is independently an unsubstituted linear Cs-Cis alkyl. In some examples of Formula I, each R² is independently an unsubstituted branched Cs-Cis alkyl. In some examples of Formula I, each R² is independently a linear Ci-Cis alkyl (e.g., C4-C6 alkyl) substituted with one or more substituents selected from the group consisting of acetal, amine, amide, ester, ether (e.g., acetal), and carbonate ester. In some examples of Formula I, each R² is independently a branched Ci-Cis alkyl (e.g., C4-C6 alkyl) substituted with one or more substituents selected from the group consisting of amine, amide, ester, ether (e.g., acetal), and carbonate ester. In some examples of Formula I, each R² is independently selected from the group consisting of wherein G and I each independently represent integers from 1 to 8.

In some examples of Formula I, each R² is independently selected from the group consisting of: wherein G and I each independently represent integers from 1 to 8. In some examples of Formula I, each R² is independently selected from the group consisting of:

In some examples of Formula I, each R² is the same. In some examples of Formula I, at least one R² is different. In some examples of Formula I, p is 0. In some examples of Formula I, p is 1.

LPAs are a collection of bioactive lipids involved in cellular signaling pathways through specific cell-surface G protein-coupled receptors (GPCRs). Currently, there are six known LPA receptors, which are designated as LPAi, LPA2, LPA3, LPA4, LPA5 and LPAe. In some examples of Formula I, the LPA receptor antagonist comprises a lysophosphatidic acid receptor 1 (LPAi) antagonist. It has been shown that LPAi plays a significant role in modulating wound healing by stimulating chemotaxis through fibroblast recruitment. In some examples of Formula I, R¹ is a Ci- Ce alkyl linker substituted with the LPA receptor antagonist (e.g., LPAi antagonist, LPA2 antagonist, LPA3 antagonist, LPA4 antagonist, LPA5 antagonist, or LPAe antagonist).

In some examples of Formula I, the LPA receptor antagonist is selected from AM966, AM095, RO-6842262, BMS-986020, BMS-986278, KH6425, and VPC12249. Several of the LPA receptor antagonists are identified by their commonly used names represented in literature, however, references to a particular compound include various pharmaceutically acceptable salts and stereoisomers thereof unless stated otherwise. Moreover, the reference to an LPA receptor antagonist can encompass derivatives thereof in which substituents, linkers, and/or covalent bonds are used to attach the LPA receptor antagonist to the main structure of Formula I. By way of nonlimiting example, LPA receptor antagonists can be coupled with the main structure of Formula I using an esterification reaction. In some examples of Formula I, the LPA receptor antagonist is selected from the group consisting of AM966, AM095, RO-6842262, and BMS-986020. In some examples of Formula I, the LPA receptor antagonist is AM966. In some examples of Formula I, the LPA receptor antagonist is AM095. In some examples of Formula I, the LPA receptor antagonist is RO-6842262. In some examples of Formula I, the LPA receptor antagonist is BMS- 986020. In some examples of Formula I, the LPA receptor antagonist is BMS-986278. In some examples of Formula I, the LPA receptor antagonist is Ki 16425. In some examples of Formula I, the LPA receptor antagonist is VPC 12249.

RO-6842262

BMS-986020

II wherein

X is N or C;

Z is N or O;

R⁴, R⁵, R⁶, R⁷, R⁸ are each independently H, OH, halogen, substituted or unsubstituted Ci-

C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or NR^xR^y, or wherein, as valence permits, R⁴, R⁵, R⁶, R⁷, R⁸, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; R^x and R^y, when present, are independently selected from substituted or unsubstituted Ci- C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, and substituted or unsubstituted C1-C20 acyl;

R⁹ is H or cyclopropyl;

R¹⁰, R¹¹, R¹² are each independently H or methyl; and

L is selected from:

In some examples of Formula II, L is: some examples of Formula II, L is: In some examples of Formula II, L is:

In some examples of Formula II, R⁴, R⁵, R⁶, R⁷, R⁸ are each H. In some examples of Formula II, at least one of R⁴, R⁵, R⁶, R⁷, R⁸ is a halogen (e.g., Cl). In some examples of Formula II, each of R¹⁰, R¹¹, R¹² is H. In some examples of Formula II, each of R¹⁰, R¹¹, R¹² is methyl. In some examples of Formula II, at least one of R¹⁰, R¹¹, R¹² is H. In some examples of Formula II, at least one of R¹⁰, R¹¹, R¹² is methyl. In some examples of Formula II, R¹⁰ and R¹² are each methyl. In some examples of Formula II, R¹¹ is H. In some examples of Formula II, R¹⁰ and R¹² are each methyl and R¹¹ is H.

III wherein

X is N or C;

Z is N or O;

R⁴, R⁵, R⁶, R⁷, R⁸ are each independently H, OH, halogen, substituted or unsubstituted Ci- C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or NR^xR^y, or wherein, as valence permits, R⁴, R⁵, R⁶, R⁷, R⁸, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms;

R^x and R^y, when present, are independently selected from substituted or unsubstituted Ci- C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, and substituted or unsubstituted C1-C20 acyl; and

R⁹ is H or cyclopropyl.

In some examples of Formula III, R⁴, R⁵, R⁶, R⁷, R⁸ are each H. In some examples of Formula III, at least one of R⁴, R⁵, R⁶, R⁷, R⁸ is a halogen (e.g., Cl).

IV wherein R⁴, R⁵, R⁶, R⁷, R⁸ are each independently H, OH, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3- C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, orNR^xR^y, or wherein, as valence permits, R⁴, R⁵, R⁶, R⁷, R⁸, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; and R^x and R^y are independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl. In some examples of Formula IV, R⁴, R⁵, R⁶, R⁷, R⁸ are each H. In some examples of

Formula IV, at least one of R⁴, R⁵, R⁶, R⁷, R⁸ is a halogen (e.g., Cl).

In some examples, the compound is selected from the group consisting of:

wherein each R² is independently selected from:

In some examples, the compound is: wherein each R² is independently selected from:

In some examples, the compound is:

R²

N.R₂

Lipid Nanoparticles

Also disclosed herein is a lipid nanoparticle (e.g., one or more nanoparticles) comprising any of the compounds disclosed herein.

In one aspect, the disclosure provides a nanoparticle comprising: a compound of Formula I; a non-cationic lipid; a polyethylene gly col-lipid; and a sterol.

The compound of Formula I, including the specific example compounds, are described in the Compounds section above. In some embodiments, the nanoparticle comprises a compound of Formula I (or any of the specific example compounds therein) in a molar ratio of 10% to 70%. In some examples, the nanoparticle comprises a compound of Formula I in a molar ratio of from 25% to 55%. In some examples, the nanoparticle comprises a compound of Formula I in a molar ratio of from 35% to 45%. In some embodiments, the nanoparticle comprises a compound of Formula I in a molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%.

In some embodiments, the nanoparticle comprises a non-cationic lipid. In some embodiments, the non-cationic lipid interacts with the lipids as a helper lipid. In some embodiments, the non-cationic lipid can include, but is not limited to, l,2-dioleoyl- w-glycero-3- phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl-sw-glycero-3 -phosphoethanolamine (POPE), l,2-distearoyl-sw-glycero-3 -phosphocholine (DSPC), l-stearoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (SOPE), DPPC (l,2-dipalmitoyl-sn-glycero-3- phosphocholine), 1,2- dioleyl-sn-glycero-3-phosphotidylcholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2- dioleoyl-5/7-glycero-3- phospho-(l'-rac-glycerol) (DOPG), or combinations thereof. In one embodiment, the non-cationic lipid is l,2-dioleoyl-sw-glycero-3 -phosphoethanolamine (DOPE). In one embodiment, the non-cationic lipid is l-palmitoyl-2-oleoyl- w-glycero-3- phosphoethanolamine (POPE), In one embodiment, the non-cationic lipid is 1,2-distearoyl- w- glycero-3 -phosphocholine (DSPC). In one embodiment, the non-cationic lipid is l-stearoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). While several non-cationic lipids are described here, additional non-cationic lipids can be used in combination with the compounds disclosed herein.

In some embodiments, the nanoparticle comprises a non-cationic lipid in a molar ratio of about 10% to about 60%. In some examples, the non-cationic lipid is present in a molar ratio of from 20% to 50%. In some embodiments, the nanoparticle comprises a non-cationic lipid in a molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%., about 45%, about 50%, about 55%, or about 60%. In one embodiment, the nanoparticle comprises a non-cationic lipid in a molar ratio of about 50%.

In some embodiments, the nanoparticle includes a polyethylene gly col-lipid (PEG- lipid). PEG-lipid is incorporated to form a hydrophilic outer layer and stabilize the particles. Nonlimiting examples of polyethylene glycol-lipids include PEG-modified lipids such as PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include DMG-PEG, DLPE-PEGs, DMPE-PEGs, DPPC-PEGs, and DSPE-PEGs. In one embodiment, the polyethylene gly col-lipid is 1,2- dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG). In one embodiment, the polyethylene glycol -lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol -2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-XXXX, wherein XXXX signifies the molecular weight of the polyethylene glycol moiety, e g. DMG-PEG2000 or DMG-PEG5000.

In some embodiments, the nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of about 0% to about 5%. In some embodiments, the nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of about 0%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 3%, about 4%, or about 5%. In one embodiment, the nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of about 0.5%.

In some embodiments, the nanoparticle includes a sterol. Sterols are well known to those skilled in the art and generally refers to those compounds having a perhydrocyclopentanophenanthrene ring system and having one or more OH substituents. Examples of sterols include, but are not limited to, cholesterol, campesterol, ergosterol, sitosterol, and the like.

In some embodiments, the sterol is selected from a cholesterol-based lipid. In some embodiments, the one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol, DC-Choi (N,N-dimethyl-N- ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino- propyl)piperazine, or combinations thereof.

The sterol can be used to tune the particle permeability and fluidity base on its function in cell membranes. In one embodiment, the sterol is cholesterol.

In some embodiments, the nanoparticle comprises a sterol in a molar ratio of about 25% to about 50%. In some embodiments, the nanoparticle comprises a sterol in a molar ratio of about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In one embodiment, the nanoparticle comprises a sterol in a molar ratio of about 40%.

In one embodiment, the nanoparticle further comprises an agent. In one embodiment, the nanoparticle further comprises a therapeutic agent. In one embodiment, the nanoparticle further comprises a diagnostic agent.

The agents delivered into cells can be a polynucleotide. Polynucleotides or oligonucleotides that can be introduced according to the methods herein include DNA, cDNA, and RNA sequences of all types. For example, the polynucleotide can be double stranded DNA, singlestranded DNA, complexed DNA, encapsulated DNA, naked RNA, encapsulated RNA, messenger RNA (mRNA), tRNA, short interfering RNA (siRNA), double stranded RNA (dsRNA), micro- RNA (miRNA), antisense RNA (asRNA) and combinations thereof. The polynucleotides can also be DNA constructs, such as expression vectors, expression vectors encoding a desired gene product (e.g., a gene product homologous or heterologous to the subject into which it is to be introduced), and the like. In one embodiment, the agent is an mRNA. In some examples, the agent comprises a polynucleotide encoding a ubiquitin-modifying enzyme. In some examples, the agent comprises Tumor Necrosis Factor a-Induced Protein 3 (TNFAIP3), Suppressor of Cytokine Signaling 1 (SOCS1), Smad7, Krtippel-like Factor 2 (KLF2), Matrix metalloproteinases (MMPs), Thrombospondin- 1 (TSP-1), Interleukin- 10 (IL-10), and/or Decorin. In some examples, the agent comprises a polynucleotide encoding Tumor Necrosis Factor a-Induced Protein 3 (TNFAIP3). In some examples, the agent is encapsulated by the nanoparticle.

The nanoparticle can be of any shape, (e.g., a sphere, a rod, a quadrilateral, an ellipse, a triangle, a polygon, etc.). In some examples, the nanoparticle can have a regular shape, an irregular shape, an isotropic shape, an anisotropic shape, or a combination thereof. In some examples, the lipid particle are substantially spherical in shape.

The lipid particles can have an average particle size. “Average particle size” and “mean particle size” are used interchangeably herein, and generally refer to the statistical mean particle size of the particles in a population of particles. For example, the average particle size for a plurality of particles with a substantially spherical shape can comprise the average diameter of the plurality of particles. For a particle with a substantially spherical shape, the diameter of a particle can refer, for example, to the hydrodynamic diameter. As used herein, the hydrodynamic diameter of a particle can refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as evaluation by scanning electron microscopy, transmission electron microscopy, and/or dynamic light scattering.

The lipid particles can, for example, have an average particle size of 30 nanometers (nm) or more (e.g., 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 275 nm or more, 300 nm or more, 325 nm or more, 350 nm or more, 375 nm or more, 400 nm or more, 425 nm or more, 450 nm or more, 475 nm or more, 500 nm or more, 550 nm or more, 600 nm or more, 650 nm or more, 700 nm or more, or 750 nm or more). In some examples, the lipid particles can have an average particle size of 800 nm or less (e.g., 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 475 nm or less, 450 nm or less, 425 nm or less, 400 nm or less, 375 nm or less, 350 nm or less, 325 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, or 40 nm or less). The average particle size of the lipid particles can range from any of the minimum values described above to any of the maximum values described above. For example, the lipid particles can have an average particle size of from 30 nm to 800 nm (e.g., from 30 nm to 425 nm, from 425 nm to 800 nm, from 30 nm to 200 nm, from 200 nm to 400 nm, from 400 nm to 600 nm, from 600 nm to 800 nm, from 50 nm to 800 nm, from 30 nm to 750 nm, or from 50 nm to 750 nm).

With respect to particle size distribution characterization, a parameter used to define the size range of the lipid particles is called the “poly dispersity index” (PDI). The term “poly dispersity” (or “dispersity” as recommended by IUPAC) is used to describe the degree of non-uniformity of a size distribution of particles. PDI is basically a representation of the distribution of size populations within a given sample. The numerical value of PDI ranges from 0.0 (for a perfectly uniform sample with respect to the particle size) to 1.0 (for a highly polydisperse sample with multiple particle size populations).

In some examples, the lipid particles can have a poly dispersity index of 0.5 or less (e.g., 0.49 or less, 0.48 or less, 0.47 or less, 0.46 or less, 0.45 or less, 0.44 or less, 0.43 or less, 0.42 or less, 0.41 or less, 0.40 or less, 0.39 or less, 0.38 or less, 0.37 or less, 0.36 or less, 0.35 or less, 0.34 or less, 0.33 or less, 0.32 or less, 0.31 or less, 0.30 or less, 0.29 or less, 0.28 or less, 0.27 or less, 0.26 or less, 0.25 or less, 0.24 or less, 0.23 or less, 0.22 or less, 0.21 or less, 0.20 or less, 0.19 or less, 0.18 or less, 0.17 or less, 0.16 or less, 0.15 or less, 0.14 or less, 0.13 or less, 0.12 or less, 0.11 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less).

In some examples, the lipid particles can be substantially monodisperse. “Monodisperse” and “homogeneous size distribution,” as used herein, and generally describe a population of particles where all of the particles are the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 80% of the distribution (e.g., 85% of the distribution, 90% of the distribution, or 95% of the distribution) lies within 25% of the median particle size (e.g., within 20% of the median particle size, within 15% of the median particle size, within 10% of the median particle size, or within 5% of the median particle size).

Compositions

Compositions, as described herein, comprising an active compound and an excipient of some sort may be useful in a variety of medical and non-medical applications. For example, pharmaceutical compositions comprising an active compound and an excipient may be useful in the delivery of an effective amount of an agent to a subject in need thereof. Nutraceutical compositions comprising an active compound and an excipient may be useful in the delivery of an effective amount of a nutraceutical, e.g., a dietary supplement, to a subject in need thereof. Cosmetic compositions comprising an active compound and an excipient may be formulated as a cream, ointment, balm, paste, film, or liquid, etc., and may be useful in the application of makeup, hair products, and materials useful for personal hygiene, etc. Compositions comprising an active compound and an excipient may be useful for non-medical applications, e.g., such as an emulsion or emulsifier, useful, for example, as a food component, for extinguishing fires, for disinfecting surfaces, for oil cleanup, etc.

In certain embodiments, the composition further comprises an agent, as described herein. For example, in certain embodiments, the agent is a small molecule, organometallic compound, nucleic acid, protein, peptide, polynucleotide, metal, targeting agent, an isotopically labeled chemical compound, drug, vaccine, immunological agent, or an agent useful in bioprocessing. In certain embodiments, the agent is a polynucleotide. In certain embodiments, the polynucleotide is DNA or RNA. In certain embodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA. In certain embodiments, the polynucleotide and the one or more active compounds are not covalently attached.

Agents

Agents to be delivered by the compounds, compositions, and systems described herein may be therapeutic, diagnostic, or prophylactic agents. Any chemical compound to be administered to a subject may be delivered using the particles or nanoparticles described herein. The agent may be an organic molecule (e.g., a therapeutic agent, a drug), inorganic molecule, nucleic acid, protein, amino acid, peptide, polypeptide, polynucleotide, targeting agent, isotopically labeled organic or inorganic molecule, vaccine, immunological agent, etc.

In certain embodiments, the agents are organic molecules with pharmaceutical activity, e.g., a drug. In certain embodiments, the drug is an antibiotic, anti-viral agent, anesthetic, steroidal agent, anti-inflammatory agent, anti -neoplastic agent, anti-cancer agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anticholinergic, analgesic, anti-depressant, anti-psychotic, f3-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non-steroidal anti-inflammatory agent, nutritional agent, etc.

In some examples, the agent comprises a polynucleotide encoding a ubiquitin-modifying enzyme. In some examples, the agent comprises Tumor Necrosis Factor a-Induced Protein 3 (TNFAIP3), Suppressor of Cytokine Signaling 1 (SOCS1), Smad7, Kriippel-like Factor 2 (KLF2), Matrix metalloproteinases (MMPs), Thrombospondin- 1 (TSP-1), Interleukin- 10 (IL-10), and/or Decorin. In some examples, the agent comprises a polynucleotide encoding Tumor Necrosis Factor a-Induced Protein 3 (TNFAIP3). In some examples, the agent is encapsulated by the nanoparticle. In certain embodiments of the present disclosure, the agent to be delivered may be a mixture of agents.

Diagnostic agents include gases; metals; commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents. Examples of suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and x-ray imaging include iodine-based materials.

Therapeutic and prophylactic agents include, but are not limited to, antibiotics, nutritional supplements, and vaccines. Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, cell extracts, and RNA encoding at least one antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide). Therapeutic and prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents include antigens of such bacterial organisms as Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainjluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens of such viruses as smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and the like; antigens of fungal, protozoan, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

In some aspects, the agent is a ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one (e.g., at least 2, 3, 4 or 5) antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide).

In some embodiments, the nucleic acids disclosed herein comprise at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof.

In one embodiment, the at least one chemically modified nucleotide is a chemically modified nucleobase.

In one embodiment, the chemically modified nucleobase is selected from 5 -formylcytidine (5fC), 5-methylcytidine (5meC), 5 -methoxy cytidine (5moC), 5-hydroxycytidine (5hoC), 5- hydroxymethylcytidine (5hmC), 5-formyluridine (5fU), 5-methyluridine (5-meU), 5- methoxyuridine (5moU), 5-carboxymethylesteruridine (5camU), pseudouridine ( ), N¹- methylpseudouridine (me¹T'), N⁶ -methyladenosine (me⁶ A), or thienoguanosine f^hG).

In some embodiments, the chemically modified nucleobase is 5-methoxyuridine (5moU). In some embodiments, the chemically modified nucleobase is pseudouridine ( ). In some embodiments, the chemically modified nucleobase is Nkmethylpseudouridine (me¹T').

The structures of these modified nucleobases are shown below:

In one embodiment, the at least one chemically modified nucleotide is a chemically modified ribose. In one embodiment, the chemically modified ribose is selected from 2'-(9-methyl (2'-O-

Me), 2'-Fluoro (2'-F), 2'-deoxy-2'-fluoro-beta-D-arabino-nucleic acid (2'F-ANA), 4'-S, 4'- SFANA, 2'-azido, UNA, 2 '-O-m ethoxy-ethyl (2'-< -ME), 2'-( -Allyl, 2'-(9-Ethylamine, 2'-O- Cyanoethyl, Locked nucleic acid (LAN), Methylene-cLAN, N-MeO-amino BNA, or N-MeO- aminooxy BNA. In one embodiment, the chemically modified ribose is 2'-O-methyl (2'-0-Me). In one embodiment, the chemically modified ribose is 2'-Fluoro (2'-F).

The structures of these modified riboses are shown below:

ethyl (2'-O-ME)

Lo

In one embodiment, the at least one chemically modified nucleotide is a chemically modified phosphodiester linkage. In one embodiment, the chemically modified phosphodiester linkage is selected from phosphorothioate (PS), boranophosphate, phosphodithioate (PS2), 3 ',5 '-amide, N3'- phosphoramidate (NP), Phosphodiester (PO), or 2', 5 '-phosphodiester (2',5'-PO). In one embodiment, the chemically modified phosphodiester linkage is phosphorothioate.

The structures of these modified phosphodiester linkages are shown below:

-PO)

Methods of Making

Also disclosed herein are methods of making any of the compounds or compositions disclosed herein. Also disclosed herein are methods of making any of the lipid particles disclosed herein. Also disclosed herein are methods of making any of the pharmaceutical compositions disclosed herein.

The compounds described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety. The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Katchem (Prague, Czech Republic), Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Suppiementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical excipients disclosed herein can be obtained from commercial sources.

Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, z.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., ^JHor ¹³C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

Methods of Use

Also disclosed herein are methods of use of any of the compounds or compositions disclosed herein.

In one aspect, provided herein is a method for the delivery of an agent (for example, a polynucleotide) into a cell comprising; introducing into the cell a composition comprising; a nanoparticle, comprising; a compound of Formula I; a non-cationic lipid; a polyethylene gly col-lipid; a sterol; and an agent.

In one aspect, disclosed herein is a method for the delivery of an agent into a cell comprising; introducing into the cell a composition comprising; a nanoparticle comprising; a compound of Formula I, or a pharmaceutically acceptable salt thereof:

R¹ is an alkyl or ether linker, wherein the alkyl or ether linker is substituted with a lysophosphatidic acid (LPA) receptor antagonist; each R² is independently a substituted or unsubstituted Ci-Cis alkyl; each R³, when present, is independently hydrogen, OH, a substituted or unsubstituted Ci- Ci8 alkyl, a substituted or unsubstituted C1-C5 alkyl alcohol; a non-cationic lipid; a polyethylene gly col-lipid; a sterol; and an agent. In some embodiments, a nanoparticle comprising any compound as described in the Compounds section above, is used in the methods herein, for delivery of an agent into a cell.

In some embodiments, the agent is a polynucleotide. In some embodiments, the agent is an RNA. In some embodiments, the agent is an mRNA. In some embodiments, the agent is a therapeutic agent, diagnostic agent, or prophylactic agent.

In some embodiments, provided herein are methods for the delivery of polynucleotides. In some embodiments, provided herein are methods for the delivery of polynucleotides (for example, mRNA) to correct a mutation in a genome. For example, mRNAs can be delivered to correct mutations that cause hemophilia (due to mutations in the genes encoding Factor VIII (F8; hemophilia A) or Factor IX (F9; hemoglobin B). In some embodiments, provided herein are methods for the delivery of polynucleotides. In some embodiments, provided herein are methods for the delivery of polynucleotides (for example, mRNA) to provide expression of the mRNA (and translation to produce a protein) in a cell. In some embodiments, provided herein are methods for the delivery of polynucleotides (for example, mRNA) to induce an immune response in a subject. In some embodiments, the RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one (e.g., at least 2, 3, 4 or 5) hMPV, PIV, RSV, MeV, and/or a BetaCoV (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKUl) antigenic polypeptide, or any combination of two or more of the antigenic polypeptides.

In one embodiment, provided herein is a method of treating a connective tissue disorder. The method comprises administering to said subject a therapeutically effective amount of a compound, a combination of compounds, or a composition provided herein, or a pharmaceutically acceptable form thereof, or a pharmaceutical composition as provided herein. In some examples, the connective tissue disorder is a fibrosis disease or disorder. Non-limiting examples of fibrotic diseases and disorders can include systemic fibrosis (i.e., radiation fibrosis), liver fibrosis and/or cirrhosis, renal fibrosis, lung fibrosis (e.g., idiopathic lung fibrosis), and/or interstitial lung disease, skin fibrosis, cardiac fibrosis, ocular fibrosis, ocular disease, myelofibrosis, cancers, and other related fibrotic diseases.

In one embodiment, provided herein is a method of treating an inflammation disorder, including autoimmune diseases in a subject. The method comprises administering to said subject a therapeutically effective amount of a compound, a combination of compounds, or a composition provided herein, or a pharmaceutically acceptable form thereof, or a pharmaceutical composition as provided herein. Examples of autoimmune diseases include but are not limited to acute disseminated encephalomyelitis (ADEM), Addison's disease, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hepatitis, autoimmune skin disease, coeliac disease, Crohn's disease, Diabetes mellitus (type 1), Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, lupus erythematosus, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, oemphigus, polyarthritis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, Reiter's syndrome, Takayasu's arteritis, temporal arteritis (also known as “giant cell arteritis”), warm autoimmune hemolytic anemia, Wegener's granulomatosis, alopecia universalis (e.g., inflammatory alopecia), Chagas disease, chronic fatigue syndrome, dysautonomia, endometriosis, hidradenitis suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, and vulvodynia. Other disorders include bone-resorption disorders and thrombosis.

Inflammation takes on many forms and includes, but is not limited to, acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, obliterative, parenchymatous, plastic, productive, proliferous, pseudomembranous, purulent, sclerosing, seroplastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and/or ulcerative inflammation.

Exemplary inflammatory conditions include, but are not limited to, inflammation associated with acne, anemia (e.g., aplastic anemia, haemolytic autoimmune anaemia), asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu's arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gout flare, gouty arthritis, reactive arthritis, rheumatoid arthritis and Reiter's arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructive pulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., type I diabetes mellitus, type 2 diabetes mellitus), a skin condition (e.g., psoriasis, eczema, bums, dermatitis, pruritus (itch)), endometriosis, Guillain- Barre syndrome, infection, ischaemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g., migraine headaches, tension headaches), ileus (e.g., postoperative ileus and ileus during sepsis), idiopathic thrombocytopenic purpura, interstitial cystitis (painful bladder syndrome), gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)), lupus, multiple sclerosis, morphea, myeasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus vulgaris, pernicious aneaemia, peptic ulcers, polymyositis, primary biliary cirrhosis, neuroinflammation associated with brain disorders (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, polymyalgia rheumatic, reperfusion injury, regional enteritis, rheumatic fever, systemic lupus erythematosus, scleroderma, scierodoma, sarcoidosis, spondyloarthopathies, Sjogren's syndrome, thyroiditis, transplantation rejection, tendonitis, trauma or injury (e.g., frostbite, chemical irritants, toxins, scarring, burns, physical injury), vasculitis, vitiligo and Wegener's granulomatosis. In certain embodiments, the inflammatory disorder is selected from arthritis (e.g., rheumatoid arthritis), inflammatory bowel disease, inflammatory bowel syndrome, asthma, psoriasis, endometriosis, interstitial cystitis and prostatistis. In certain embodiments, the inflammatory condition is an acute inflammatory condition (e.g., for example, inflammation resulting from infection). In certain embodiments, the inflammatory condition is a chronic inflammatory condition (e.g., conditions resulting from asthma, arthritis and inflammatory bowel disease). The compounds can also be useful in treating inflammation associated with trauma and non-infl ammatory myalgia.

Immune disorders, such as auto-immune disorders include, but are not limited to, arthritis (including rheumatoid arthritis, spondyloarthopathies, gouty arthritis, degenerative joint diseases such as osteoarthritis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, undifferentiated spondylitis, Behcet's disease, haemolytic autoimmune anaemias, multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute painful shoulder, psoriatic, and juvenile arthritis), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin condition (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itch)), enuresis, eosinophilic disease, gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)), relapsing polychondritis (e.g., atrophic polychondritis and systemic polychondromalacia), and disorders ameliorated by a gastroprokinetic agent (e.g., ileus, postoperative ileus and ileus during sepsis; gastroesophageal reflux disease (GORD, or its synonym GERD); eosinophilic esophagitis, gastroparesis such as diabetic gastroparesis; food intolerances and food allergies and other functional bowel disorders, such as non-ulcerative dyspepsia (NUD) and non-cardiac chest pain (NCCP, including costo-chondritis)).

In some examples, the compound or composition can be administered to the subject in an amount of 1 microgram (pg) per kilogram (kg) of body weight of the subject per day (pg/kg/day) or more (e.g., 2 pg/kg/day or more, 3 pg/kg/day or more, 4 pg/kg/day or more, 5 pg/kg/day or more, 10 pg/kg/day or more, 15 pg/kg/day or more, 20 pg/kg/day or more, 25 pg/kg/day or more, 30 pg/kg/day or more, 35 pg/kg/day or more, 40 pg/kg/day or more, 45 pg/kg/day or more, 50 pg/kg/day or more, 60 pg/kg/day or more, 70 pg/kg/day or more, 80 pg/kg/day or more, 90 pg/kg/day or more, 100 pg/kg/day or more, 125 pg/kg/day or more, 150 pg/kg/day or more, 175 pg/kg/day or more, 200 pg/kg/day or more, 225 pg/kg/day or more, 250 pg/kg/day or more, 300 pg/kg/day or more, 350 pg/kg/day or more, 400 pg/kg/day or more, 450 pg/kg/day or more, 500 pg/kg/day or more, 600 pg/kg/day or more, 700 pg/kg/day or more, 800 pg/kg/day or more, 900 pg/kg/day or more, 1 milligram (mg)/kg/day or more, 2 mg/kg/day or more, 3 mg/kg/day or more, 4 mg/kg/day or more, 5 mg/kg/day or more, 6 mg/kg/day or more, 7 mg/kg/day or more, 8 mg/kg/day or more, or 9 mg/kg/day or more). In some examples, the compound or composition can be administered to the subject in an amount of 10 milligrams (mg) per kilogram (kg) of body weight of the subject per day (mg/kg/day) or less (e.g., 9 mg/kg/day or less, 8 mg/kg/day or less, 7 mg/kg/day or less, 6 mg/kg/day or less, 5 mg/kg/day or less, 4 mg/kg/day or less, 3 mg/kg/day or less, 2 mg/kg/day or less, 1 mg/kg/day or less, 900 pg/kg/day or less, 800 pg/kg/day or less, 700 pg/kg/day or less, 600 pg/kg/day or less, 500 pg/kg/day or less, 450 pg/kg/day or less, 400 pg/kg/day or less, 350 pg/kg/day or less, 300 pg/kg/day or less, 250 pg/kg/day or less, 225 pg/kg/day or less, 200 pg/kg/day or less, 175 pg/kg/day or less, 150 pg/kg/day or less, 125 pg/kg/day or less, 100 pg/kg/day or less, 90 pg/kg/day or less, 80 pg/kg/day or less, 70 pg/kg/day or less, 60 pg/kg/day or less, 50 pg/kg/day or less, 45 pg/kg/day or less, 40 pg/kg/day or less, 35 pg/kg/day or less, 30 pg/kg/day or less, 25 pg/kg/day or less, 20 pg/kg/day or less, 15 pg/kg/day or less, 10 pg/kg/day or less, 5 pg/kg/day or less, 4 pg/kg/day or less, 3 pg/kg/day or less, or 2 pg/kg/day or less). The amount of the compound or composition administered to the subject can range from any of the minimum values described above to any of the maximum values described above. For example, the compound or composition can be administered to the subject in an amount of from 1 microgram (pg) per kilogram (kg) of body weight of the subject per day to 10 milligrams (mg)/kg/day (e.g., from 1 pg/kg/day to 100 pg/kg/day, from 100 pg/kg/day to 10 mg/kg/day, from 1 pg/kg/day to 10 pg/kg/day, from 10 pg/kg/day to 100 pg/kg/day, from 100 pg/kg/day to 1 mg/kg/day, from 1 mg/kg/day to 10 mg/kg/day, from 5 pg/kg/day to 10 mg/kg/day, from 1 pg/kg/day to 5 mg/kg/day, or from 5 to 5 mg/kg/day).

It is understood, however, that the specific dose level for any particular subject will depend upon a variety of factors. Such factors include the age, body weight, general health, sex, and diet of the subject. Other factors include the time and route of administration, rate of excretion, drug combination, and the type and severity of the particular disease or disorder.

Compositions, Formulations, Methods of Administration, and Kits

In vivo application of the disclosed compounds, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the disclosed compounds or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

The compounds disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The compounds can also be administered in their salt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington ’s Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable excipient in order to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and application. The compositions can also include conventional pharmaceutically- acceptable carriers and diluents which are known to those skilled in the art.

Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired application, compositions disclosed herein can comprise between about 0.1% and 100% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the excipients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can be delivered to a cell either through direct contact with the cell or via a carrier means. Carrier means for delivering compounds and compositions to cells are known in the art.

For the treatment of oncological disorders, the compounds or compositions disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anticancer substances and/or with radiation and/or photodynamic therapy and/or with surgical treatment to remove a tumor. These other substances or treatments can be given at the same as or at different times from the compounds or compositions disclosed herein. For example, the compounds or compositions disclosed herein can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5 -fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, anti angiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and/or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or an immunotherapeutic such as ipilimumab and bortezomib.

In certain examples, compounds and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin growth, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. Compounds and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient’s diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; diluents such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices.

Compounds and compositions disclosed herein, including pharmaceutically acceptable salts thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

Pharmaceutical compositions disclosed herein suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some examples, the final injectable form can be sterile and can be effectively fluid for easy syringability. In some examples, the pharmaceutical compositions can be stable under the conditions of manufacture and storage; thus, they can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

Sterile injectable solutions are prepared by incorporating a compound and/or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

Pharmaceutical compositions disclosed herein can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, solution, tincture, and the like. In some examples, the compositions can be in a form suitable for use in transdermal devices. In some examples, it will be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. Compounds and agents and compositions disclosed herein can be applied topically to a subject’s skin. These formulations can be prepared, utilizing any of the compounds disclosed herein or pharmaceutically acceptable salts thereof, via conventional processing methods.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Pharmaceutical compositions disclosed herein can be in a form suitable for rectal administration wherein the carrier is a solid. In some examples, the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carriers) followed by chilling and shaping in molds.

In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing any of the compounds disclosed herein, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

Also disclosed are kits that comprise a compound disclosed herein in one or more containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and/or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a compound and/or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a compound and/or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing a compound and/or agent disclosed herein in liquid or solution form. In some examples, the kit further comprises at least one agent, wherein the compound and the agent are co-formulated.

In some examples, the compound and the agent are co-packaged.

The kits can also comprise compounds and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient.

It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using, and/or the disclosed compositions.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.

EXAMPLES

The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measurement conditions, e.g., component concentrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process.

Example 1 -LPAi antagonist-derived LNPs deliver A20 mRNA and promote anti- fibrotic activities

Idiopathic pulmonary fibrosis (IPF) is a chronic condition marked by an abnormal buildup of extracellular matrix (ECM) in the lung interstitial section, leading to pulmonary dysfunction [1,2], Globally, approximately 3 million people are affected by IPF with a median survival duration of less than 5 years if left untreated [3], Although the precise cause of IPF is undetermined, it has been linked to different factors including environmental exposure, family history, infections as well as medication [4], The mechanism of IPF development is complicated, but immune cells and inflammatory signals are believed to play a role in the pathogenesis and progression of fibrosis [5], Due to challenges in precise diagnosis, poor prognosis, limited medication options, and high mortality, developing new treatment regimens for IPF has reached unprecedented interest levels. Currently, only two anti-fibrotic medications are approved for IPF treatment by the US Food and Drug Administration (FDA), pirfenidone and nintedanib. However, both drugs showed notable side effects and are only palliative [6], Therefore, the development of novel treatments to slow down or possibly reverse the IPF progression is of urgent need.

The pathogenesis of IPF starts from lung epithelial micro-injuries caused by the aforementioned factors, triggering abnormal healing processes and immune activation, which leads to excessive myofibroblast activation and proliferation [7], Therapeutic agents have been developed for suppressing immune activation and fibroblast proliferation by targeting one of these steps in the pathogenesis of IPF [1], Both pirfenidone and nintedanib function by targeting fibroblasts, which are the primary effector cells that engender the development of IPF. While pirfenidone exerts anti-inflammatory effects and acts as an antioxidant, nintedanib inhibits the PDFG receptor which reduces the chemotaxis and proliferation of myofibroblasts [8,9], Another proposed mechanism of modulating fibroblasts is through the lysophosphatidic acid receptor 1 (LPAi), which is a G protein-coupled receptor (GPCR) that binds to lysophosphatidic acid (LPA) [10], Evidence suggests that LPAi plays an important role in modulating wound healing by stimulating chemotaxis through fibroblast recruitment [11], AM095 and AM966, two potent LPAi antagonists, have demonstrated efficacy in reducing tissue injury, inflammation, and fibrosis in IPF mouse models [12,13],

Aberrant immune responses have been associated with the pathophysiology of autoimmune and inflammatory disorders. The pleiotropic ubiquitin-modifying enzyme A20, encoded by the Tumor necrosis factor alpha-induced protein 3 TNFAIPS) gene, has attracted much attention for its anti-inflammatory signaling that closely regulates multiple immune cell functions in diverse human fibrotic diseases [14], Studies have reported that A20 regulates several inflammatory signaling cascades, notably the canonical nuclear factor-KB (NF-KB) signaling pathway [14], The suppression of A20 enzymatic activities has been shown to play a role in the advancement of lung fibrosis, making A20 a potential target in IPF treatments [15,16].

The present study explored whether the integration of LPAi antagonist mediated anti- fibrotic activities with A20 mediated anti-inflammatory activities could suppress fibroblast activation, and thereby limit the fibrotic responses and IPF development. Lipid nanoparticles (LNPs) exhibit significant potential in exogenous gene expression through the delivery of mRNA, thus presenting a promising approach to transiently elevate A20 levels in lung fibroblasts [17-25], To explore this strategy, the study synthesized LPAi antagonists (AM095 and AM966)-derived amino lipids (LA lipids) and formulated them into LNPs (LA-LNPs) for A20 mRNA delivery to activated fibroblasts (Figure 1). The results showed that LA5-LNPs, with AM966 as the head group and branched biodegradable acetal lipid tails, can efficiently deliver A20 mRNA to lung fibroblasts both in vitro and ex vivo. This approach effectively inhibits fibroblast proliferation, migration, and collagen synthesis in primary mice lung fibroblasts (MLF). This study underscores the potential of integrating multiple anti -fibrotic pathways for IPF treatment.

Results and discussion

To produce LPAi antagonist (AM095 and AM966)-derived lipids (LA), the experiment first synthesized three different hydroxylated ionizable lipids tails with either saturated carbon chains or bioresponsive acetal-containing chains [19], These hydroxylated ionizable lipids were then coupled with AM095 and AM966 using Mitsunobu esterification to generate LA lipids. LA lipids contain LPAi antagonist-based heads, amino cores, and various lipid tails. The structures of LA lipids were validated using ^JH NMR and mass spectrum.

The synthesized LA lipids were then formulated with the addition of 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), cholesterol (Choi), and DMG-PEG2000 (PEG) to obtain LA-derived lipid nanoparticles (LA-LNPs). To test the mRNA delivery efficiency, the different LA -LNPs were formulated with firefly luciferase (FLuc) mRNA and then treated to MLg cells, a lung fibroblast cell line [19,26-28], All formulated LA-LNPs have a hydrodynamic diameter under 300 nm with a poly dispersity index (PDI) below 0.3 (Figure 2, panel B). FLuc mRNA encapsulated in LA5-LNPs showed over a 100-fold higher luminescence intensity compared to that of the other LA-LNPs, including the D-Lin-MC3-DMA (MC3) lipid formulation utilized in the FDA-approved siRNA-LNP therapy, ONPATTRO® (Figure 3, panel C) [29], The luminescence intensity serves as an indicator for mRNA delivery efficiency, which can be analyzed to evaluate the structure- activity relationship of LA lipids for mRNA delivery. Lipids derived from two distinct LPAi antagonists, AM095 and AM966, exhibited varying luminescence intensities in MLg cells, suggesting that the headgroup properties of LA lipids may impact mRNA delivery efficiency. Meanwhile, the lipid tail structure can also affect mRNA delivery efficiency. For example, LAI- and LA4-LNPs with fully saturated 12 hydrocarbon chain yielded lower mRNA delivery efficiency compared to lipid tails with an acetal groups. Without wishing to be bound by theory, the enhanced delivery efficiency could stem from variations in the critical packing parameters of the lipid tails [28], The acetal groups may facilitate the formation of the hexagonal phase upon acidification in the endosome to promote endosomal escape of the mRNA. Notably, LA5 with an AM096 head group and three tails containing formaldehyde acetal groups exhibited over 100-fold greater luminescence intensity than MC3. These findings indicate that the mRNA delivery efficiency of LA lipids can be improved by both the headgroup characteristics and the functional groups in the lipid tails.

To further optimize the formulation of LA5, the study conducted a Design of Experiment (DoE) based on the Lie (4)⁴ orthogonal table and obtained 16 LA5 FLuc-LNPs formulations with different lipid molar ratios (Figure 3, panel A; Table 2) [26], Luminescence intensity was measured after the delivery of FLuc mRNA in 16 formulated LA5-LNPs in MLg cells (Figure 3, panel B). Four levels of each component were plotted based on the observed luminescence intensity shown in Figure 3, panels C-F. The trend of LA5 showed peak luminescence intensity at a molar ratio of 40, so the molar ratio of LA5 was increased from 20 to 40 (Figure 3, panel C). As shown in Figure 3, panel D, increased DOPE levels facilitated mRNA delivery efficiency, therefore the study increased the molar ratio of DOPE from 30 to 50. The levels of Choi and PEG were decreased from 40 to 30 and 0.75 to 0.5 based on the trends shown in Figure 3, panel E and Figure 3, panel F, respectively. The top performing formulation of lipid molar ratios based on the DoE optimization was LA5:DOPE:Chol:PEG=40:50:30:0.5. The optimized LA5 LNPs result in a comparable delivery efficiency to LNPs formulated with ALC-0315 lipid, which is used for the Pfizer/BioNTech Covidl9 vaccine (Figure 6, panel A).

The experiment then characterized the physiochemical properties of the optimized formulation. The LA5-LNPs have a particle size of 110.1 ± 1.1 nm with a PDI of 0.066 ± 0.006 (Figure 3, panel G). The particles have an RNA encapsulation rate of around 86.4 ± 3.0%, and they were positively charged at around 7.0 mV in a lightly acidic formulation buffer (Figure 3, panel H). Images from cryogenic transmission electron microscopy (cryo-TEM) showed that the LA5-LNPs have an elliptical structure (Figure 4, panel A). Once endocytosed into the cell, the LA5-LNPs encapsulated mRNA needs to escape from the endosomes in order to reach the cytoplasm for translation into corresponding proteins. To study the internalization mechanisms of LA5-LNPs, Alexa-Fluor 647 (AF647) labeled RNA were encapsulated in the LNPs. Before the LNPs were administered, pre-treatment with 5-(N-Ethyl-N-isopropyl) amiloride (EIP A), chlorpromazine (CPZ), and methyl-P-cyclodextrin (MpCD) were conducted to inhibit the macropinocytosis, clathrin, and caveolae endocytic pathways, respectively. Cells pre-treated with MpCD significantly blocked LA5-LNP uptake, indicating that MLg cells primarily internalized LA5-LNPs through the caveolae-mediated endocytic pathway (Figure 4, panel B). To evaluate if the RNA can escape from the endosome, cells were co-incubated with LA5 AF647-LNPs together with calcein, a membrane-impermeable fluorophore that is typically retained in the endosome. The study observed that while the cells treated with calcein alone showed punctate green-fluorescent signals in the endosome, the cells treated with both the calcein and LA5 AF647-LNPs showed diffused green-fluorescent signals throughout the cytoplasm, suggesting endosomal membrane rupture and subsequent release of AF647 RNA into the cytoplasm (Figure 4, panel C).

To assess whether the LA5-LNPs can target lung fibroblasts with enhanced LPAi receptor expression, the study treated MLg cells with I-R2 GFP-LNPs, where I-R2 is LA5 without AM966 conjugation, or added excess amount of AM966 (10 pM) to saturate and block LPAi receptor on lung fibroblast before adding LA5 GFP-LNPs. The experiment observed that groups with no LPAi targeting (I-R2 GFP-LNPs) or LPAi blockage showed lower GFP intensity compared to LA5 GFP-LNPs, indicating that AM966 conjugation in LA5-LNPs could increase particle uptake in lung fibroblasts, mediated through LPAi interaction (Figure 4, panel D). To assess whether it was possible to increase A20 expression levels in lung fibroblasts, the experiment delivered A20 mRNA using LA5-LNPs and found significantly higher A20 expression in the MLg cell line (80.2 ± 1.5%) compared to PBS treated cells (3.0 ± 0.8%) (Figure 4, panel E).

Next, the experiment investigated the safety, delivery efficiency, and anti-fibrotic functions of LA5 A20-LNPs in isolated primary mouse lung fibroblasts (MLF). Compared to other FDA-approved LNPs formulations based on ALC-0315 or MC3 ionizable lipids, LA5 LNPs showed comparable cytotoxic profiles as measured by an MTT assay (Figure 6, panel B). As depicted in Figure 5, panel A, LA5 A20-LNPs notably elevated A20 protein expression in MLF (17.2 ± 0.4%), 20-fold of that of PBS (0.9 ± 0.3%) and 2-fold of free LA5-LNPs without A20 mRNA encapsulation (6.9 ± 0.9%). It was found that free LA5-LNPs without A20 mRNA encapsulated could also increase A20 protein expression to some extent. Without wishing to be bound by theory, it was speculated that this occurred due to the suppression of LPAi mediated profibrotic pathway, which can restore the activities of A20 in lung fibroblasts. Fibroblasts can differentiate into a myofibroblast phenotype, often characterized by the overexpression of a- SMA upon stimulation with TGF-01 [30], Upon myofibroblast differentiation, fibroblasts become proliferative, migratory, and increase the production of ECM components, such as collagen. Therefore, the study further investigated if LA5 A20-LNPs could inhibit TGF-01- induced A20 downregulation and myofibroblast differentiation in MLFs [15], MLFs were initially pre-treated with TGF-01, followed by treatment with PBS, free LA5-LNPs, or LA5 A20-LNPs. While TGF-01 treatment caused the suppression of Tnfaip3 gene expression level, LA5 A20-LNPs could significantly increase Tnfaip3 gene expression in MLFs to exert its anti- fibrotic activities (Figure 5, panel B). The anti-fibrotic activity is demonstrated by the reduced Collal mRNA levels, where TGF-01 treatment alone increased collagen synthesis while LA5 A20-LNPs treatment reduced it to inactivated levels (Figure 5, panel C). Furthermore, the study evaluated the effect of LA5 A20-LNPs on fibroblast migration using in vitro scratch assay, which was conducted by creating a straight-line scratch across the MLF monolayer. The fibroblast migration was significantly decelerated in the LA5 A20-LNP group compared to the other two groups treated with TGF-pi (Figure 5, panels D-E).

Conclusion

Herein, a platform was developed to slow down IPF progression using LA5-LNPs. As the LPAi receptor is highly expressed on fibroblast cell surface membrane, the lipidized LPAi antagonist in the LA5-LNP formulation enhances mRNA uptake in lung fibroblasts and reduces chemotaxis and proliferation of lung fibroblast upon LPAi blockade. On the other hand, A20 plays an important role in the downregulation of fibrotic responses by suppressing the NF-KB signaling pathway [16], A strong effect was observed on blocking the LPAi signaling pathway and restoring A20 enzymatic activities. The findings indicate that LA5-LNPs encapsulated with A20 mRNA exhibit stronger antifibrotic activities compared to free LA5-LNPs in mice lung fibroblasts, as evidenced by a slower migration process and reduced collagen synthesis. The precise targeting of lung tissues in vivo is important for the effective utilization of LA5 A20-

LNPs in the treatment of pulmonary fibrosis. Local administration methods, such as intratracheal injection, oropharyngeal aspiration, and intranasal delivery, can direct LNPs specifically to the lung [31-33], Furthermore, the manipulation of the LNPs formulation, whether through design of the ionizable lipid or modulating the charge characteristics of LNPs, can tune the LNP tropism towards specific organs [34,35], For instance, the incorporation of a cationic lipid, such as Dioleoyl-3 -trimethylammonium propane (DOTAP), within the LNP formulation has been demonstrated to enable selective mRNA delivery to the lungs [34], These approaches may be applied to LA5-LNPs for future in vivo studies to advance the therapeutic potential of LA5- LNPs in pulmonary fibrosis. In summary, the findings provide proof of concept that effective delivery of A20 mRNA with LA5-LNPs can mitigate fibrotic activities in IPF.

Experimental details

Materials. All chemicals and solvents were purchased from Fisher Scientific unless otherwise listed. AM095 and AM966 were obtained from MedChemExpress (NJ, USA). DOPE was purchased from Avanti Polar Lipids (AL, USA). DMG-PEG2000 was purchased from NOF America Corporation (NY, USA).

Cell lines and maintenance. MLg [Mlg 2908] (ATCC® CCL-206™) cells were purchased from ATCC (VA, USA), and cultured in Eagle's Minimum Essential Medium (Thermo Fisher, MA, USA) containing 10% fetal bovine serum (Invitrogen, MA, USA).

Isolation of primary mouse lung fibroblast (MLF). MLF was isolated from C57BL/6J mice and cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin. In brief, the lung was gathered, minced, and enzymatically digested in serum-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with Liberase (Roche, Switzerland) and 1% penicillinstreptomycin for 30 min at 37°C. Digestion was stopped by adding a complete DMEM medium containing 10% FBS. The resulting cell suspension was centrifuged at 500 x g for 5 min, resuspended in complete DMEM medium and filtered through 70-pm strainer. MLFs were cultured for 3 days before being passaged. MLFs were treated with TGF-pi (R&D Systems, USA) at a concentration of 10 ng/mL in DMEM medium to induce myofibroblast differentiation.

Characterization of Nanoparticle formulation. mRNA encapsulated LNPs were formulated with LAs and helper lipids 2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), cholesterol, l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). All lipid components were dissolved in ethanol at desired concentrations, while mRNA was dissolved in aqueous citrate buffer. The ethanol and aqueous phases were mixed rapidly together at a volume ratio of 1 :3 (10: 1 weight ratio of LA: mRNA) using a rapid nanomedicine system INano L+ microfluidics instrument from Micro & Nano Biologies Technology Ltd. The N/P ratio of the formulated LA-LNPs is shown in Table 1. The mRNA encapsulated LNPs underwent an 80-min dialysis in PBS buffer using Slide-A-Lyzer Dialysis Cassettes (Life Technologies, NY, USA) and were then filtered through 0.22 pm PES filter (Millipore Sigma, MA, USA). The particle size and zeta potential of LA-LNPs were determined using Zetasizer NanoZS (Malvern, United Kingdom). Additionally, the mRNA encapsulation efficacy (EE%) was determined by Quant-it™ RiboGreen RNA Assay (ThermoFisher Scientific, MA, USA) [26], The morphology of LA5-LNPs was assessed using a Glacios Cryo-TEM device (Thermo Fisher Scientific, MA, USA) using the methods described previously [28], For the luminescence readout, a dose of 50 ng FLuc RNA encapsulated in LA-LNPs were treated to MLg cells, and luminescence readout was conducted after 18h of co-culture.

Endosomal escape. A total of 6 * 10⁴ MLg cells in 300 pL of complete medium were plated in each chamber of the imaging dish (Ibidi USA Inc, WI, USA) and cultured overnight at 37 °C with 5% CO2. Calcein with or without LA5-LNPs containing Alexa-Fluro 647 RNA were added to each chamber. Cells were washed twice with PBS after 2 hours of co-incubation, and imaged using Leica DMi8 Brightfield (Leica, Germany).

A20 mRNA Preparation. The A20/Tnfaip3 gene cDNA sequence was retrieved from GenBank (Reference number: U19463.1). Linearized A20 dsDNA sequence was acquired from IDT and integrated into pUC19 vector containing T7 promoter and optimized UTRs using NEBuilder® HiFi DNA Assembly (New England Biolabs, MA, USA), and the correct plasmid was confirmed by sanger sequencing [22], The 120 A tail structure was added to genomic DNA template was amplified using polymerase chain reaction (PCR). Uncapped A20 mRNA was synthesized using AmpliScribe T7-Flash Transcription Kit (Lucigen, WI, USA). The mRNA cap structure was added using the Vaccinia Capping System and Cap 2'-O-Methyltransferase system (NEB, MA, USA). All mRNAs were purified with RNA Clean & Concentrator (Zymo Research, CA, USA), and mRNA concentration was measured using a NanoDrop 2000 Spectrophotometer (ThermoFisher, MA, USA).

In vitro and ex vivo delivery of A20 mRNA to lung fibroblasts. The study evaluated the delivery of A20 mRNA to MLg cells or mouse lung fibroblasts (MLF) using FITC-labeled anti- A20/TNFAIP3 monoclonal antibody (Novus Biologicals™, Cat# NBP177533F) via flow cytometry. Initially, 1 x 10⁵ MLg or MLF cells were seeded in a 24-well plate and incubated at 37°C in 5% CO2 incubator for 24h. Subsequently, the cells were treated with LA5-LNPs containing 500 ng A20 mRNAs for 18h. Following the treatment, the cells underwent fixation and permeabilization using eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher, MA, USA). The cells were then incubated with FITC labeled anti- A20/TNFAIP3 monoclonal antibody (clone 59A426, 1 :50 dilution) in cold PBS containing 1% FBS for 30 min at 4 °C. Cellular uptake was subsequently assessed using a BD LSR Fortessa or BD LSR II flow cytometer. To study the endocytic pathway of the LA5-LNPs, cells were pretreated with various endocytosis inhibitors 30 minutes before exposure to LA5-LNPs containing Firefly luciferase (Luc) mRNA and Alexa-Fluor 647-labeled RNA at a 1 : 1 weight ratio. After 3h of co-incubation, the cellular uptake was analyzed on a BD LSR Fortessa or BD LSR II flow cytometer.

In vitro scratch assay. 1.2 x 10⁵ MLFs were seeded per well in a clear 12-well plate and cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin overnight at 37°C. MLFs were treated with LA5-LNP with or without A20 mRNA in the presence of 10 ng/ml TGF-pi, followed by incubation at 37°C for 24 hours. A line was drawn on the cell monolayer using a sterile 200 pl pipette tip and images of each well were captured after 6h incubation in serum-free medium using the BioTek Cytation 5 Cell Imaging Multimode Reader (Agilent, USA).

Quantitative RT-PCR. Total RNA was extracted from MLF with RNeasy kit (Qiagen, USA). Subsequently, cDNA was synthesized using SuperScript™ IV VILO™ Master Mix with ezDNase (Invitrogen, USA). For qRT-PCR gene expression analysis, TaqMan™ Fast Advanced Master Mix for qPCR (Applied Biosystems™, USA) was utilized with TaqMan™ Gene Expression Assay ID Mm00437121_ml for mouse Tnfaipl. Mm00801666_gl for Collal (Applied Biosystems™, USA) on a QuantStudio™ 6 Pro Real-Time PCR system (Applied Biosystems™, USA). qRT-PCR data were normalized to Gapdh analyzed with TaqMan™ Gene Expression Assay ID Mm99999915_Gl as a housekeeping gene standard. Fold changes of target mRNAs were analyzed using the 2 ''^CT method.

Data Analysis. No collected experimental data were excluded for the quantitative analysis. The number of repetitions in each group and the error bar definitions were specified in the figure legend. Statistical significance was determined using unpaired, two-tailed Student's t- tests for two groups or one-way analysis of variance (ANOVA) with Dunnett' s multiple comparison test for multiple groups. *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 were considered statistically significant. All data analysis was conducted in Prism 8 (GraphPad). Synthesis of LP A i-angonist derived lipids. All LPAi-angonist derived lipids were purified by column chromatography using a CombiFlash Rf system with a RediSep Gold Resolution silica column (Teledyne Isco) with gradient elution. All ¹ H NMR spectra were run on a Bruker Avance 400 MHz instrument. Mass spectrometric measurements were performed by Acquity SQD UPLC/MS (Waters) or microflex LRF MALDI-TOF mass spectrometer (Bruker) at Icahn School of Medicine at Mount Sinai.

Aldehydes and [1-R] were synthesized according to previously reported procedures. [19]

To a solution of LPAi-angonist AM095 or AM966 (0.1 mmol), 3 mL of THF and Triphenylphosphine (0.3 mmol), diethyl azodi carb oxy late (DEAD, 0.3 mmol) was added dropwise. The solution was stirred for 10 min at 0 °C, [1-R] (O.lmmol) was added, then kept stirred at room temperature overnight. The resulting mixture was diluted with DCM, washed three times with brine (50 mL), and dried over anhydrous ISfeSC After and the solvent was removed under reduced pressure, the residue was purified was further purified by Combiflash column chromatography using a silica column (Buchi) with gradient elution from 100% CH2Q2 to 10% CH2C12/MeOH/NH4OH (75/25/3, v/v/v) to give compound LPAi -antagonist derived lipids.

LL1 (30mg, 29%): *HNMR (400 MHz, CDCI3) 8 7.76 (m, 2H), 7.69 - 7.58 (m, 3H), 7.54 (m, 2H), 7.49 -7.18 (m, 6H), 6.21 (q, 1H), 4.26 - 4.09 (t, 2H), 3.69 (s, 2H), 2.73 (m, 4H), 2.57 - 2.35 (m, 4H), 2.27 (s, 3H), 1.78-1.33 (m, 63H), 0.87 (t, J = 6.7 Hz, 9H). MS for C67H106N4O5 ([M+H]⁺) Calculated: 1047.8, Found: 1047.8.

LL2 (25mg, 22%): 'HNMR (400 MHz, CDCI3) 6 7.75 - 7.55 (m, 7H), 7.49 -7.18 (m, 6H), 6.21 (q, 1H), 4.65 (s, 6H), 4.26 - 4.09 (t, 2H), 3.69 - 3.50 (m, 14H), 2.73 (m, 4H), 2.57 - 2.35 (s, 3H), 2.27 (m, 4H), 1.78-1.33 (m, 72H), 0.88 (t, J= 6.7 Hz, 9H). MS for C70H112N4O11 ([M+H]⁺) Calculated: 1185.8, Found: 1185.8.

LL3 (28mg, 23%): 'HNMR (400 MHz, CDCI3) 6 7.75 - 7.55 (m, 7H), 7.49 -7.18 (m, 6H), 6.21 (q, 1H), 4.66 (q, J= 5.6 Hz, 3H), 4.20 (m, 2H), 3.70 - 3.39 (m, 14H), 2.70 (m, 4H), 2.57 - 2.35 (m, 4H), 2.21 (s, 3H), 1.78-1.33 (m, 70H), 0.87 (t, J = 6.7 Hz, 9H). MS for C73H118N4O11 ([M+H]⁺) Calculated: 1227.9, Found: 1227.9.

LL4 (38mg, 35%): 'H NMR (400 MHz, CDCI3) 6 7.95 - 7.75 (m, 2H), 7.69 - 7.58 (m, 3H), 7.54 (m, 2H), 7.47 - 7.29 (m, 5H), 6.20 (q, 1H), 4.38 - 4.21 (m, 2H), 3.77 - 3.60 (s, 2H), 2.68 (m, 4H), 2.29 (m, 4H), 2.27 (s, 3H), 1.59-1.03 (m, 69H), 0.89 (t, J = 6.7 Hz, 9H). MS for C67H105CIN4O5 ([M+H]⁺) Calculated: 1081.8, Found: 1081.8. LL5 (23mg, 19%): 'HNMR (400 MHz, CDCh) 6 7.75 - 7.52 (m, 7H), 7.49 -7.33 (m, 5H), 6.24 (q, 1H), 4.65 (s, 6H), 4.18 (m, 6H), 3.65 - 3.42 (m, 12H), 2.87 - 2.37 (m, 8H), 2.24 (s, 3H), 1.78 - 1.04 (m, 65H), 0.87 (t, J = 6.7 Hz, 9H). MS for C7OHIIIC1N₄OII([M+H]⁺) Calculated: 1219.8, Found: 1219.8. LL6 (27mg, 22%): 'HNMR (400 MHz, CDCh) 8 7.75 - 7.52 (m, 7H), 7.49 -7.33 (m, 5H),

6.24 (q, 1H), 4.66 (q, J= 5.6 Hz, 3H), 4.21 (m, 6H), 3.70 - 3.42 (m, 14H), 2.80 - 2.35 (m, 8H), 2.21 (s, 3H), 1.78 - 1.04 (m, 68H), 0.88 (t, J = 6.7 Hz, 9H). MS for C73HII₇C1N₄0II([M+H]⁺) Calculated: 1261.8, Found: 1261.8.

Table 1. N/P ratio of LA-LNPs

N/P ratio (mol/mol)

Table 2. Design of orthogonal table Lie References

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The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended as illustrations of a few aspects of the claims and any methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

CLAIMS What is claimed is:

1. A compound defined by Formula I, or a pharmaceutically acceptable salt thereof:

2. The compound of claim 1, wherein each R² is independently an unsubstituted linear Cs- Cis alkyl.

3. The compound of claim 1, wherein each R² is independently an unsubstituted branched C₈-Ci8 alkyl.

4. The compound of claim 1, wherein each R² is independently a linear Ci-Cis alkyl (e.g., C4-C6 alkyl) substituted with one or more substituents selected from the group consisting of acetal, amine, amide, ester, ether (e.g., acetal), and carbonate ester.

5. The compound of claim 1, wherein each R² is independently a branched Ci-Cis alkyl (e.g., C4-C6 alkyl) substituted with one or more substituents selected from the group consisting of amine, amide, ester, ether (e.g., acetal), and carbonate ester.

6. The compound of any one of claims 1-5, wherein each R² is independently selected from the group consisting of: wherein G and I each independently represent integers from 1 to 8.

7. The compound of any one of claims 1-6, wherein each R² is independently selected from the group consisting of:

wherein G and I each independently represent integers from 1 to 8.

8. The compound of any one of claims 1-7, wherein each R² is independently selected from the group consisting of:

9. The compound of any one of claims 1-8, wherein each R² is the same.

10. The compound of any one of claims 1-8, wherein at least one R² is different.

11. The compound of any one of claims 1-10, wherein p is 0.

12. The compound of any one of claims 1-10, wherein p is 1.

13. The compound of any one of claims 1-12, wherein the LPA receptor antagonist comprises a lysophosphatidic acid receptor 1 (LPAi) antagonist.

14. The compound of any one of claims 1-13, wherein R¹ is a Ci-Ce alkyl linker substituted with the LPA receptor antagonist.

15. The compound of any one of claims 1-14, wherein the LPAreceptor antagonist is represented by Formula II:

II wherein

X is N or C;

Z is N or O;

R^x and R^y, when present, are independently selected from substituted or unsubstituted Ci- C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl;

R⁹ is H or cyclopropyl;

R¹⁰, R¹¹, R¹² are each independently H or methyl; and L is selected from:

16. The compound of any one of claims 1-15, wherein the LPAreceptor antagonist is represented by Formula III:

III wherein

X is N or C;

Z is N or O;

R^x and R^y, when present, are independently selected from substituted or unsubstituted Ci- C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl; and R⁹ is H or cyclopropyl.

17. The compound of any one of claims 1-16, wherein the LPA receptor antagonist is represented by Formula IV:

R^x and R^y are independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

18. The compound of any one of claims 1-17, wherein the LPAreceptor antagonist is selected from the group consisting of AM966, AM095, RO-6842262, and BMS-986020.

19. The compound of any one of claims 1-18, wherein the compound is selected from the group consisting of:

20. A composition comprising: the compound of any of claims 1-19; and an agent.

21. The composition of claim 20, wherein the agent is a polynucleotide.

22. The composition of any of claims 20-21, wherein the agent is an RNA.

23. The composition of any of claims 20-22, wherein the agent is an mRNA.

24. A method of making the compound of any one of claims 1-19.

25. A lipid nanoparticle comprising: the compound of any one of claims 1-19; a non-cationic lipid; a polyethylene glycol (PEG)-lipid; and a sterol.

26. The lipid nanoparticle of claim 25, further comprising an agent.

27. The lipid nanoparticle of claim 26, wherein the agent comprises a polynucleotide.

28. The lipid nanoparticle of any one of claims 26-27, wherein the agent comprises an RNA.

29. The lipid nanoparticle of any one of claims 26-28, wherein the agent comprises an mRNA.

30. The lipid nanoparticle of any one of claims 26-29, wherein the agent comprises a polynucleotide encoding a ubiquitin-modifying enzyme.

31. The lipid nanoparticle of any one of claims 26-30, wherein the agent comprises Tumor Necrosis Factor a- Induced Protein 3 (TNFAIP3), Suppressor of Cytokine Signaling 1 (SOCS1), Smad7, Kriippel-like Factor 2 (KLF2), Matrix metalloproteinases (MMPs), Thrombospondin- 1 (TSP-1), Interleukin- 10 (IL-10), and/or Decorin.

32. The lipid nanoparticle of any one of claims 26-31, wherein the agent comprises a polynucleotide encoding Tumor Necrosis Factor a-Induced Protein 3 (TNFAIP3).

33. The lipid nanoparticle of any one of claims 26-32, wherein the agent is encapsulated by the nanoparticle.

34. The lipid nanoparticle of any one of claims 25-33, wherein the non-cationic lipid comprises l,2-dioleoyl-sw-glycero-3 -phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl- w- glycero-3 -phosphoethanolamine (POPE), l,2-distearoyl- w-glycero-3 -phosphocholine (DSPC), 1 -stearoyl -2-oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn- glycero-3- phosphocholine), l,2-dioleyl-sn-glycero-3-phosphotidylcholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), l,2-dioleoyl-5/7-glycero-3- phospho-(l'-rac-glycerol) (DOPG), or combinations thereof.

35. The lipid nanoparticle of any one of claims 25-34, wherein the sterol comprises a cholesterol-based lipid.

36. The lipid nanoparticle of any one of claims 25-35, wherein a molar ratio of the noncationic lipid is from 20% to 50%.

37. The lipid nanoparticle of any one of claims 25-36, wherein a molar ratio of the compound is from 5% to 60%.

38. The lipid nanoparticle of any one of claims 25-37, wherein a molar ratio of the sterol is from 20% to 50%.

39. The lipid nanoparticle of any one of claims 25-38, wherein a molar ratio of the PEG-lipid is from 0.1% to 2%.

40. The lipid nanoparticle of any one of claims 25-39, wherein a molar ratio of the compound is from 35% to 45%, a molar ratio of the non-cationic lipid is from 45% to 55%, a molar ratio of the sterol is from 35% to 45%, and a molar ratio of the PEG-lipid is from 0.1% to 1%.

41. The lipid nanoparticle of any one of claims 26-40, wherein a weight fraction of the agent is from 5% to 20%.

42. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the compounds of any one of claims 1-19, the composition of any one of claims 20-23, or the lipid nanoparticle of any one of claims 25-41.

43. A method of treating a disease or disorder in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 42.

44. The method of claim 43, wherein the disease or disorder comprises a connective tissue disorder.

45. The method of any one of claims 43-44, wherein the disease or disorder comprises fibrosis.

46. The method of claim 45, wherein the fibrosis is lung fibrosis.