Formulated and / or co-formulated liposomal compositions containing tgf beta antagonist prodrugs useful in the treatment of cancer and methods thereof

Abstract

Disclosed herein are formulated and / or co-formulated lipid nanoparticles (LNPs) and solid lipid nanoparticles (SLNPs) comprising TB prodrugs and methods of making the LNPs and the SLNPs. TB prodrug compositions comprising a drug moiety, a lipid moiety, and a linking unit, inhibit ALK5. The TB prodrugs can be formulated and / or co-formulated into liposomes or solid lipid nanoparticles to provide methods of treating cancer, immune disorders, and other diseases by taking advantage of targeted drug delivery vehicles.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 995,887, filed on February 19, 2020, the contents of which are incorporated herein by reference in their entirety.

[0003] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0004] not applicable. Technical Field

[0005] The present invention described herein relates to prodrug compositions and nanoformulations comprising prodrugs that inhibit signal transduction induced by transforming growth factor beta 1, 2, or 3 ("TGFβ") proteins after release of the active inhibitor from the prodrug. Specifically, the present invention relates to prodrug compositions formulated within nanocarriers (e.g., liposomes) and used as vehicles for the treatment of human cancer. The present invention also relates to co-formulations of such prodrugs with other immunomodulators or prodrugs. The present invention further relates to the treatment of cancer and other immune disorders and diseases. Background Art

[0006] Cancer is the second leading cause of death worldwide, second only to coronary heart disease. Millions die from cancer each year, and in the United States alone, over half a million people die annually from cancer, with 1,688,780 new cancer cases diagnosed in 2017 (American Cancer Society). While deaths from heart disease have been declining significantly, deaths from cancer are generally increasing. Unless medical advances change current trends, cancer is predicted to become the leading cause of death by the beginning of the next century.

[0007] The mortality rate of several cancers is high. Specifically, lung cancer (accounting for 18.4% of all cancer deaths), breast cancer (accounting for 6.6% of all cancer deaths), colorectal cancer (accounting for 9.2% of all cancer deaths), liver cancer (accounting for 8.2% of all cancer deaths) and gastric cancer (accounting for 8.2% of all cancer deaths) are the leading causes of cancer death in both sexes of all ages worldwide (GLOBOCAN 2018). These cancers share a common fatal feature with almost all other cancers, namely that they metastasize to sites distant from the primary tumor, and with very few exceptions, metastatic disease is fatal. In addition, even for those cancer patients who initially survive the primary cancer, common experience has shown that the life of the cancer patient is greatly changed. Many cancer patients experience intense anxiety driven by the awareness of the possibility of recurrence or treatment failure. Many cancer patients also experience physical weakness after treatment. In addition, many cancer patients experience recurrence of the disease.

[0008] Although cancer therapies have improved over the past few decades and survival rates have increased, the heterogeneity of cancer still requires new therapeutic strategies that utilize multiple treatment modalities. This is particularly true when treating solid tumors at anatomically critical sites (e.g., glioblastoma, head and neck squamous cell carcinoma, and lung adenocarcinoma), which are sometimes limited to standard radiotherapy and / or chemotherapy. However, a deleterious effect of these therapies is chemoresistance and radioresistance, which, in addition to severe side effects that reduce the patient's quality of life, promote local recurrence, distant metastasis, and second primary tumors.

[0009] TGFβ refers to a subset of polypeptides of the cytokine transforming growth factor β superfamily. It is a secreted protein that performs many cellular functions, including controlling cell growth, cell proliferation, cell differentiation and apoptosis. In the human body, TGFβ1 is encoded by the TGFB1 gene. Functionally, TGFβ synergizes with TGF-α when inducing transformation. It also acts as a negative autocrine growth factor. Disorders of TGFβ activation and signal transduction may lead to apoptosis. Many cells synthesize TGFβ, and almost all cells have specific receptors for these cytokines. TGF-β1, TGF-β2 and TGF-β3 all play a role through the same receptor signal transduction system. TGFβ plays an important role in controlling the immune system and shows different activities on different types of cells or cells at different developmental stages. Most immune cells (or leukocytes) secrete TGFβ. See LETTERIO et al., Regulation of Immune Responses by TGF-beta, Annu. Rev. Immunol, 16: pp. 137-161 (1998). It is taught that some T cells (e.g., regulatory T cells) release TGFβ to inhibit the action of other T cells. For example, interleukin 1-dependent proliferation and interleukin 2-dependent proliferation of activated T cells, as well as activation of resting helper T cells and cytotoxic T cells, are prevented by the activity of TGF-β1. See GILBERT et al., Transforming growth factor-beta 1induces antigen-specific unresponsiveness in naive T cells. T cells), Immunol. Invest., 26(4): 459-472 (1997) and WAHL et al., TGF-β: a mobile purveyor of immune privilege, Immunol. Rev., 213: 213-227 (2006). Similarly, TGFβ can inhibit the secretion and activity of many other cytokines, including interferon-γ, tumor necrosis factor-α (TNF-α) and various interleukins. It can also reduce the expression level of cytokine receptors such as IL-2 receptor to downregulate the activity of immune cells. In addition, TGFβ has similar effects on B cells, which also vary depending on the differentiation state of the cells. It inhibits the proliferation of B cells and stimulates apoptosis, and plays a role in controlling the expression of antibodies, transferrin and MHC class II proteins on immature and mature B cells. LEBMAN et al., The role of TGF-beta in growth, differentiation, and maturation of b lymphocytes, Microbes Infect., 1(15) pp. 1297-1304 (1999). Finally, the effects of TGF-beta on macrophages and monocytes are primarily inhibitory; this cytokine inhibits the proliferation of these cells and prevents their production of reactive oxygen species (e.g., superoxide (O2 - )) and nitrogen (e.g., nitric oxide (NO)) intermediates. However, as with other cell types, TGFβ can also have the opposite effect on cells of myeloid origin. See Wall et al., supra. It has also been shown that TGFβ reduces the efficacy of MHC II in astrocytes and dendritic cells, which in turn reduces the activation of appropriate helper T cell populations. See Tang et al., The Smad3 protein is involved in TGF-beta inhibition of class II transactivator and class II MHC expression, J. of Immun, 167(1): 311-319 (2001).

[0010] TGFβ initiates intracellular signaling by first binding to TGFβ receptor II (TGFβRII), which then recruits a second receptor, TGFβRI (TGFβRI), also known as activin-like kinase 5 (ALK5). After recruitment to the TGFβ receptor:ligand complex, TGFβRII phosphorylates and activates ALK5, which mediates downstream signaling, leading to transcriptional regulation. It is taught that ALK5 deletion or mutation in tumors appears to be a common form of pathway alteration. However, efforts aimed at inhibiting the ALK5 cascade have shown promise. See Loomas et al., Activin receptor-like kinases: a diverse family playing an important role in cancer, Am. J. Cancer Res. 6(11): 2431-2447 (2016).

[0011] Currently, a variety of small molecule kinase inhibitors that selectively inhibit ALK5 have been identified and developed into clinical stage testing as anticancer agents. Galunisertib (LY2157229) and Vactosortib (TEW-7197) are the most advanced therapeutic candidates in clinical trials and have demonstrated anticancer efficacy in humans as single agents and in combination with approved drugs, thus validating this mechanism as a clinically meaningful approach. However, doses are limited by the cardiotoxicity induced by these molecules, and the combination with other treatment risks increases systemic off-target toxicity.

[0012] Prodrug is the medicine or compound that is metabolized (i.e., converted in vivo) into pharmacologically active drug after administration. Instead of directly administering the drug, corresponding prodrug is used to improve the absorption, distribution, metabolism and / or excretion mode of the drug. For example, when the drug itself is poorly absorbed in the gastrointestinal tract, prodrug is usually designed to improve bioavailability. Prodrug can be used to improve how the drug selectively interacts with cells or processes that are not its intended target. This reduces the adverse effects or unexpected effects of the drug, which is particularly important in treatments such as chemotherapy, because the treatment may produce serious unexpected and undesirable side effects. Therefore, prodrug can be considered as the medicine containing specialized non-toxic protective groups, which are used to change or eliminate the undesirable characteristics in the parent molecule in a transient manner.

[0013] Finally, a nanocarrier is a nanomaterial used to transport another substance, such as a drug. There are many different types of nanocarriers. For example, nanocarriers include polymer conjugates, polymer nanoparticles, lipid-based carriers, and dendrimers, to name a few. The different types of nanomaterials used in nanocarriers allow for the delivery of both hydrophobic and hydrophilic drugs throughout the body. Since the human body is primarily composed of water, the ability to deliver hydrophobic drugs within the body is a major therapeutic benefit of nanocarriers. Nanocarriers have shown promise in drug delivery because they can deliver drugs to site-specific targets, allowing them to be delivered to certain organs or cells but not others. Site-specificity is a major therapeutic benefit because it prevents drugs from being delivered to the wrong place. Furthermore, nanocarriers show particular promise in chemotherapy because they can help reduce the adverse, more widespread toxicity of chemotherapy to healthy, fast-growing cells around the body. Since chemotherapy drugs can be extremely toxic to human cells, it is important that they are delivered to tumors without being released into other parts of the body.

[0014] From the foregoing, it will be apparent to those skilled in the art that new therapeutic paradigms are needed in the treatment of cancer and other immune diseases. New disease treatments can be achieved by combining novel prodrugs with modern nanodelivery modalities, with the overall goal of more effective treatment, reduced side effects, and greater therapeutic utility in cancer treatment, particularly in solid tumors.

[0015] In view of the current deficiencies associated with cancer treatment, it is an object of the present invention to provide new and improved methods for treating cancer, immune disorders and other diseases using prodrugs encapsulated within nanocarriers. Summary of the Invention

[0016] The present invention provides ALK5 inhibitor prodrug ("TB prodrug") compositions comprising an ALK5 inhibitor, a lipid, and a biocleavable linker. In certain embodiments, nanocarriers comprising TB prodrugs are formulated for use as a delivery mode for treating human diseases, such as cancer, including solid tumor cancers, and other immune disorders. In certain embodiments, the nanocarrier comprises a lipid bilayer that can be incorporated into a drug delivery vehicle (i.e., a liposome). In another preferred embodiment, the liposome comprises cholesterol hemisuccinate ("CHEMS"). In another preferred embodiment, the liposomes of the present invention comprise stearic acid.

[0017] In further embodiments, the TB prodrugs of the present disclosure include TB4 prodrugs.

[0018] In further embodiments, the present invention includes a method of delivering an ALK5 inhibitor to a tumor, comprising: (i) synthesizing a TB prodrug; (ii) formulating the TB prodrug of the invention in a nanocarrier of the invention; and (iii) administering the nanocarrier to a patient.

[0019] In another embodiment, the present invention includes a method for delivering an ALK5 inhibitor with one or more additional immunomodulators to a tumor, the method comprising (i) synthesizing a TB prodrug; (ii) co-formulating the TB prodrug of the invention with one or more additional immunomodulators of the invention in a nanocarrier; and (iii) administering the nanocarrier to a patient.

[0020] In another embodiment, the immunomodulatory agent includes an immunogenic cell death-inducing chemotherapeutic agent, a PD-1 antagonist, a toll receptor agonist, a STING agonist, an IDO inhibitor, a CTLA4 inhibitor, a CD1D agonist, and / or a prodrug thereof.

[0021] In another embodiment, the present disclosure teaches methods for synthesizing TB prodrugs.

[0022] In another embodiment, the present disclosure teaches methods for synthesizing TB4 prodrugs.

[0023] In another embodiment, the present disclosure teaches methods of formulating TB prodrugs within nanocarriers, including but not limited to liposomes.

[0024] In another embodiment, the present disclosure teaches methods of formulating TB4 prodrugs within nanocarriers, including but not limited to liposomes.

[0025] In another embodiment, the present disclosure teaches methods of using the disclosed nanocarriers to treat one or more cancers, immune disorders, and other diseases in a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Chemical synthesis of TB4 prodrug.

[0027] Figure 2 : Chemical synthesis of protecting group intermediates on the way to the final TB4 prodrug.

[0028] Figure 3 : Chemical synthesis of protecting group intermediates on the way to the final TB4 prodrug.

[0029] Figure 4 : Synthesis scheme of ALK5 inhibitor prodrugs using carboxylic acid functionality.

[0030] Figure 5 : Synthesis scheme of ALK5 inhibitor prodrugs using the alcohol functionality.

[0031] Figure 6 : Synthetic schemes for ALK5 inhibitor prodrugs using secondary amine functionality, secondary amide functionality, or secondary aniline functionality.

[0032] Figure 7 : Chemical synthesis of TB4 prodrugs involving stearic acid.

[0033] Figure 8 : Characterization of LNP-TB4 liposomes.

[0034] Figure 9 : Characterization of LNP-TB4 liposomes (zeta potential).

[0035] Figure 10 : Characterization of LNP-TB4-ID3 liposomes.

[0036] Figure 11 : Characterization of LNP-TB4-ID3 liposomes (zeta potential).

[0037] Figure 12 : Characterization of SLNP-TB4 solid lipid nanoparticles.

[0038] Figure 13 : Characterization of SLNP-TB4 solid lipid nanoparticles (zeta potential).

[0039] Figure 14 : Characterization of SLNP-TB4-ID3 solid lipid nanoparticles.

[0040] Figure 15 : Characterization (zeta potential) of SLNP-TB4-ID3 solid lipid nanoparticles.

[0041] Figure 16 : In vivo tumor inhibition by the combination of SLNP-TB4 and LNP-MTO using B16F10 cells.

[0042] Figure 17 : In vivo tumor inhibition by LNP-TB4 in various combinations using B16F10 cells.

[0043] Figure 18 : In vitro verification of the mechanism of action of LNP-TB4 and SLNP-TB4. DETAILED DESCRIPTION

[0044] Chapter Summary

[0045] I.) Definition

[0046] II.) Prodrugs

[0047] III.) Chemical compounds

[0048] IV.) Lipids

[0049] V.) Attachment Unit ("LU")

[0050] VI.) Nanocarriers

[0051] VII.) Liposomes

[0052] VIII.) Pharmaceutical Formulations

[0053] IX.) Combination therapy

[0054] X.) Method of delivering liposomes comprising prodrugs to cells

[0055] XI.) Methods of treating cancer and other immune disorders

[0056] XII.) Kits / Products

[0057] I.) Definition:

[0058] Unless otherwise defined, all technical terms, symbols and other scientific terms or expressions used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs, unless the context clearly indicates otherwise. In some cases, for the sake of clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from what is generally understood in the art.

[0059] When a trade name is used herein, reference to the trade name also refers to the product formulation, generic drug, and active pharmaceutical ingredient(s) of the trade name product, unless the context indicates otherwise.

[0060] As used herein, the term "about" when referring to a value or amount of size (i.e., diameter), weight, concentration, or percentage is meant to encompass variations from the specified amount of ±20% or ±10% in one example, ±5% in another example, ±1% in another example, and ±0.1% in still another example, as such variations are suitable for performing the disclosed methods.

[0061] As used herein, when used in the context of a list of entities, the term "and / or" refers to the entities as being present individually or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

[0062] The recitation of numerical ranges herein by endpoints include all numbers and fractions subsumed within that range (eg, 1 to 5, including but not limited to 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5).

[0063] As used herein, the phrase "consisting essentially of" limits the scope of a solution to the specified materials or steps, and to those materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0064] The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" mean cancer that has extended through the relevant tissue capsule and are intended to encompass stage C disease according to the American Urological Association (AUA) system, stage C1-C2 disease according to the Whitmore-Jewett system, and stage T3-T4 and N+ disease according to the TNM (tumor, node, metastasis) system. Surgery is generally not recommended for patients with locally advanced disease, and these patients have significantly more unfavorable outcomes than patients with clinically localized (organ-confined) cancer.

[0065] As used herein, the term "alkyl" may refer to a C1-C 20 (Inclusive) straight chain (i.e., "straight chain"), branched chain, or cyclic, saturated or at least partially and in some cases unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, vinyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl, or propyl, is attached to a straight chain alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms (i.e., C1-C8 alkyl). "Higher alkyl" refers to an alkyl group having 10 to about 20 carbon atoms, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" specifically refers to a C1-C8 straight chain alkyl group. In other embodiments, "alkyl" specifically refers to a C 1-8 Branched chain alkyl.

[0066] An alkyl group may be optionally substituted with one or more alkyl substituents which may be the same or different ("substituted alkyl"). The term "alkyl substituent" includes, but is not limited to, alkyl, substituted alkyl, halo, arylamino, acyl, hydroxy, aryloxy, alkoxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxyl, alkoxycarbonyl, oxy, and cycloalkyl. In some embodiments, one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms may be optionally inserted along the alkyl chain, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl"), or aryl.

[0067] Thus, as used herein, the term "substituted alkyl" includes alkyl groups as defined herein in which one or more atoms or functional groups are replaced by another atom or functional group, including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxy, hydroxy, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0068] The term "aryl" is used herein to refer to an aromatic substituent, which can be a single aromatic ring or multiple aromatic rings fused together, covalently linked, or linked to common groups such as, but not limited to, a methylene or vinyl moiety. Common linking groups can also be a carbonyl as in benzophenone or an oxygen as in diphenyl ether or a nitrogen as in diphenylamine. The term "aryl" specifically encompasses heterocyclic aromatic compounds. Aromatic rings can include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, and benzophenone, among others. In specific embodiments, the term "aryl" means a cyclic aromatic compound comprising from about 5 to about 10 carbon atoms, such as 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered aromatic rings and heteroaromatic rings. An aryl group can be optionally substituted with one or more aryl substituents ("substituted aryl") which can be the same or different, wherein "aryl substituents" include alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkyloxy, carboxy, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R", wherein R' and R" can each independently be hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl. Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

[0069] As used herein, "heteroaryl" refers to an aromatic group containing one or more non-carbon atoms (e.g., O, N, S, Se, etc.) in the backbone of the ring structure. Nitrogen-containing heteroaryl moieties include, but are not limited to, pyridine, imidazole, benzimidazole, pyrazole, pyrazine, triazine, pyrimidine, and the like.

[0070] The terms "anticancer drug," "chemotherapeutic agent," and "anticancer prodrug" refer to drugs (i.e., chemical compounds) or prodrugs that are known or suspected to be able to treat cancer (i.e., kill cancer cells, prevent cancer cell proliferation, or treat cancer-related symptoms). In some embodiments, as used herein, the term "chemotherapeutic agent" refers to a non-PS molecule used to treat cancer and / or having cytotoxic properties. More traditional or conventional chemotherapeutic agents can be described by mechanism of action or class of chemical compounds and can include, but are not limited to, alkylating agents (e.g., melphalan), anthracyclines (e.g., doxorubicin), cytoskeletal disruptors (e.g., paclitaxel), epothilones, histone deacetylase inhibitors (e.g., vorinostat), topoisomerase I or II inhibitors (e.g., irinotecan or etoposide), kinase inhibitors (e.g., bortezomib), nucleotide analogs or their precursors (e.g., methotrexate), peptide antibiotics (e.g., bleomycin), platinum-based agents (e.g., cisplatin or oxaliplatin), retinoids (e.g., tretinoin), and vinca alkaloids (e.g., vinblastine).

[0071] "Aralkyl" refers to an -alkyl-aryl group, optionally wherein the alkyl and / or aryl portions are substituted.

[0072] "Alkylene" refers to a straight or branched divalent aliphatic hydrocarbon radical having 1 to about 20 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group can also be optionally unsaturated and / or substituted with one or more "alkyl substituents". One or more oxygen atoms, sulfur atoms or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl") can be optionally inserted along the alkylene group, wherein the nitrogen substituent is an alkyl group as previously described. Exemplary alkylene groups include methylene (-CH2-), ethylene (-CH2-CH2-), propylene (-(CH2)3-), cyclohexylene (-CH6H 10 -), -CH=CH—CH=CH-, -CH=CH-CH2-, -(CH2) q-N(R)-(CH2)-, wherein each of q is an integer from 0 to about 20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and R is hydrogen or a lower alkyl group, methylenedioxy (-O-CH2-O-) and ethylenedioxy (-O-(CH2)2-O-). The alkylene group may have from about 2 to about 3 carbon atoms and may further have from 6 to 20 carbon atoms.

[0073] The term "arylene" refers to a divalent aromatic group, such as a divalent phenyl or naphthyl group. An arylene group may be optionally substituted with one or more aryl substituents and / or contain one or more heteroatoms.

[0074] The term "amino" refers to the group -N(R)2, where each R is independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, or substituted aralkyl. The terms "aminoalkyl" and "alkylamino" may refer to the group -N(R)2, where each R is H, alkyl, or substituted alkyl, and where at least one R is alkyl or substituted alkyl. "Arylamine" and "aminoaryl" refer to the group -N(R)2, where each R is H, aryl, or substituted aryl, and where at least one R is aryl or substituted aryl, for example, aniline (i.e., -NHC6H5).

[0075] "Bulk drug" (also known as drug product) refers to a drug product or pharmaceutical product that has not yet been filled into its final container for distribution. Final compounded bulk drug product typically refers to a drug product that has been compounded and stored or held prior to filling. Prior to compounding into a drug product, drug products may be stored or held in bulk or concentrated bulk form.

[0076] The terms "carboxylate" and "carboxylic acid" may refer to the group -C(=O)O - and -C(=O)OH. The term "carboxyl" may also refer to a -C(=O)OH group.

[0077] As used herein, the terms "conjugate" and "conjugated" can refer to two or more components (e.g., chemical compounds, polymers, biomolecules, particles, etc.) being linked (e.g., covalently linked) to each other. In some embodiments, the conjugate can include a monovalent moiety (e.g., an optionally substituted alkylene or arylene) derived from two different chemical compounds covalently linked by a bivalent linker moiety. In some embodiments, the linker can contain one or more biodegradable bonds such that one or more bonds in the linker can be broken when the prodrug is exposed to a particular physiological environment or enzyme (e.g., an esterase).

[0078] The term "compound" refers to and encompasses the chemical compound (e.g., a prodrug) itself, and whether or not explicitly stated, and unless the context clearly indicates otherwise, excludes: amorphous and crystalline forms of the compound, including polymorphic forms, where these forms may be part of a mixture or exist alone; free acid and free base forms of the compound, which are generally the forms shown in the structures provided herein; isomers of the compound, which refers to optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers as well as mixtures of optical isomers, including racemic and non-racemic mixtures, where an isomer may be in isolated form or in mixtures with one or more other isomers; isotopes of the compound, including deuterated and Compounds containing tritium, and compounds containing radioisotopes, including therapeutically and diagnostically effective radioisotopes; polymeric forms of the compounds, including dimers, trimers, and the like; salts of the compounds, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts, including salts with organic counterions and inorganic counterions, and including zwitterionic forms, wherein if a compound is associated with two or more counterions, the two or more counterions may be the same or different; and solvates of the compounds, including hemisolvates, monosolvates, and disolvates, including organic solvates and inorganic solvates, including hydrates, wherein if a compound is associated with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some cases, references herein to compounds of the invention will include explicit reference to one or more of the above-mentioned forms, such as salts and / or solvates; however, this reference is for emphasis only and should not be interpreted as excluding other forms identified above.

[0079] "Drug product" means the final formulation containing the active pharmaceutical ingredient (i.e., liposomes containing an ALK5 inhibitor prodrug), typically but not necessarily associated with inactive ingredients. The term also includes finished dosage forms that do not contain an active ingredient but are intended for use as a placebo.

[0080] The term "disulfide" may refer to an SS- group.

[0081] The term "empty vesicle" refers to a lipid vesicle that is itself unloaded.

[0082] As used herein, the term "ester" means a chemical compound derived from an acid (organic or inorganic) in which at least one -OH hydroxyl group is replaced by an -O-alkyl (alkoxy) or O-aryl (aryloxy) group.

[0083] As used herein, the term "esterase" is a hydrolase that breaks down esters into acids and alcohols.

[0084] "Excipient" refers to an inactive substance used as a carrier for the active ingredient in pharmaceuticals, such as vaccines. Excipients are also sometimes used to bulk up formulations containing very potent active ingredients to facilitate convenient and accurate dosing. Examples of excipients include, but are not limited to, antiadherents, binders, coatings, disintegrants, fillers, diluents, flavorings, pigments, lubricants, and preservatives.

[0085] As used herein, the term "halo," "halide," or "halogen" refers to fluoro, chloro, bromo, and iodo groups.

[0086] The terms "hydroxyl" and "hydroxy" refer to an -OH group.

[0087] As used herein, the term "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent completely.

[0088] As used in the context of the present disclosure, the terms "individual" or "patient" are used interchangeably.

[0089] As used herein, the term "ligand" generally refers to a species, such as a molecule or ion, that interacts with, e.g., binds to, another species in some manner. See Martell, AE and Hancock, RP, Metal Complexes in Aqueous Solutions, Plenum: New York, (1996), which is incorporated herein by reference in its entirety.

[0090] As used herein, the term "lipid" refers to a class of naturally occurring (organic) compounds that are insoluble in polar solvents. In the context of the present disclosure, lipid refers to conventional lipids, phospholipids, cholesterol, chemically functionalized lipids for linking PEG and ligands, etc.

[0091] The term "lipid bilayer" or "LB" refers to any bilayer of amphiphilic lipid molecules oriented with the hydrocarbon tails facing inward to form a continuous non-polar phase.

[0092] The terms "liposome" or "lipid vesicle" or "vesicle" are used interchangeably to refer to an aqueous compartment surrounded by a lipid bilayer as conventionally defined (see Stryer (1981) Biochemistry, 2nd ed., WH Freeman & Co., 213).

[0093] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one embodiment of the present invention, the mammal is a mouse. In another embodiment of the present invention, the mammal is a human.

[0094] The term "mercapto" or "thiol" refers to a -SH group.

[0095] The terms "metastatic cancer" and "metastatic disease" mean cancer that has spread to regional lymph nodes or distant sites and are meant to encompass stage D disease according to the AUA system and stage TxNxM+ according to the TNM system.

[0096] The terms "nanocarrier," "nanoparticle," and "nanoparticle drug carrier" are used interchangeably and refer to nanostructures having an aqueous, solid, or polymeric interior core. In certain embodiments, the nanocarrier comprises a lipid bilayer that encapsulates (or surrounds or encapsulates) a porous particle core. In certain embodiments, the nanocarrier is a liposome, a lipid nanoparticle ("LNP"), or a solid lipid nanoparticle ("SLNP").

[0097] The terms "nanoscale particle," "nanomaterial," "nanocarrier," and "nanoparticle" refer to a structure having at least one region having a dimension (e.g., length, width, diameter, etc.) less than about 1,000 nm. In some embodiments, the dimension is smaller (e.g., less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, or even less than about 20 nm). In some embodiments, the size is between about 20 nm and about 250 nm (e.g., about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, or 250 nm).

[0098] The term "nanovesicle" refers to a "lipid vesicle" having a diameter (or a population of vesicles having an average diameter) in the range of about 20 nm, or about 30 nm, or about 40 nm, or about 50 nm to about 500 nm, or to about 400 nm, or to about 300 nm, or to about 200 nm, or to 150 nm, or to about 100 nm, or to about 80 nm. In certain embodiments, the diameter of the nanovesicle ranges from about 40 nm to about 80 nm or from about 50 nm to about 70 nm.

[0099] "Pharmaceutically acceptable" refers to non-toxic, inert and / or compositions that are physiologically compatible with humans or other mammals.

[0100] Drug compounding refers to the process of combining different chemical substances into pure drug products to produce the final drug product.

[0101] The term "phosphonate" refers to a -P(=O)(OR)2 group, wherein each R can independently be H, alkyl, aralkyl, aryl, or negatively charged (i.e., wherein there is no R group bonded to the oxygen atom, resulting in an unshared electron pair on the oxygen atom). Thus, in other words, each R can be present or absent, and when present, is selected from H, alkyl, aralkyl, or aryl.

[0102] The term "phosphate" refers to a -OP(=O)(OR')2 group, where R' is H or a negative charge.

[0103] The term "prodrug" means a drug or compound that is metabolized into a pharmacologically active drug after administration. For the purposes of this disclosure, the prodrugs of the present invention include three (3) components: (i) a drug moiety; (ii) a lipid moiety; and (iii) a linking unit ("LU").

[0104] The term "TB prodrug" refers to a prodrug of the present invention wherein the drug moiety comprises an ALK5 inhibitor.

[0105] The term "pyrolipid" refers to a conjugate of a lipid with a porphyrin, a porphyrin derivative, or a porphyrin analog. In some embodiments, the pyrolipid may include a lipid conjugate in which a porphyrin, or a derivative or analog thereof, is covalently attached to a lipid side chain. See, for example, U.S. Patent Application Publication No. 2014 / 0127763.

[0106] As used herein, the terms "specifically," "specifically binds," and "binds specifically" refer to the selective binding of the nanocarrier of the present invention to the target TGFβ1 or related family members.

[0107] The term "supported lipid bilayer" means the lipid bilayer surrounding the porous particle core. This definition as shown in the present disclosure is because the lipid bilayer is positioned on the surface and supported by the porous particle core. In certain embodiments, the thickness of the lipid bilayer can be about 6 nm to about 7 nm, which includes a 3-4 nm thickness of the hydrophobic core, plus a layer of hydrated hydrophilic head groups (each about 0.9 nm) plus two partially hydrated regions of about 0.3 nm each. In various embodiments, the lipid bilayer surrounding the liposome comprises a continuous bilayer or a substantially continuous bilayer that effectively encapsulates and seals the ALK5 inhibitor.

[0108] The term "thioalkyl" may refer to a group -SR, where R is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl. Similarly, the terms "thioaralkyl" and "thioaryl" refer to a -SR group, where R is aralkyl and aryl, respectively.

[0109] As used herein, "for the treatment of" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of the disease, such as prolonged survival, reduced morbidity and / or reduced side effects, which are by-products of alternative treatment modalities; as is readily understood in the art, complete eradication of the disease is preferred, but nonetheless is not a requirement for therapeutic action.

[0110] The term "therapeutically effective amount" refers to the amount of active prodrug, nanoencapsulated prodrug, or pharmaceutical agent that elicits a biological or medicinal response in a tissue, system, animal, individual, or human.

[0111] The term "unsupported lipid bilayer" refers to the uncoated lipid bilayer in a lipid vesicle or liposome.

[0112] II.) Prodrugs

[0113] As shown in this disclosure, and for the purposes of the present invention, suitable prodrugs are formed by conjugating a drug moiety of the present invention (see the section entitled "Drug Moiety") to a lipid moiety of the present invention (see the section entitled "Lipid") via a LU of the present disclosure (see the section entitled "Linker Unit"). For the purposes of this disclosure, several strategies can be utilized for the formation of TB prodrugs. (See, e.g., Figure 4 、 Figure 5 and Figure 6 ).

[0114] Thus, in some embodiments, a prodrug is a drug lipid portion that includes an ALK5 inhibitor of the present disclosure.

[0115] In one embodiment, the prodrug comprises the following chemical structure represented by Formula I:

[0116]

[0117] Wherein in the exemplary embodiment of Formula I:

[0118] R1=C 11 -C 21 saturated alkyl; and

[0119] R2=H, CH3.

[0120] In further embodiments, the prodrug comprises the following chemical structure represented by Formula II:

[0121]

[0122] Wherein in the exemplary embodiment of Formula II:

[0123]

[0124] R1=C 11 -C 21 saturated alkyl; and

[0125] R2=H, CH3.

[0126] In further embodiments, the prodrug comprises the following chemical structure represented by Formula III:

[0127]

[0128] Wherein in the exemplary embodiment of Formula III:

[0129]

[0130] R1=C 11 -C 21 saturated alkyl; and

[0131] R2=H, CH3.

[0132] Thus, in one embodiment, the prodrug is a drug lipid portion comprising an ALK5 inhibitor of Formula I.

[0133] In further embodiments, the prodrug is a drug lipid portion comprising an ALK5 inhibitor of Formula II.

[0134] In further embodiments, the prodrug is a drug lipid portion comprising an ALK5 inhibitor of Formula III.

[0135] In one embodiment, the prodrug comprises Figure 4 The drug lipid portion of the ALK5 inhibitors shown.

[0136] In one embodiment, the prodrug comprises Figure 5 The drug lipid portion of the ALK5 inhibitors shown.

[0137] In one embodiment, the prodrug comprises Figure 6 The drug lipid portion of the ALK5 inhibitors shown.

[0138] In further embodiments, the TB prodrug is a drug lipid moiety comprising a lipid of the present disclosure.

[0139] In further embodiments, the TB prodrug is a drug lipid moiety whereby the lipid is CHEMS.

[0140] In further embodiments, the TB prodrug is a drug lipid moiety whereby the lipid is stearic acid.

[0141] In further embodiments, the TB prodrug is a drug lipid moiety comprising the LU of the present disclosure.

[0142] In further embodiments, the TB prodrug is a drug lipid moiety whereby LU is a hydroxymethylcarbamate linker.

[0143] In further embodiments, the prodrug is a drug lipid portion comprising an ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises the chemical composition represented as TB4.

[0144] In further embodiments, the prodrug is a drug lipid portion comprising an ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and has the following chemical structure:

[0145]

[0146] In further embodiments, the prodrug is a drug lipid portion comprising an ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises a lipid of the present disclosure having the following formula:

[0147]

[0148] (“TB4 prodrug”)

[0149] In further embodiments, the prodrug is a drug lipid moiety comprising an ALK5 inhibitor of the invention, wherein the ALK5 inhibitor comprises TB4 and further comprises CHEMS.

[0150] In further embodiments, the prodrug is a drug lipid moiety comprising an ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises stearic acid.

[0151] In further embodiments, the prodrug is a drug lipid moiety comprising an ALK5 inhibitor of the invention, wherein the ALK5 inhibitor comprises TB4 and further comprises CHEMS, and whereby LU is a hydroxymethylcarbamate linker.

[0152] In further embodiments, the prodrug is a drug lipid moiety comprising an ALK5 inhibitor of the invention, wherein the ALK5 inhibitor comprises TB4 and further comprises stearic acid, and whereby LU is a hydroxymethylcarbamate linker.

[0153] In further embodiments, the prodrug is a drug lipid moiety comprising an ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises stearic acid having the following structure:

[0154]

[0155] In further embodiments, the prodrug is a drug lipid moiety comprising an ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises stearic acid having the following structure:

[0156]

[0157] In another embodiment of the present disclosure, the present subject matter provides an ALK5 inhibitor prodrug comprising a lipid-conjugated therapeutic agent parent drug. In some embodiments, the prodrug comprises: (a) a monovalent drug portion; (b) a monovalent lipid portion; and (c) a divalent linker portion, the divalent linker portion comprising a linking unit that will degrade in vivo, such as a disulfide bond, wherein the monovalent drug portion and the monovalent lipid portion are connected (e.g., covalently linked) via a linker. The monovalent drug portion and the monovalent lipid portion can be monovalent derivatives of a chemical compound and a lipid, respectively. For example, the monovalent derivative can be a deprotonated derivative of a chemical compound or lipid comprising a hydroxyl group, a thiol group, an amino group, or a carboxylic acid group.

[0158] In another embodiment of the present disclosure, the present subject matter provides an ALK5 inhibitor prodrug comprising a lipid-conjugated therapeutic agent parent drug. In some embodiments, the prodrug comprises: (a) a bivalent drug portion; (b) a bivalent lipid portion; and (c) a bivalent linker portion comprising a bond that will degrade in vivo, wherein the bivalent drug portion and the bivalent lipid portion are connected (e.g., covalently linked) via a linker. The bivalent drug portion and the bivalent lipid portion can be divalent derivatives of chemical compounds and lipids, respectively. For example, the bivalent derivative can be a deprotonated derivative of a chemical compound or lipid comprising a hydroxyl group, a thiol group, an amino group, or a carboxylic acid group.

[0159] Those skilled in the art will understand the disclosed embodiments and will enable those skilled in the art to make changes and modifications to the disclosed embodiments without changing the functions and purposes of the present invention disclosed herein. Such changes and modifications are intended to be within the scope of the present disclosure.

[0160] III.) Drug part

[0161] Another aspect of the present invention provides novel TB prodrug compounds, including an ALK5 inhibitor having the formula TB4.

[0162] Those skilled in the art will understand that the compound can be used as an ALK5 signaling inhibitor (e.g., inhibiting ALK5 and other family members). As a brief background, ALK5, also known as TGFβ receptor I (TGFβRI), is a membrane-bound receptor belonging to the cytokine superfamily, which acts on protein kinase receptors at the plasma membrane to induce excessive biological signals, regulating cell growth and death, differentiation, immune response, angiogenesis and inflammation. The disorder of its pathway leads to a variety of pathologies, including cancer. TGFβ is an important regulatory tumor suppressor in epithelial cells, where it inhibits early proliferation and induces apoptosis. See FABREGAT et al., TGF-β Signaling in Cancer Treatment, Current Drug Design (Curr.Pharm.Des.), 20(17): pp. 2934-2947 (2014). Studies have shown that the development of therapeutic compounds that target TGFβ production or block its effects may help treat cancer. See HAQUE et al., Transforming growth factor-β: A Therapeutic Target for Cancer, Hum. Vaccin. Immunother., 13(8): 1741-1750 (2017).

[0163] Based on the foregoing, the present disclosure describes a class of TGFβ inhibitors.

[0164] In one embodiment, the drug moiety of the present disclosure includes a compound having the following chemical structure (denoted as TB4):

[0165]

[0166] In one embodiment, the drug moiety of the present disclosure includes Figure 2 Protecting group intermediates en route to the final TB4 prodrug are shown.

[0167] In further embodiments, the drug moiety of the present disclosure includes Figure 3 Protecting group intermediates en route to the final TB4 prodrug are shown.

[0168] Those skilled in the art will understand the disclosed embodiments and will enable those skilled in the art to make changes and modifications to the disclosed embodiments without changing the functions and purposes of the present invention disclosed herein. Such changes and modifications are intended to be within the scope of the present disclosure.

[0169] IV.) Lipids

[0170] In general, and for the purposes of this disclosure, the term "lipid" is used in its broadest sense and includes several subclasses of lipids, including but not limited to phospholipids / fatty acids. As will be appreciated by those skilled in the art, phospholipids represent a class of lipids that are the main components of all cell membranes. Phospholipids can form lipid bilayers because they have amphipathic properties. The structure of a phospholipid molecule is typically composed of two hydrophobic fatty acid "tails" and a hydrophilic "head" composed of a phosphate group, which can be modified with simple organic molecules such as choline, ethanolamine or serine. These two components are typically linked together by a glycerol molecule. A representative list of phospholipids / fatty acids of the present invention is shown in Table III.

[0171] As brief background, on the most basic level, the characteristic of liposome depends on the subtle physicochemical interaction between various lipid species in its composition.Each lipid can be combined to form countless superstructures, comprises double layer, and can adjust double layer characteristics to regulate drug release and membrane stability.In simplified double layer model, acyl chain length determines double layer thickness and phase transition temperature (Tm), acyl chain saturation controls double layer fluidity, and head group interaction affects the molecular force between lipid and lipid.Liposome behavior can be by synthetic lipid, as lipid prodrug, fusion lipid and functionalized lipid are incorporated into double layer and regulated.Referring to Ke Li (KOHLI) et al., controlled release magazine (J.Control Release), 0: 274-287 pages (September 28, 2014).

[0172] In one embodiment of the present disclosure, the TB prodrug includes a monovalent lipid moiety.

[0173] In one embodiment, the TB prodrug includes a bivalent lipid moiety.

[0174] In one embodiment, the lipid comprises cholesterol having the following chemical structure:

[0175]

[0176] In one embodiment, the lipid comprises DPPG having the following chemical structure:

[0177]

[0178] In one embodiment, the lipid comprises DMPG having the following chemical structure:

[0179]

[0180] In one embodiment, the lipid comprises Lyso PC having the following chemical structure:

[0181]

[0182] In one embodiment, the lipid comprises (Δ9-cis)PG having the following chemical structure:

[0183]

[0184] In one embodiment, the lipid comprises Soy Lyso PC having the following chemical structure:

[0185]

[0186] In one embodiment, the lipid comprises PG having the following chemical structure:

[0187]

[0188] In one embodiment, the lipid comprises C16 PEG2000 having the following chemical structure:

[0189]

[0190] In one embodiment, the lipid comprises cholesterol hemisuccinate ("CHEMS") having the following chemical structure:

[0191]

[0192] For reference, a complete listing of the chemical formulas and abbreviations of the lipids disclosed herein is shown in Table 1.

[0193] In another embodiment, the lipid comprises the phospholipids / fatty acids disclosed herein and shown in Table III.

[0194] In further embodiments, the lipid comprises stearic acid.

[0195] In addition, the TB prodrugs and / or liposomes of the present disclosure may include one or more helper lipids, also referred to herein as "helper lipid components." The helper lipid components are preferably selected from the group consisting of phospholipids and steroids. The phospholipids are preferably diesters and monoesters of phosphoric acid. Preferred members of the phospholipid class are phosphoglycerides and sphingolipids. As used herein, steroids are naturally occurring and synthetic compounds based on partially hydrogenated cyclopenta[a]phenanthrene. Preferably, the steroid contains 21 to 30 C atoms. A particularly preferred steroid is cholesterol.

[0196] It should be noted that, although not wishing to be bound by any theory, surprising effects can be achieved due to the specific molar percentage of helper lipids contained in the lipid composition according to the present invention, which can be PEG-free helper lipids or specifically PEG-containing helper lipids, more particularly if the content of any such helper lipids is contained within the concentration range specified herein.

[0197] In another aspect of the invention, the lipid composition, preferably present as a lipoplex or liposome, preferably exhibits a neutral or overall anionic charge. The anionic lipid is preferably any neutral or anionic lipid described herein. The lipid composition comprises, in a preferred embodiment, any helper lipid or combination of helper lipids and any ALK5 inhibitor described herein (e.g., TB4). In another embodiment, the composition containing nucleic acids according to the present invention forms a lipoplex. In a preferred embodiment, the term lipoplex as used herein refers to a composition consisting of a neutral or anionic lipid of the present invention, a neutral helper lipid, and an ALK5 inhibitor. For references to the use of helper lipids in the art, see, for example, U.S. Patent Application Publication No. 2011 / 0178164; OJEDA et al., Int. J. of Pharmaceutics (March 2016); DABKOWSKA et al., JRSoc. Interface 9, pp. 548-561 (2012); and MOCHIZUKI et al., Biochimica et. Biophysica Acta, 1828, pp. 412-418 (2013).

[0198] In preferred embodiments, the helper lipids of the present invention include the helper lipids shown in Table II.

[0199] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is CHEMS and wherein the drug moiety is TB4.

[0200] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is CHEMS and wherein the drug moiety is TB4, further comprising LU and wherein LU is a hydroxymethylcarbamate linker.

[0201] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is CHEMS and wherein the drug moiety is TB4, further comprising LU and wherein LU is a hydroxymethylcarbamate linker, further comprising a helper lipid component, wherein the helper lipid component comprises a helper lipid in Table II.

[0202] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is CHEMS and wherein the drug moiety is TB4, and wherein the CHEMS is monovalent.

[0203] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is stearic acid and wherein the drug moiety is TB4.

[0204] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is stearic acid and wherein the drug moiety is TB4 and wherein the stearic acid is monovalent.

[0205] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is stearic acid and wherein the drug moiety is TB4, further comprising LU and wherein LU is a hydroxymethylcarbamate linker.

[0206] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is stearic acid and wherein the chemical composition is TB4, further comprising LU and wherein LU is a hydroxymethylcarbamate linker, further comprising a helper lipid component, wherein the helper lipid component comprises a helper lipid in Table II.

[0207] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is stearic acid and wherein the drug moiety is TB4 and wherein the TB prodrug has the following chemical structure:

[0208]

[0209] Those skilled in the art will understand the disclosed embodiments and will enable those skilled in the art to make changes and modifications to the disclosed embodiments without changing the functions and purposes of the present invention disclosed herein. Such changes and modifications are intended to be within the scope of the present disclosure.

[0210] V.) Linking Unit ("LU")

[0211] In some embodiments, the presently disclosed subject matter provides prodrugs including drug-lipid conjugates comprising biodegradable linkages, such as esters, thioesters, and other linkers known in the art.

[0212] Exemplary examples of ester chemistry are shown herein:

[0213]

[0214] In some embodiments, the prodrug is a drug-lipid conjugate, whereby the drug-lipid conjugate is cleaved by an esterase.

[0215] In one embodiment, the prodrugs of the present invention include LU via a secondary amine, secondary amide, or secondary aniline using the following scheme:

[0216]

[0217] An exemplary synthesis is as follows:

[0218]

[0219] Cleavage of the prodrug structure comprising a secondary amine, secondary amide, or secondary aniline is achieved by esterase hydrolysis of a secondary amine prodrug, secondary amide prodrug, or secondary aniline prodrug in the following exemplary synthesis:

[0220]

[0221] in:

[0222] R1-NH-R2 can be any molecule containing a secondary amine, secondary amide or secondary aniline.

[0223] In one embodiment, the secondary amide nitrogen of the TB4 drug moiety is conjugated to CHEMS via a hydroxymethylcarbamate linker.

[0224] Those skilled in the art will understand the disclosed embodiments and will enable those skilled in the art to make changes and modifications to the disclosed embodiments without changing the functions and purposes of the present invention disclosed herein. Such changes and modifications are intended to be within the scope of the present disclosure.

[0225] VI.) Nanocarriers

[0226] In general, and for the purposes of this disclosure, nanocarriers are within the scope of the present invention. Nanocarriers are nanomaterials used as another substance, such as transport molecules of drugs. Conventional nanocarriers include micelles, polymers, carbon-based materials, liposomes and other substances. Due to their small size, nanocarriers can deliver drugs to parts of the body that are otherwise difficult to access. Nanocarriers can include polymer conjugates, polymer nanoparticles, lipid-based carriers, dendrimers, carbon nanotubes and gold nanoparticles. Lipid-based carriers all include liposomes, solid lipid nanoparticles and micelles. In certain embodiments, nanocarriers are liposomes, lipid nanoparticles ("LNPs") or solid lipid nanoparticles ("SLNPs").

[0227] In addition, nanocarriers are very useful in drug delivery because they can deliver drugs to site-specific targets, allowing drugs to be delivered to certain organs or cells rather than other organs or cells. Site-specificity brings major therapeutic benefits because it can prevent drugs from being delivered to the wrong place. In addition, nanocarriers show application prospects in chemotherapy because they can help reduce the adverse and more widespread toxicity of chemotherapy to healthy, fast-growing cells around the body. Since chemotherapy drugs can be extremely toxic to human cells, it is important that they are delivered to tumors without being released into other parts of the body.

[0228] Generally speaking, nanocarriers can deliver drugs through four (4) methods, and the methods include passive targeting, active targeting, pH specificity, and temperature specificity.

[0229] Passive targeting refers to the ability of nanocarriers to travel down the tumor vasculature, become trapped, and accumulate in the tumor. This accumulation is caused by enhanced permeability and retention effects. The leaky vasculature of a tumor is a network of blood vessels that forms within the tumor and contains numerous small pores. These pores allow nanocarriers to enter, but also contain numerous tortuosities that allow nanocarriers to become trapped. As more nanocarriers are trapped, the drug accumulates at the tumor site. This accumulation results in the delivery of large doses of drug directly to the tumor site.

[0230] Active targeting involves incorporating targeting moieties, such as ligands or antibodies, onto the surface of nanocarriers that are specific for certain types of cells around the body. Typically, nanocarriers have a high surface area to volume ratio, allowing for the incorporation of multiple ligands on their surface.

[0231] In addition, certain nanocarriers will only release the drug they contain within a specific pH range. pH specificity also allows nanocarriers to deliver drugs directly to the tumor site. This is because tumors are generally more acidic than normal human cells, with a pH of about 6.8. The pH of normal tissue is about 7.4. Therefore, nanocarriers that release drugs only within certain pH ranges can be used to release drugs only in the acidic tumor environment. Since the acidic environment degrades the structure of the nanocarrier, the highly acidic environment causes drug release. Generally, these nanocarriers will not release drugs in neutral or alkaline environments, thereby effectively targeting the acidic environment of the tumor while leaving normal body cells unaffected. This pH sensitivity can also be induced in micellar systems by adding copolymer chains to micelles, which have been determined to function in a pH-independent manner. See Wu et al., Biomaterials, 34(4):1213-1222 (2012). These micellar polymer complexes also help prevent cancer cells from developing multidrug resistance. The low pH environment triggers rapid release from the micellar polymer, resulting in the release of most of the drug all at once, rather than gradually as with other drug therapies.

[0232] In addition, it has been shown that some nanocarriers can deliver drugs more effectively at certain temperatures. Since tumor temperatures are generally higher than those of other parts of the body (approximately 40°C), this temperature gradient helps ensure tumor-specific delivery. See REZAEI et al., Polymer, 53(16): 3485-3497 (2012).

[0233] As disclosed herein, lipid-based nanocarriers, such as liposomes, are within the scope of the present invention. Lipid-based nanoparticles (LBNP or LNP), such as liposomes, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) can transport hydrophobic molecules and hydrophilic molecules, showing minimal toxicity or no toxicity, and increasing the drug action time by extending the half-life and controlling drug release. Lipid nanoparticles can include chemical modifications to avoid detection by the immune system (gangliosides or polyethylene glycol (PEG)) or to improve the solubility of the drug. In addition, it can be prepared into a formulation that is sensitive to pH to promote the release of the drug in an acidic environment, and can also be associated with small molecules or antibodies that recognize tumor cells or their receptors (such as folic acid (FoA)). Nanomedicines can also be used in combination with other treatment strategies to improve the patient's response. See Garcia-Pinell et al., Nanomaterials 9 (639) (2019).

[0234] In various embodiments, the silicasome drug carriers described herein include porous silica (or other materials) nanoparticles (e.g., silica bodies having a surface and defining a plurality of holes suitable for receiving molecules therein) coated with a lipid bilayer. The fact that the nanoparticles are referred to as silica nanoparticles does not exclude that materials other than silica are also incorporated into the silica nanoparticles. In some embodiments, the silica nanoparticles can be substantially spherical, with a plurality of pores opening through the surface, thereby providing access to the holes. However, in various embodiments, the silica nanoparticles can have a shape other than substantially spherical. Thus, for example, in certain embodiments, the silica nanoparticles can be substantially oval, rod-shaped, substantially regular polygonal, irregular polygonal, etc.

[0235] Typically, the silica nanoparticles include a silica body that defines an outer surface between pore openings and sidewalls within the pores. The pores may extend through the silica body to another pore opening, or the pores may extend only partially through the silica body such that they have a bottom surface defined by the silica body.

[0236] In some embodiments, the silica body is mesoporous. In other embodiments, the silica body is microporous. As used herein, "mesoporous" means a pore having a diameter between about 2 nm and about 50 nm, while "microporous" means a pore having a diameter less than about 2 nm. Generally, the pores can be of any size, but in typical embodiments are large enough to contain one or more therapeutic compounds therein. In such embodiments, the pores allow small molecules, such as therapeutic compounds, such as anticancer compounds, to adhere to or bind to the inner surface of the pores and be released from the silica body when used for therapeutic purposes. In some embodiments, the pores are substantially cylindrical.

[0237] In certain embodiments, the nanoparticles include pores with a pore diameter between about 1 nm and about 10 nm, or between about 2 nm and about 8 nm. In certain embodiments, the nanoparticles include pores with a pore diameter between about 1 nm and about 6 nm, or between about 2 nm and about 5 nm. Other embodiments include particles with a pore diameter less than 2.5 nm.

[0238] In other embodiments, the pore size is between 1.5nm and 2.5nm. Silica nanoparticles with other pore sizes can be prepared, for example, by using different surfactants or swelling agents during the preparation of silica nanoparticles. In various embodiments, nanoparticles can include particles as large as (such as average diameter or median diameter (or another characteristic size)) about 1000nm. However, in various embodiments, nanoparticles are typically less than 500nm or less than about 300nm, because particles typically greater than 300nm may not be very effective when entering living cells or vascular fenestrations. In certain embodiments, the size range of nanoparticles is about 40nm, or about 50nm, or about 60nm to about 100nm, or to about 90nm, or to about 80nm, or to about 70nm. In certain embodiments, the size range of nanoparticles is about 60nm to about 70nm. Some embodiments include nanoparticles with an average maximum size between about 50nm and about 1000nm. Other embodiments include nanoparticles with an average maximum size between about 50nm and about 500nm. Other embodiments comprise nanoparticles having an average maximum dimension between about 50 nm and about 200 nm.

[0239] In certain embodiments, the average maximum dimension is greater than about 20nm, greater than about 30nm, greater than about 40nm or greater than about 50nm. Other embodiments include nanoparticles having an average maximum dimension less than about 500nm, less than about 300nm, less than about 200nm, less than about 100nm or less than about 75nm. As used herein, the size of nanoparticles refers to the average size or median size of the primary particles as measured by transmission electron microscopy (TEM) or similar visualization techniques known in the art. Other examples of mesoporous silica nanoparticles include but are not limited to MCM-41, MCM-48 and SBA-15. See Katiyar et al., Journal of Chromatography (J. Chromotog.) 1122 (1-2): 13-20 (2006).

[0240] Methods for preparing porous silica nanoparticles are well known to those skilled in the art. In certain embodiments, mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate (TEOS) with a template made of micellar rods. The result is a collection of nanometer-sized spheres or rods filled with regularly arranged pores. The template can then be removed by washing with a solvent adjusted to an appropriate pH (see, for example, Trewyn et al., (2007) Chem. Eng. J. 137(1):23-29).

[0241] In some embodiments, mesoporous particles can also be synthesized using a simple sol-gel method (see, for example, Nandiyanto et al. (2009) Microporous and Mesoporous Mat. 120(3):447-453). In some embodiments, tetraethyl orthosilicate can also be used as a template together with other polymer monomers. In some embodiments, (3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS.

[0242] In certain embodiments, the mesoporous silica nanoparticles are cores synthesized by modifying the sol / gel procedure described in: MENG et al., (2015) ACS Nano, 9(4): 3540-3557.

[0243] Although the methods described herein have been demonstrated with respect to porous silica nanoparticles (e.g., mesoporous silica), it will be appreciated by those skilled in the art that similar methods can be used for other porous nanoparticles. Many other mesoporous materials that can be used for drug delivery nanoparticles are known to those skilled in the art. For example, in certain embodiments, mesoporous carbon nanoparticles can be utilized.

[0244] Mesoporous carbon nanoparticles are well known to those skilled in the art (see, for example, Huang et al., (2016) Carbon, 101: 135-142; Zhu et al., (2014) Asian J. Pharm. Sci., 9(2): 82-91; etc.).

[0245] Similarly, in certain embodiments, mesoporous polymer particles can be utilized. The synthesis of highly ordered mesoporous polymers and carbon frameworks from organic-organic assembly of triblock copolymers with soluble low molecular weight phenolic resin precursors via an evaporation-induced self-assembly strategy has been reported as follows: Meng et al., (2006) Chem. Mat. 6(18):4447-4464.

[0246] The nanoparticles described herein are illustrative and non-limiting. Using the teachings provided herein, one skilled in the art will be able to obtain many other lipid bilayer-coated nanoparticles.

[0247] In one embodiment, the present invention teaches nanocarriers comprising TB prodrugs.

[0248] In one embodiment, the present invention teaches nanocarriers comprising liposomes, wherein the lipids comprise CHEMS.

[0249] In one embodiment, the present invention teaches nanocarriers comprising liposomes, wherein the lipid comprises stearic acid.

[0250] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises CHEMS and whereby the liposome further comprises a TB prodrug.

[0251] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises CHEMS and whereby the liposome further comprises TB4.

[0252] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid and whereby the liposome further comprises an ALK5 inhibitor.

[0253] In one embodiment, the present invention teaches nanocarriers comprising liposomes, wherein the lipids comprise stearic acid and whereby the liposomes further comprise TB4.

[0254] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid and whereby the liposome further comprises TB4 (denoted as LNP-TB4).

[0255] In further embodiments, the present invention teaches nanocarriers comprising liposomes, wherein the lipids comprise stearic acid and whereby the liposomes further comprise TB4 and whereby the liposomes are co-formulated with ID3 (denoted as LNP-TB4-ID3).

[0256] In a preferred embodiment, the lipid particles comprise solid lipid nanoparticles (SLNPs) comprising liposomes comprising a TB4 prodrug.

[0257] In one embodiment, the present invention teaches a nanocarrier comprising solid lipid nanoparticles (SLNPs), wherein the solid lipid nanoparticles comprise stearic acid and whereby the solid lipid nanoparticles further comprise TB4 (denoted as SLNP-TB4).

[0258] In further embodiments, the present invention teaches nanocarriers comprising solid lipid nanoparticles (SLNPs), wherein the solid lipid nanoparticles comprise stearic acid and whereby the solid lipid nanoparticles further comprise TB4 and whereby the SLNPs are co-formulated with ID3 (denoted as SLNP-TB4-ID3).

[0259] In another preferred embodiment, the solid lipid nanoparticles of the present invention comprise a composition having the following ratios:

[0260] Composition of SLNP Amount(%w / w) Lipid 1 (lipid-prodrug) 5-80 Lipid 2 (lipid-prodrug) 0-40 Helper lipids 0-80 DSPE-PEG2000 0-10 stabilizer 0.5-20

[0261] Whereby lipid 1 comprises a TB4 prodrug, wherein the lipid portion comprises stearic acid, and whereby the helper lipid is a helper lipid shown in Table II, and whereby the stabilizer is selected from the group consisting of polyvinyl alcohol (e.g., Moliwol 488), poloxamer (e.g., Pluronic F127), Tween 80, PEG400, and Kolliphor RH 40, and whereby lipid 2 (lipid prodrug) comprises a lipid prodrug of the present disclosure or a lipid prodrug selected from the group consisting of ID3, AR5, TR3, an ID1 inhibitor (e.g., ID3-STEA, ID3-CHEM, AR5-STEA, TR3-STEA, ID1-CHOL, etc.), MPLA, and Telratolimod.

[0262] Those skilled in the art will understand the disclosed embodiments and will enable those skilled in the art to make changes and modifications to the disclosed embodiments without changing the functions and purposes of the present invention disclosed herein. Such changes and modifications are intended to be within the scope of the present disclosure.

[0263] Additionally, the scope of this disclosure teaches three (3) possible therapeutic modes using the formulated prodrugs of the present invention. See PCT Patent Publication No. WO2018 / 213631.

[0264] The first treatment modality involves combining a TB prodrug with another therapeutic agent (e.g., another formulated prodrug that inhibits ALK5 and other family members, a chemotherapeutic agent (such as ICD-induced chemotherapy), etc.) into a single liposome that allows systemic (or local) biodistribution and drug delivery to the tumor site. The dual delivery approach achieves a synergistic enhancement of adaptive and innate immunity, thereby significantly improving animal survival. In certain embodiments, the nanocarrier comprises a vesicle (i.e., a lipid bilayer surrounding a fluid).

[0265] A second treatment modality involves local delivery to the tumor or peritumoral area of ​​an agent that inhibits ALK5 and other family members in combination with lipids (eg, liposomes) that include an ALK5 signaling inhibitor.

[0266] The third treatment modality involves vaccination with dead cancer cells (e.g., KPC cells) in which inhibition of ALK5 is induced ex vivo. This vaccination has been found to generate a systemic immune response that may interfere with tumor growth at distant sites and allow adoptive transfer to non-immune animals. One skilled in the art will understand and be able to perform the methods of the treatment modalities provided herein.

[0267] VII.) Liposomes

[0268] In one aspect, the presently disclosed subject matter is based on a method for providing TB prodrugs of the present disclosure (see the section entitled "Prodrugs") suitable for incorporation into nanocarriers comprising a lipid coating to provide enhanced delivery of the corresponding prodrug and to provide combination therapies comprising the prodrug. Advantages of using the prodrugs of the present invention include facilitating the formulation of controlled formulations into the disclosed LNPs (e.g., liposomes). This allows the prodrug to remain in an inactive form during systemic circulation, which allows the liposomes to release the active agent after being engulfed by cells, for example, within a tumor.

[0269] In certain embodiments, one or more TB prodrugs (e.g., any one or more of the TB prodrug inhibitors taught in Formula I, Formula II, Formula III, and / or TB4 prodrugs) (see the section entitled "Prodrugs") are formulated as lipid moieties that form vesicle (e.g., liposome) structures in aqueous solution or can form components of lipid bilayers comprising liposomes. The liposomes can be used directly or provided as a component in a combination formulation (e.g., in combination with another drug moiety or therapeutic modality disclosed herein).

[0270] In certain embodiments, liposomes formulated with TB prodrugs include lipids, PHGP, vitamin E, cholesterol, and / or fatty acids.

[0271] In one embodiment, the liposomes include cholesterol.

[0272] In one embodiment, the liposomes include DSPC.

[0273] In one embodiment, the liposomes comprise HSPCs.

[0274] In one embodiment, the liposomes include DSPE-PEG 2000 .

[0275] In one embodiment, the liposomes comprise DPPG.

[0276] In one embodiment, the liposomes comprise DMPG.

[0277] In one embodiment, the liposomes comprise Lyso PC.

[0278] In one embodiment, the liposomes comprise (Δ9-cis)PG.

[0279] In one embodiment, the liposomes comprise Soy Lyso PC.

[0280] In one embodiment, the liposomes comprise PG.

[0281] In one embodiment, the liposomes comprise PA-PEG3-mannose.

[0282] In one embodiment, the liposomes comprise C16 PEG2000 ceramide.

[0283] In one embodiment, the liposomes comprise MPLA.

[0284] In one embodiment, the liposomes comprise CHEMS.

[0285] In one embodiment, the liposomes comprise stearic acid.

[0286] In one embodiment, the liposomes comprise the phospholipids shown in Table III.

[0287] In one embodiment, the liposome comprises TB4, and further comprises CHEMS, and further comprises LU, wherein the LU is a hydroxymethylcarbamate linker.

[0288] In one embodiment, the liposome comprises TB4, and further comprises stearic acid, and further comprises LU, wherein the LU is a hydroxymethylcarbamate linker.

[0289] In one embodiment, the liposome comprises TB4, and further comprises CHEMS, and further comprises LU, wherein the LU is a hydroxymethylcarbamate linker, and further comprises a helper lipid as shown in Table II.

[0290] In one embodiment, the liposome comprises TB4, and further comprises stearic acid, and further comprises LU, wherein the LU is a hydroxymethylcarbamate linker, and further comprises a helper lipid as shown in Table II.

[0291] In one embodiment, the liposomes of the present disclosure include a TB prodrug co-formulated with one or more additional immunomodulatory agents, whereby the immunomodulatory agents include, but are not limited to, immunogenic cell death-inducing chemotherapeutic agents, toll receptor agonists, sting agonists, IDO inhibitors, CTLA4 inhibitors, PD-1 inhibitors, and / or prodrugs thereof.

[0292] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with an ICD-inducing chemotherapeutic agent.

[0293] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with an ICD-inducing chemotherapeutic agent selected from the following list: doxorubicin (DOX), mitoxantrone (MTO), oxaliplatin (OXA), cyclophosphamide (CP), bortezomib, carfilzimib, or paclitaxel.

[0294] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a Toll receptor TLR agonist / prodrug.

[0295] In further preferred embodiments, the Toll receptor TLR agonist / prodrug is selected from the group consisting of TR3, TR4, TR5 and TR6.

[0296] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a Toll receptor (TLR) agonist / prodrug selected from the following list: Resiquimod (R848), Gardiquimod, 852A, DSR 6434, Tiramid, CU-T12-9, monophosphoryl lipid A (MPLA), 3D (6-acyl)- SMU127, Pam3CSK4 or 3D- or a prodrug thereof.

[0297] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a PD-1 inhibitor / prodrug.

[0298] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a PD-1 inhibitor / prodrug selected from the following list: AUNP12, CA-170, or BMS-986189 or a prodrug thereof.

[0299] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with an IDO-1 inhibitor / prodrug.

[0300] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with an IDO-1 inhibitor / prodrug selected from the following list: epacadostat, L-1-methyltryptophan (Indoximod), D-1-methyltryptophan, Linrodostat mesylate (BMS 986205), MK-7162, LY-3381916, KHK-2455, HTI-1090, DN-1406131, or BGB-5777.

[0301] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with doxorubicin (DOX).

[0302] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with mitoxantrone (MTO).

[0303] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with doxorubicin (DOX) and a PD-1 prodrug.

[0304] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with mitoxantrone (MTO) and a PD-1 prodrug.

[0305] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with doxorubicin (DOX) and an IDO-1 prodrug.

[0306] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with mitoxantrone (MTO) and an IDO-1 prodrug.

[0307] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with doxorubicin (DOX) and a TLR agonist / prodrug.

[0308] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with mitoxantrone (MTO) and a TLR agonist / prodrug.

[0309] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with doxorubicin (DOX) and a PD-1 prodrug and a TLR agonist / prodrug.

[0310] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with mitoxantrone (MTO) and a PD-1 prodrug and a TLR agonist / prodrug.

[0311] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a TLR agonist / prodrug.

[0312] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with an IDO antagonist / prodrug.

[0313] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a CD1D agonist / prodrug.

[0314] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a TLR agonist / prodrug and a PD-1 prodrug.

[0315] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a TLR agonist / prodrug and an IDO-1 prodrug.

[0316] In a preferred embodiment, the liposomes include a TB4 prodrug co-formulated with doxorubicin (DOX).

[0317] In a preferred embodiment, the liposomes include a TB4 prodrug co-formulated with mitoxantrone (MTO).

[0318] In a preferred embodiment, the liposomes include a TB4 prodrug co-formulated with doxorubicin (DOX) and an IDO prodrug and / or a TLR agonist / prodrug.

[0319] In preferred embodiments, the liposomes include a TB4 prodrug co-formulated with mitoxantrone (MTO) and / or an IDO prodrug and / or a TLR agonist / prodrug.

[0320] Those skilled in the art will recognize and understand that solubility is one of the most common problems faced by technicians during drug development. Chemical conjugation of drugs / anticancer agents by lipid molecules (i.e., lipid-based prodrugs) provides a platform for solving the problem of formulating drugs in aqueous suspensions. The main advantage of using lipid conjugates (lipid-based prodrugs) to deliver drugs is their ability to improve pharmacokinetics / half-life and targeted delivery.

[0321] By appropriately selecting lipid molecules, lipid-based prodrugs can be integrated / deployed in liposome formulations using techniques known in the art, which has more advantages than conventional drug delivery systems. (KOHLI et al., J. Controlled Release, 0: pp. 274-287 (September 28, 2014); and Garcia-Pinel et al., Nanomaterials 9: 638 (2019)). Combining lipid prodrugs with liposomes has dual advantages: (i) liposomes containing lipid prodrugs not only increase the solubility of the drug / prodrug itself, but also (ii) have the ability to encapsulate multiple drugs (both hydrophilic and lipophilic drugs) (see section entitled "Nanocarriers").

[0322] For the purposes of this disclosure, the main advantages of liposomal formulations are as follows:

[0323] i) biocompatibility / biodegradability of the liposomal formulation and lack of general toxicity;

[0324] ii) flexibility and manipulation of size and surface charge depending on the desired purpose. For the purposes of this disclosure, liposome formulations may range in size from 40-150 nm in diameter and have a surface charge range of -40 to +40 mV; and

[0325] iii) The liposomes of the present invention have single or multiple lipid prodrugs as the constituent lipid portion of the liposomes. In addition, multiple drugs with different solubility profiles (hydrophilicity or lipophilicity) (e.g., acting with different mechanisms of action) can be formulated in these liposomes (in the lipid bilayer or in the hydrophilic core).

[0326] As will be understood by one of ordinary skill in the art, all methods of preparing liposomes involve four (4) basic stages:

[0327] (i) drying lipids from organic solvents;

[0328] (ii) dispersing the lipid in an aqueous solution;

[0329] (iii) purifying the obtained liposomes; and

[0330] (iv) Analyze the final product.

[0331] See AKBARZADEH et al., Nanoscale Research Letters, 8: 102 (2013).

[0332] Another aspect of the present invention discloses liposome encapsulation technology (LET), which is a delivery technology for transporting drugs. LET is a method of producing submicroscopic bubbles called liposomes that encapsulate a variety of materials. These "liposomes" form a barrier around their contents that resists enzymes in the mouth and stomach, alkaline solutions, digestive juices, bile salts, and intestinal flora produced in the human body, as well as free radicals. As a result, the contents of the liposomes are protected from oxidation and degradation. This protective phospholipid shield or barrier remains intact until the contents of the liposomes are delivered to the exact target gland, target organ, or target system where the contents will be used (see the section entitled "Nanocarriers").

[0333] In one embodiment, the liposomes of the present disclosure are synthesized using a variety of different ratios of TB prodrugs, lipids and / or lipid prodrugs. As disclosed herein, the TB prodrug may include a helper lipid as disclosed herein (see, e.g., Table II).

[0334] In one embodiment, the liposomes of the present disclosure are synthesized using a variety of different ratios of TB prodrug, lipid and / or lipid prodrug.As disclosed herein, the TB prodrug may further include DSPE-PEG.

[0335] In a preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios:

[0336] Liposome composition Amount(%w / w) Lipid 1 (lipid-prodrug) 5-60 Lipid 2 (lipid-prodrug) 0-40 Helper lipids 50-80 DSPE-PEG 2000 2-5

[0337] In another preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios:

[0338] Liposome composition Amount(%w / w) Lipid 1 (lipid-prodrug) 5-60 Helper lipids 50-80 DSPEG-PEG 2000 2-5

[0339] In another preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios:

[0340] Liposome composition Amount(%w / w) Lipid 1 (lipid-prodrug) 5-60 Helper lipids 50-80 DSPEG-PEG 2000 2-5

[0341] Lipid 1 thus comprises a TB4 prodrug wherein the lipid portion comprises CHEMS.

[0342] In another preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios:

[0343] Liposome composition Amount(%w / w) Lipid 1 (lipid-prodrug) 5-60 Helper lipids 50-80 DSPEG-PEG 2000 2-5

[0344] Lipid 1 thus comprises a TB4 prodrug wherein the lipid portion comprises stearic acid.

[0345] Those skilled in the art will understand the disclosed embodiments and will enable those skilled in the art to make changes and modifications to the disclosed embodiments without changing the functions and purposes of the present invention disclosed herein. Such changes and modifications are intended to be within the scope of the present disclosure.

[0346] VIII.) Pharmaceutical Formulations

[0347] As used herein, the term "drug" is synonymous with "pharmaceutical." In certain embodiments, the liposomes of the present disclosure are formulated into encapsulated dosage forms and administered to a patient to treat a disease.

[0348] Generally speaking, drug formulation is the process of combining different chemical substances into a pure drug product to produce the final drug product. Formulation studies involve developing a drug formulation that is both stable and well-accepted by patients. For oral medications, this typically involves incorporating the drug into a tablet or capsule. It's important to recognize that dosage forms contain a variety of other substances in addition to the drug itself, and studies must be performed to ensure compatibility with these other substances.

[0349] Excipients are inactive substances that serve as carriers for the active ingredient in a pharmaceutical product, in this case, liposomes containing a TB prodrug. Excipients can also aid in the manufacturing process of a pharmaceutical product. The active substance is then dissolved or mixed with the excipient. Excipients are also sometimes used to bulk up formulations containing very potent active ingredients to facilitate convenient and accurate dosing. Once an active ingredient is purified, it cannot remain purified over extended periods of time. In many cases, it denatures, falls out of solution, or sticks to the sides of the container.

[0350] To stabilize the active ingredient, excipients are added to ensure that the active ingredient remains active and stable for a sufficiently long period of time, thereby enabling the product to have a shelf life competitive with other products and be safe for the end user. Examples of excipients include, but are not limited to, antiadherents, binders, coatings, disintegrants, fillers, diluents, flavorings, pigments, lubricants, and preservatives. The final formulation, comprising the active ingredient and excipients, is then encapsulated in a pharmaceutical dosage form.

[0351] Pre-formulation involves characterizing the physical, chemical, and mechanical properties of the drug in order to select the other ingredients that should be used in the formulation. Formulation studies then consider factors such as stability, particle size, polymorphism, pH, and solubility, as all of these factors may affect bioavailability and, therefore, the activity of the drug. The drug must be combined with inactive additives in a way that ensures that the amount of drug present in each dosage unit (e.g., each vial) is consistent. The dose should have a uniform appearance.

[0352] These studies are unlikely to be completed at the start of clinical trials. This means that the simple formulations initially developed are used for Phase I clinical trials. These are typically composed of vials, manually filled capsules containing a small amount of drug and diluent. There is no need to prove the long-term stability of these formulations because they will be used (tested) within a few days. However, long-term stability is crucial in supply chain management because the final formulation may take months or years from packaging to the time it arrives in the hands of the patient. The so-called drug loading (i.e., the ratio of active drug to total dose content) must be considered. Low drug loading may cause homogeneity problems. If the bulk density of the compound is low, high drug loading may cause flow problems or require large capsules. By the time Phase III clinical trials are completed, the formulation of the drug should have been developed to a formulation close to the one that will eventually be used on the market.

[0353] At this stage, understanding stability is crucial, and conditions must be developed to ensure that the drug is stable in the formulation. If the drug proves to be unstable, it will invalidate the results from clinical trials because it is impossible to know what dose will actually be administered. Stability studies are conducted to test for any effects of temperature, humidity, oxidation, or photolysis (UV or visible light), and the formulation is analyzed to see if any degradation products are formed. It is also important to check for any unwanted interactions between the formulation and the container. If a plastic container is used, tests should be conducted to see if any ingredients adsorb to the plastic and if any plasticizers, lubricants, pigments, or stabilizers leach from the plastic into the formulation. Even the adhesive on the container label needs to be tested to ensure that it does not leach through the plastic container into the formulation. The way the drug is formulated can avoid some of the problems associated with oral administration. Drugs are typically taken orally in the form of tablets or capsules. The drug (active substance) itself needs to be soluble in aqueous solution at a controlled rate. Factors such as particle size and crystal form can significantly affect dissolution. Rapid dissolution is not always ideal. For example, a slow dissolution rate may prolong the duration of action or avoid initial high plasma levels.

[0354] In some embodiments, nanocarriers (e.g., liposomes comprising TB prodrugs) and / or liposomes comprising TB prodrugs and co-formulated with immunomodulators are administered alone or in admixture with a physiologically acceptable carrier (e.g., saline or phosphate buffered saline) selected according to the route of administration and standard pharmaceutical practice. For example, when used as an injection, the nanocarrier can be formulated into a sterile suspension, dispersion, or emulsion together with a pharmaceutically acceptable carrier. In certain embodiments, saline can be used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, 5% glucose, etc., including glycoproteins for enhancing stability, such as albumin, lipoproteins, globulins, etc. In compositions comprising saline or other saline-containing carriers, the carrier is preferably added after the nanocarrier is formed. Therefore, after the nanocarrier is formed and loaded with the appropriate drug, the nanocarrier can be diluted into a pharmaceutically acceptable carrier, such as saline. Similarly, TB prodrug liposomes can be introduced into a vehicle (eg, emulsion, diluent, etc.) that facilitates suspension of the nanomaterial.

[0355] The pharmaceutical composition can be sterilized by conventional known sterilization techniques. The resulting aqueous solution, suspension, dispersion, emulsion, etc. can be packaged for use or filtered under aseptic conditions. In certain embodiments, the drug delivery nanocarrier (for example, nanoparticles coated with LB) is lyophilized, and the lyophilized preparation is combined with a sterile aqueous solution before administration. The pharmaceutical composition can also contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH adjusting agents and buffers, tension regulators, etc., for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0356] In addition, in certain embodiments, the pharmaceutical formulation can include a lipid protective agent that protects lipids from free radicals and lipid peroxidation damage during storage. Lipophilic free radical quenchers, such as α-tocopherol and water-soluble iron-specific chelating agents, such as ferric oxamine are suitable and contemplated herein. The concentration of the nanocarrier (e.g., liposomes comprising TB prodrugs) in the pharmaceutical formulation can vary greatly, for example, from less than about 0.05 weight %, typically at least about 2-5 weight % to as high as 10 weight % to 50 weight %, or to 40 weight %, or to 30 weight %, and is selected primarily by fluid volume, viscosity, etc. according to the selected specific mode of administration. For example, the concentration can be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients suffering from congestive heart failure or severe hypertension associated with atherosclerosis. Alternatively, the nanocarrier consisting of irritating lipids can be diluted to a low concentration to alleviate inflammation at the application site. The amount of nanocarrier administered will depend on the specific drug used, the disease state being treated, and the judgment of the clinician, but will generally be between about 0.01 and about 50 mg per kilogram body weight, preferably between about 0.1 and about 5 mg per kilogram body weight.

[0357] Those skilled in the art will appreciate that the exact dosage will vary depending on such factors as the particular TB prodrug and any co-formulated immunomodulator and the desired medical effect, as well as patient factors such as age, sex, general condition, etc. Those skilled in the art can readily take these factors into account and use them to establish effective therapeutic concentrations without undue experimentation.

[0358] In order to be administered to a human (or non-human mammal) in the curative, palliative, delayed or preventive treatment of the diseases described herein, the prescribing physician will ultimately determine the appropriate drug dosage for a given human (or non-human) subject, and this can be expected to vary according to the age, weight and response of the individual and the nature and severity of the patient's disease. In certain embodiments, the dose of the drug provided by the nanocarrier can be approximately equal to the dose of the free drug employed. However, as described above, the nanocarriers described herein can significantly reduce the toxicity of the drug administered thereby and significantly increase the therapeutic window. Therefore, in some cases, a prescribed dose exceeding the free drug will be used.

[0359] Those skilled in the art will understand the disclosed embodiments and will enable those skilled in the art to make changes and modifications to the disclosed embodiments without changing the functions and purposes of the present invention disclosed herein. Such changes and modifications are intended to be within the scope of the present disclosure.

[0360] IX.) Combination therapy

[0361] As will be recognized and appreciated by those skilled in the art, the growth and survival of cancer cells may be affected by a variety of signaling pathways. Therefore, it is useful to combine different enzyme / protein / receptor inhibitors that exhibit different preferences for the targets of their modulatory activity to treat such conditions. Targeting more than one signaling pathway (or more than one biomolecule involved in a given signaling pathway) can reduce the likelihood of drug resistance in a cell population and / or reduce the toxicity of the treatment.

[0362] Thus, liposomes comprising the TB prodrugs of the present disclosure can be used in combination with one or more other enzyme / protein / receptor inhibitors or one or more therapies for treating diseases, such as cancer or infection. Examples of diseases and indications that can be treated with combination therapies include those disclosed herein. Examples of cancers include, but are not limited to, solid tumors and liquid tumors, such as blood cancers. Examples of infections include viral, bacterial, fungal, or parasitic infections.

[0363] For example, liposomes comprising the disclosed TB prodrugs can be combined with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, PI3K (α, β, γ, δ), CSFIR, KIT, FLK-II, KDR / F LK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, TAM kinase (Axl, Mer, Tyro3), FLT3, VE GFR / Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf.

[0364] In further embodiments, liposomes comprising the TB prodrugs of the present disclosure can be combined with one or more of the following inhibitors for the treatment of cancer or infection. Non-limiting examples of inhibitors that can be combined with the compounds of the present disclosure for the treatment of cancer and infection include FGFR inhibitors (FGFR1, FGFR2, FGFR3, or FGFR4, e.g., INCB54828, INCB62079, and INCB63904), JAK inhibitors (JAK1 and / or JAK2, e.g., ruxolitinib, baricitinib, or INCB39110), IDO inhibitors (e.g., epacadostat, NLG919, or BMS-986205), LSD1 inhibitors (e.g., INCB59872 and INCB60003), TDO inhibitors, PI3K-δ inhibitors (e.g., INCB50797 and INCB50465), PI3K-γ inhibitors, such as PI3K-γ selective inhibitors, Pim inhibitors (e.g., INCB53914), CSF1R inhibitors, TAM receptor tyrosine kinases (Tyro-3, Axl and Mer), adenosine receptor antagonists (e.g., A2a / A2b receptor antagonists), HPK1 inhibitors, histone deacetylase inhibitors (HDAC), such as HDAC8 inhibitors, angiogenesis inhibitors, interleukin receptor inhibitors, bromodomain and extra-terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors, such as INCB54329 and INCB57643), poly ADP ribose polymerase (PARP) inhibitors, such as rucaparib, olaparib, niraparib, veliparib or talazoparib, arginase inhibitors (INCB01158), PD-1 inhibitors, PD-1 / L-1 inhibitors, PD-1 / L-2 inhibitors, CTLA-4 antagonists and adenosine receptor antagonists, or a combination thereof.

[0365] Additionally, liposomes comprising the disclosed TB prodrugs can be further used in combination with other methods of treating cancer, such as by chemotherapy, radiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, or surgery.

[0366] Examples of immunotherapy include cytokine therapy (e.g., interferon, GM-CSF, g-CSF, and IL-2), CRS-207 immunotherapy, cancer vaccines, monoclonal antibodies, adoptive T cell transfer, Toll receptor agonists, STING agonists, oncolytic virus therapy, and immunomodulatory small molecules, including thalidomide or JAK1 / 2 inhibitors.

[0367] The liposomes comprising the TB prodrug can be administered in combination with one or more anticancer drugs, such as chemotherapeutic agents. Exemplary chemotherapeutic agents include any of the following: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezomib, bortezomib, busulfan intravenous, busulfan oral, orally administered. oral), calusterone, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate propionate, eculizumab, epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citratecitrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine hydrochloride, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenylpropionate phenpropionate), nelarabine, nofetumomab, olaparib, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodiumdisodium, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, ruxolitinib, rucaparib, sorafenib, streptozocin, sunitinib, sunitinib maleate maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, niraparib, veliparib, talazoparib, and zoledronate.

[0368] Other anti-cancer agents include antibody therapeutics, such as trastuzumab (Herceptin), antibodies to costimulatory molecules, such as CTLA-4 (e.g., ipilimumab), 4-1BB (e.g., urelumab, utomilumab), antibodies to PD-1 and PD-L1 / L2, or cytokine antibodies (IL-10, TGF-β, etc.).

[0369] Examples of antibodies against PD-1 and / or PD-L1 / L2 that can be combined with the compounds of the present disclosure for the treatment of cancer or infections, such as viral, bacterial, fungal, and parasitic infections, include, but are not limited to, nivolumab, pembrolizumab, MPDL3280A, MEDI-4736, and SHR-1210.

[0370] In addition, liposomes including TB prodrugs of the present disclosure can be combined with one or more immune checkpoint inhibitors for the treatment of diseases such as cancer or infection. Exemplary immune checkpoint inhibitors include inhibitors for immune checkpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, PD-1, PD-L1 and PD-L2.

[0371] In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from the following: CD27, CD28, CD40, ICOS, OX40, GITR and CD137. In other embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from the following: A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3 and VISTA. In other embodiments, the liposomes comprising the TB prodrug provided herein can be used in combination with one or more agents selected from the following: KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors and TGFβ inhibitors.

[0372] X.) Methods for delivering nanocarriers comprising TB prodrugs to cells expressing ALK5

[0373] As is known in the art, various compositions and methods for killing tumor cells using prodrugs and / or nanocarriers are known in the art. In the context of cancer, a typical method entails administering a biologically effective amount of a TB prodrug of the present disclosure and / or a nanocarrier comprising a TB prodrug of the present disclosure to a mammal having a tumor.

[0374] An exemplary embodiment is a method of delivering a therapeutic agent to a cell expressing ALK5, the method comprising conjugating a drug moiety of the present disclosure to a lipid of the present disclosure via a linker unit to form a TB prodrug, and exposing the cell to the TB prodrug.

[0375] In one embodiment, the TB prodrug comprises a drug moiety of Formula I and CHEMS conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0376] In one embodiment, the TB prodrug comprises a drug moiety of Formula I and stearic acid conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0377] In one embodiment, the TB prodrug comprises a drug moiety of Formula II and CHEMS conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0378] In one embodiment, the TB prodrug comprises a drug moiety of Formula II and stearic acid conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0379] In one embodiment, the TB prodrug comprises a drug moiety of Formula III and CHEMS conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0380] In one embodiment, the TB prodrug comprises a drug moiety of Formula III and stearic acid conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0381] In one embodiment, the TB prodrug comprises a TB4 prodrug wherein the lipid moiety comprises CHEMS conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0382] In one embodiment, the TB prodrug comprises a TB4 prodrug wherein the lipid moiety comprises stearic acid conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0383] Another illustrative embodiment is a method of treating an individual suspected of having metastatic cancer, comprising the steps of parenterally administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of a TB prodrug produced by conjugating a drug moiety to a lipid of the present disclosure via a linker unit, and exposing cells to the TB prodrug.

[0384] In one embodiment, the TB prodrug comprises a drug moiety of Formula I and CHEMS conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0385] In one embodiment, the TB prodrug comprises a drug moiety of Formula I and stearic acid conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0386] In one embodiment, the TB prodrug comprises a drug moiety of Formula II and CHEMS conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0387] In one embodiment, the TB prodrug comprises a drug moiety of Formula II and stearic acid conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0388] In one embodiment, the TB prodrug comprises a drug moiety of Formula III and CHEMS conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0389] In one embodiment, the TB prodrug comprises a drug moiety of Formula III and stearic acid conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0390] In one embodiment, the TB prodrug comprises a TB4 prodrug wherein the lipid moiety comprises CHEMS conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0391] In one embodiment, the TB prodrug comprises a TB4 prodrug wherein the lipid moiety comprises stearic acid conjugated via a LU comprising a hydroxymethylcarbamate linker.

[0392] The TB prodrugs, liposomes, and co-formulated liposomes of the present disclosure inhibit the activity of TGFβ protein / protein interactions and are therefore useful in treating diseases and conditions associated with TGFβ activity and diseases and conditions associated with kinase inhibition. In further embodiments of the present disclosure, the TB prodrugs, liposomes, or pharmaceutically acceptable salts or stereoisomers thereof, may be used for therapeutic administration to enhance, stimulate, and / or increase immunity in cancer, chronic infection, or sepsis, including enhancing responses to vaccination.

[0393] In another embodiment, the present disclosure provides a method for inhibiting ALK5 T cell function. The method comprises administering to an individual or patient a TB prodrug, liposome, SLNP and / or any formula as described herein (e.g., TB4 and / or TB4 prodrug), or a TB prodrug, liposome, SLNP and nanoencapsulated ALK5 inhibitor prodrug according to any of the technical solutions and described herein, or a pharmaceutically acceptable salt or stereoisomer thereof. The TB prodrug, liposome, SLNP and nanoencapsulated ALK5 inhibitor prodrug disclosed herein can be used alone, in combination with other agents or therapies, or as an adjuvant or new adjuvant for the treatment of a disease or condition, including cancer and other diseases. For the uses and methods described herein, any of the TB prodrugs, liposomes and nanoencapsulated TB prodrugs disclosed herein, including any embodiment thereof, can be used.

[0394] Additionally, the TB prodrugs, liposomes, SLNPs and nanoencapsulated TB prodrugs of the present disclosure inhibit ALK5 and / or T cell function, leading to TGFβ pathway blockade.

[0395] In further embodiments, the present disclosure provides in vivo treatment to an individual or patient using TB prodrugs, liposomes and nanoencapsulated TB prodrugs or salts or stereoisomers thereof, such that the growth of cancerous tumors is inhibited.

[0396] TB prodrugs, liposomes and nanoencapsulated TB prodrugs, or any formula as described herein (e.g., TB4 prodrug), or TB prodrugs, liposomes, SLNPs and nanoencapsulated TB prodrugs according to any of the technical solutions and described herein, or salts or stereoisomers thereof can be used to inhibit the growth of cancerous tumors.

[0397] Alternatively, the TB prodrugs, liposomes, SLNPs and nanoencapsulated TB prodrugs of the present disclosure, or any formula as described herein, or compounds described according to any of the technical embodiments and described herein (e.g., TB4 prodrugs), or salts or stereoisomers thereof can be used in combination with other agents or standard cancer treatments as described in the present disclosure.

[0398] In another embodiment, the present disclosure provides a method for inhibiting tumor cell growth in vitro, comprising contacting tumor cells in vitro with a TB prodrug, liposome, or nanoencapsulated TB prodrug of the present disclosure, or any formula as described herein (e.g., TB4 prodrug), or a TB prodrug, liposome, SLNP, or nanoencapsulated TB prodrug according to any of the technical solutions and described herein, or a salt or stereoisomer thereof.

[0399] In another embodiment, the present disclosure provides a method for inhibiting tumor cell growth in a patient, comprising contacting the tumor cells with a TB prodrug, liposome, or nanoencapsulated TB prodrug of the present disclosure, or any formula as described herein (e.g., TB4 prodrug), or a TB prodrug, liposome, SLNP, or nanoencapsulated TB prodrug according to any of the technical solutions and described herein, or a salt or stereoisomer thereof.

[0400] XI.) Methods of treating cancer and other immune disorders

[0401] Another embodiment of the present disclosure is a method for treating cancer. The method comprises administering to a patient a therapeutically effective amount of liposomes comprising a TB prodrug herein (i.e., a TB4 prodrug), a compound described in any of the technical solutions and described herein, or a salt thereof. Examples of cancers include those cancers whose growth can be inhibited using the ALK5 inhibitors of the present disclosure and the TB prodrugs of the present disclosure, as well as cancers that are generally responsive to immunotherapy.

[0402] In some embodiments, the present disclosure provides a method for enhancing, stimulating, and / or increasing an immune response in a patient, comprising administering to the patient a therapeutically effective amount of a TB prodrug and / or a nanocarrier comprising the prodrug (i.e., a TB4 prodrug), a compound or composition according to any of the technical solutions and described herein, or a salt thereof.

[0403] In one embodiment, the method comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0404] In further embodiments, the method comprises administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0405] Non-limiting examples of cancers that can be treated using the liposomes, TB prodrugs, and co-formulated liposomes comprising the present disclosure include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, carcinoma of the endometrium, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, and ovarian cancer. The compounds of the present invention are also useful for treating metastatic cancers, particularly those expressing ALK5.

[0406] In some embodiments, cancers that can be treated with the liposomes or TB prodrugs of the present disclosure include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), squamous cell head and neck cancer, urothelial carcinoma (e.g., bladder cancer), and microsatellite instability-high (MSI) cancer. 高 ). Additionally, the present disclosure encompasses refractory or recurrent malignancies whose growth can be inhibited using the liposomes or TB prodrugs or co-formulated liposomes of the present disclosure.

[0407] In further embodiments, cancers that can be treated using the formulated and / or co-formulated liposomes or TB prodrugs of the present disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., lymphomas, leukemias such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin lymphoma, or multiple myeloma), and combinations of such cancers.

[0408] In further embodiments, cancers that can be treated using the formulated and / or co-formulated liposomes or TB prodrugs of the present disclosure include, but are not limited to, cholangiocarcinoma, bile duct cancer, triple negative breast cancer, rhabdomyosarcoma, small cell lung cancer, leiomyosarcoma, hepatocellular carcinoma, Ewing's sarcoma, brain cancer, brain tumor, astrocytoma, neuroblastoma, neurofibroma, basal cell carcinoma, chondrosarcoma, epithelioid sarcoma, eye cancer, fallopian tube cancer, gastrointestinal cancer, gastrointestinal stromal tumor, hairy cell leukemia, intestinal cancer, islet cell carcinoma, oral cancer, mouth cancer, throat cancer, cancer), laryngeal cancer, lip cancer, mesothelioma, neck cancer, nasal cavity cancer, eye cancer, ocular melanoma, pelvic cancer, rectal cancer, renal cell carcinoma, salivary gland cancer, paranasal sinus cancer, spinal cancer, tongue cancer, tubule cancer, urethra cancer, and ureter cancer.

[0409] Additionally, in some embodiments, the formulated and / or co-formulated liposomes or TB prodrugs of the present disclosure may be used to treat sickle cell disease and sickle cell anemia.

[0410] Furthermore, in some embodiments, diseases and indications that can be treated using the formulated and / or co-formulated liposomes or TB prodrugs of the present disclosure include, but are not limited to, hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.

[0411] Exemplary hematological cancers include lymphomas and leukemias, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, non-Hodgkin lymphoma (including relapsed or refractory NHL and relapsed follicular NHL), Hodgkin lymphoma, myeloproliferative disorders (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocythemia (ET)), myelodysplastic syndrome (MDS), T-cell acute lymphoblastic lymphoma (T-ALL), and multiple myeloma (MM).

[0412] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdomyosarcoma, fibroma, lipoma, hematoma, and teratoma.

[0413] Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, enchondromatous hamartoma, and mesothelioma.

[0414] Exemplary gastrointestinal cancers include esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastric cancer (carcinoma, lymphoma, leiomyosarcoma), pancreatic cancer (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid, vasodilatory peptide tumor), small intestine cancer (adenocarcinoma, lymphoma, carcinoid, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), colorectal cancer (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), and colorectal cancer.

[0415] Exemplary genitourinary tract cancers include renal cancer (adenocarcinoma, Wilm's tumor [Nephroblastoma]), bladder and urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), and testicular cancer (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatoid tumor, lipoma).

[0416] Exemplary liver cancers include liver cancer (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0417] Exemplary bone cancers include, e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell chordoma, osteochondroma (osteocartilaginous exostosis), benign enchondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, and giant cell tumor.

[0418] Exemplary nervous system cancers include cancers of the skull (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meningeal cancers (meningiomas, meningiosarcomas, gliosis), brain cancers (astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors (pinealomas), glioblastomas, glioblastomas multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), and spinal cord cancers (neurofibromas, meningiomas, gliomas, sarcomas), as well as neuroblastoma and Lhermitte-Duclos disease.

[0419] Exemplary gynecological cancers include uterine cancer (endometrial cancer), cervical cancer (cervical cancer, preneoplastic cervical atypical hyperplasia), ovarian cancer (ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulvar cancer (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), and fallopian tube (carcinoma).

[0420] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, nevus dysplasia, lipoma, hemangioma, dermatofibroma, and nevus dysplasia. In some embodiments, diseases and indications that can be treated using the compounds of the present disclosure include, but are not limited to, sickle cell disease (e.g., sickle cell anemia), triple-negative breast cancer (TNBC), myelodysplastic syndrome, testicular cancer, bile duct cancer, esophageal cancer, and urothelial cancer.

[0421] Additionally, blocking TGFβ, ALK5 and / or kinase pathways using formulated and / or co-formulated liposomes or TB prodrugs of the present disclosure can also be used to treat infections, such as viral, bacterial, fungal and parasitic infections.

[0422] The present disclosure provides a method for treating an infection such as a viral infection. The method comprises administering to a patient a therapeutically effective amount of formulated and / or co-formulated liposomes or TB prodrugs or any formula as described herein according to any of the technical solutions and described herein (i.e., TB4 prodrugs), or salts thereof.

[0423] In one embodiment, the method comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0424] In further embodiments, the method comprises administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0425] Examples of viruses that can be treated by the methods of the present invention include, but are not limited to, human immunodeficiency virus, human papillomavirus, influenza, hepatitis A, B, C, or D, adenovirus, poxvirus, herpes simplex virus, human cytomegalovirus, severe acute respiratory syndrome virus, Ebola virus, and measles virus. In some embodiments, viruses that can be treated by the methods of the present invention include, but are not limited to, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, human herpes virus type 4), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0426] In addition, the present disclosure provides a method for treating a bacterial infection, comprising administering to a patient a therapeutically effective amount of formulated and / or co-formulated liposomes or a TB prodrug, or any formula as described herein according to any of the technical solutions and described herein (i.e., a TB4 prodrug), or a salt thereof.

[0427] Examples of pathogenic bacteria that cause infections that can be treated by the methods of the present disclosure include, but are not limited to, Chlamydia, Rickettsial bacteria, Mycobacteria, Staphylococci, Streptococci, Pneumococci, Meningococci and Gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, and Lyme's disease bacteria.

[0428] In addition, the present disclosure provides a method for treating fungal infection, comprising administering to a patient a therapeutically effective amount of formulated and / or co-formulated liposomes or TB prodrugs, or any formula as described herein according to any of the technical solutions and described herein (i.e., TB4 prodrugs), or salts thereof.

[0429] In one embodiment, the method comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0430] In further embodiments, the method comprises administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0431] Examples of pathogenic fungi that cause infections that can be treated by the methods of the present disclosure include, but are not limited to, Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Mucor, Abreus, such as Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0432] In addition, the present disclosure provides a method for treating parasitic infections, comprising administering to a patient a therapeutically effective amount of formulated and / or co-formulated liposomes or TB prodrugs, or any formula as described herein according to any of the technical solutions and described herein (i.e., TB4 prodrugs), or salts thereof.

[0433] In one embodiment, the method comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0434] In further embodiments, the method comprises administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0435] Examples of pathogenic parasites that cause infections that can be treated by the methods of the present disclosure include, but are not limited to, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia Zambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0436] In another set of embodiments within the scope of the present disclosure, the formulated and / or co-formulated nanocarriers, liposomes, SLNPs or TB prodrugs, or any formula as described herein (i.e., TB4 prodrugs) can be used to prevent or reduce the risk of developing any of the diseases mentioned in the present disclosure; for example, to prevent or reduce the risk of developing a disease, condition or disorder in an individual who may be susceptible to the disease, condition or disorder but has not yet experienced or displayed the pathology or symptomology of the disease.

[0437] In one embodiment, the method comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0438] In further embodiments, the method comprises administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0439] XII.) Kits / Products

[0440] For use in laboratory, prognosis, prevention, diagnosis and treatment applications described herein, kits are within the scope of the present invention. Such kits may include carriers, packaging or containers that are separated to accommodate one or more containers such as bottles, test tubes, etc., each of which includes a separate element for the separate elements in the method and a label or insert including instructions for use as described herein. For example, the container may include a nanocarrier that is formulated and / or co-formulated, and the nanocarrier is or can be detectably labeled and / or loaded with a TB prodrug of the present disclosure. The kit may include a container that contains drug units. The kit may include all or part of the nanocarriers and / or TB prodrugs formulated and / or co-formulated.

[0441] The kits of the present invention will generally include the container described above and one or more other containers associated therewith, said one or more other containers containing materials desirable from a commercial and user standpoint, including: buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or test tube labels listing the contents and / or instructions for use; and package inserts with instructions for use.

[0442] The label can be presented on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application such as prognosis, prevention, diagnosis or laboratory application and can also indicate in vivo or in vitro use instructions, such as the purposes described herein. Instructions and or other information can also be included on one or more inserts or one or more labels included with the kit or on the kit. The label can be on or associated with the container. When the letters, numbers or other characters forming the label are molded or etched into the container itself, the label can be on the container; when the label is present in a receiver or carrier that also holds the container, for example as a package insert, the label can be associated with the container. The label can indicate that the composition is used for diagnosis, treatment, prevention or prediction of a condition, such as cancer or other immune disorders.

[0443] The terms "kit" and "article of manufacture" may be used synonymously.

[0444] In another embodiment of the present invention, an article containing a composition, such as a formulated and / or co-formulated nanocarrier and / or TB prodrug, is within the scope of the present disclosure. The article generally comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed from a variety of materials such as glass, metal, or plastic. The container can hold the formulated and / or co-formulated nanocarrier loaded with the TB prodrug.

[0445] The container may alternatively contain a composition effective for treating, diagnosing, prognosing and preventing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle). The active agent in the composition may be formulated and / or co-formulated into a nanocarrier loaded with a TB prodrug and / or a TB prodrug as disclosed herein.

[0446] The article of manufacture may further include a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes, and / or package inserts with instructions for use and / or instructions for use.

[0447] In one embodiment, the kit or article of manufacture includes LNP-TB4 and / or a therapeutically effective amount of LNP-TB4.

[0448] In one embodiment, the kit or article of manufacture includes SLNP-TB4 and / or a therapeutically effective amount of SLNP-TB4.

[0449] Exemplary Embodiments

[0450] Examples provided include:

[0451] 1) A TB prodrug composition comprising:

[0452] (i) drug component;

[0453] (ii) a lipid portion; and

[0454] (iii) Linking Unit ("LU"),

[0455] The drug moiety thereby comprises a TGF[beta] antagonist, and the LU thereby conjugates the drug moiety to the lipid moiety.

[0456] 2) The TB prodrug according to technical solution 1, further comprising a chemical structure as shown in Formula I.

[0457] 3) The TB prodrug according to technical solution 1 further comprises a chemical structure as shown in Formula II.

[0458] 4) The TB prodrug according to technical solution 1, further comprising a chemical structure as shown in Formula III.

[0459] 5) The TB prodrug according to technical solution 1, wherein the drug moiety comprises the chemical structure shown in TB4.

[0460] 6) The TB prodrug according to technical solution 1, wherein the LU is a hydroxymethylcarbamate linker.

[0461] 7) The TB prodrug according to technical solution 1, wherein the lipid portion comprises a lipid as shown in Table 1.

[0462] 8) The TB prodrug according to technical solution 1, wherein the lipid portion comprises a lipid as shown in Table III.

[0463] 9) The TB prodrug according to technical solution 1, wherein the lipid portion comprises CHEMS.

[0464] 10) The TB prodrug according to technical solution 1, wherein the lipid portion comprises stearic acid.

[0465] 11) The TB prodrug according to technical solution 1, wherein the drug portion comprises the chemical structure shown in TB4, and wherein the lipid portion comprises stearic acid, and wherein the compound has the following chemical structure:

[0466]

[0467] 12) A TB prodrug composition comprising:

[0468] (i) a drug moiety, whereby the drug moiety comprises TB4;

[0469] (ii) a lipid moiety, whereby the lipid moiety comprises CHEMS; and

[0470] (iii) LU, whereby the LU comprises a hydroxymethylcarbamate linker.

[0471] 13) A TB prodrug composition comprising:

[0472] (i) a drug moiety, whereby the drug moiety comprises TB4;

[0473] (ii) a lipid fraction, whereby the lipid fraction comprises stearic acid; and

[0474] (iii) LU, whereby the LU comprises a hydroxymethylcarbamate linker.

[0475] 14) The TB prodrug composition according to technical solution 13 has the following chemical structure:

[0476]

[0477] 15) A nanocarrier comprising a TB prodrug, whereby the nanocarrier releases an active ALK5 inhibitor upon cleavage of LU.

[0478] 16) The nanocarrier according to technical solution 15, wherein the LU is a hydroxymethyl carbamate linker.

[0479] 17) The nanocarrier according to technical solution 15 further comprises an auxiliary lipid, wherein the auxiliary lipid is shown in Table II.

[0480] 18) A nanocarrier according to technical solution 15, wherein the TB prodrug includes TB4.

[0481] 19) The nanocarrier according to technical solution 15, wherein the nanocarrier is a liposome.

[0482] 20) The liposome according to technical solution 19, wherein the TB prodrug includes TB4 and is expressed as LNP-TB4.

[0483] 21) The liposome according to technical solution 19, wherein the liposome is further co-formulated with one or more immunomodulators or lipid prodrugs thereof, wherein the immunomodulator is selected from the group consisting of: immunogenic cell death-inducing chemotherapeutics, toll receptor agonists, STING agonists, CTLA-4 inhibitors, IDO inhibitors, PD-1 / PD-L1 inhibitors, CD1D agonists and / or their prodrugs.

[0484] 22) The liposome according to technical solution 19, wherein the liposome is further co-formulated with an ICD-inducing chemotherapy agent, wherein the ICD-inducing chemotherapy agent is selected from the group consisting of: DOX, MTO, OXA, CP, bortezomib, carfilzomib or paclitaxel.

[0485] 23) The liposome according to technical solution 19 further comprises DOX.

[0486] 24) The liposome according to technical solution 19 further comprises MTO.

[0487] 25) The liposome according to technical solution 22 further comprises DOX.

[0488] 26) The liposome according to technical solution 22 further comprises MTO.

[0489] 27) The liposome according to technical solution 19, wherein the liposome is further co-formulated with a toll receptor agonist or a lipid prodrug thereof, wherein the toll receptor agonist is selected from the group consisting of: resiquimod (R848), gademod, 852A, DSR 6434, tiramod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D (6-acyl)- SMU127, Pam3CSK4 or 3D-

[0490] 28) The liposome according to technical solution 19, wherein the liposome is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid prodrug thereof, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of: AUNP12, CA-170 or BMS-986189.

[0491] 29) A kit comprising the liposome according to any one of technical solutions 15 to 28.

[0492] 30) The nanocarrier according to technical solution 15, wherein the nanocarrier is solid lipid nanoparticles (SLNP).

[0493] 31) The SLNP according to technical solution 30, wherein the TB prodrug includes TB4 and is represented as SLNP-TB4.

[0494] 32) The SLNP according to technical solution 30, wherein the SLNP is further co-formulated with one or more immunomodulators or lipid prodrugs thereof, wherein the immunomodulator is selected from the group consisting of: immunogenic cell death-inducing chemotherapeutics, toll receptor agonists, STING agonists, CTLA-4 inhibitors, IDO inhibitors, PD-1 / PD-L1 inhibitors, CD1D agonists and / or their prodrugs.

[0495] 33) The SLNP according to technical solution 30, wherein the SLNP is further co-formulated with an ICD-inducing chemotherapy agent, wherein the ICD-inducing chemotherapy agent is selected from the group consisting of: DOX, MTO, OXA, CP, bortezomib, carfilzomib or paclitaxel.

[0496] 34) The SLNP according to technical solution 30 further includes DOX.

[0497] 35) The SLNP according to technical solution 30 further includes MTO.

[0498] 36) The SLNP according to technical solution 33 further includes DOX.

[0499] 37) The SLNP according to technical solution 33 further includes MTO.

[0500] 38) The SLNP according to technical solution 30, wherein the liposome is further co-formulated with a toll receptor agonist or a lipid prodrug thereof, wherein the toll receptor agonist is selected from the group consisting of: resiquimod (R848), gademod, 852A, DSR 6434, tiramod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D (6-acyl)- SMU127, Pam3CSK4 or 3D-

[0501] 39) The SLNP according to technical solution 30, wherein the liposome is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid prodrug thereof, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of: AUNP12, CA-170 or BMS-986189.

[0502] 40) A kit comprising the SLNP according to any one of technical solutions 30 to 39.

[0503] 41) A method of treating a subject having or diagnosed with cancer, the method comprising:

[0504] (i) administering to a subject in need of such treatment an effective amount of a nanocarrier, wherein the nanocarrier comprises a TB prodrug; and

[0505] (ii) a pharmaceutically acceptable salt thereof.

[0506] 42) A method according to technical solution 41, wherein the TB prodrug includes a TB4 prodrug.

[0507] 43) A method according to technical solution 41, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an ICD-inducing chemotherapy agent.

[0508] 44) A method according to technical solution 41, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an immunomodulator.

[0509] 45) A method according to technical solution 41, wherein the nanocarrier is a liposome.

[0510] 46) The method according to technical solution 45, wherein the liposome is LNP-TB4.

[0511] 47) A method according to technical solution 41, wherein the nanocarrier is solid lipid nanoparticles.

[0512] 48) The method according to technical solution 47, wherein the liposome is SLNP-TB4.

[0513] 49) A method of treating a subject having or diagnosed with cancer, the method comprising:

[0514] (iii) administering to a subject in need of such treatment an effective amount of a nanocarrier, wherein the nanocarrier comprises a TB prodrug; and

[0515] (iv) a pharmaceutically acceptable salt thereof.

[0516] 50) A method according to technical solution 49, wherein the TB prodrug includes a TB4 prodrug.

[0517] 51) A method according to technical solution 49, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an ICD-inducing chemotherapy agent.

[0518] 52) A method according to technical solution 49, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an immunomodulator.

[0519] 53) A method according to technical solution 49, wherein the nanocarrier is solid lipid nanoparticles ("SLNPs").

[0520] 54) The method according to technical solution 53, wherein the SLNP is SLNP-TB4.

[0521] 55) The method according to technical solution 49, wherein the nanocarrier is a liposome.

[0522] 56) The method according to technical solution 55, wherein the liposome is LNP-TB4.

[0523] 57) A TB4 prodrug having the following chemical structure:

[0524]

[0525] 58) A liposome comprising the TB4 prodrug according to technical solution 57.

[0526] 59) A liposome comprising the TB4 prodrug according to technical solution 57, which further comprises an auxiliary lipid.

[0527] 60) A liposome according to technical solution 59, wherein the auxiliary lipid is shown in Table II.

[0528] 61) A solid lipid nanoparticle (SLNP), comprising the TB4 prodrug according to technical solution 57.

[0529] 62) A TB4 prodrug having the following chemical structure:

[0530]

[0531] 63) A liposome comprising the TB4 prodrug according to technical solution 62.

[0532] 64) A liposome comprising the TB4 prodrug according to technical solution 62, which further comprises an auxiliary lipid.

[0533] 65) A liposome according to technical solution 64, wherein the auxiliary lipid is shown in Table II.

[0534] 66) A liposome according to technical solution 62, which is represented by LNP-TB4.

[0535] 67) A solid lipid nanoparticle (SLNP), comprising the TB4 prodrug according to technical solution 62.

[0536] 68) The SLNP according to technical solution 67 is represented by SLNP-TB4.

[0537] 69) The liposome according to technical solution 63, which is co-formulated with AR5.

[0538] 70) The liposome according to technical solution 63, which is co-formulated with TR6.

[0539] 71) The liposome according to technical solution 63, which is co-formulated with ID3.

[0540] 72) The liposome according to technical solution 63, which is co-formulated with PD3.

[0541] 73) The liposome according to technical solution 63 is co-formulated with MTO.

[0542] 74) The liposome according to technical solution 63, which is co-formulated with MTO and ID3.

[0543] 75) The liposome according to technical solution 63, which is co-formulated with MTO and AR5.

[0544] 76) The SLNP according to technical solution 68 is co-formulated with MTO.

[0545] 77) The SLNP according to technical solution 68 is co-formulated with AR5.

[0546] 78) The SLNP according to technical solution 68 is co-modulated with ID3.

[0547] 79) The SLNP according to technical solution 68 is co-formulated with PD3.

[0548] 80) The SLNP according to technical solution 68 is co-formulated with MTO and ID3.

[0549] 81) The SLNP according to technical solution 68 is co-formulated with MTO and AR5.

[0550] Examples:

[0551] Various aspects of the present invention will be further described and illustrated by the following several examples, which are not intended to limit the scope of the present invention.

[0552] Example 1: Chemical synthesis of TB4 prodrugs including stearic acid.

[0553] The chemical synthesis of the TB4 prodrug comprising stearic acid was synthesized using the following scheme. First, compound (1) was treated with compound (2) and KHMDS to produce intermediate 3. Intermediate 3 was then treated sequentially with DMF dimethyl acetal and then treated with hydrazine hydrate to produce intermediate 4. Intermediate 4 was then treated with triphenylmethane chloride to produce intermediate 5. Next, intermediate 5 was treated with reagent (6) and triphenylphosphine palladium and then hydrolyzed with sodium hydroxide to produce intermediate 7. Intermediate 7 was then treated with reagent (8) and EDCI / HOBt to produce intermediate 9. Intermediate 9 was then treated with lithium hexamethyldisilazide and then treated with chloromethyl chloroformate (10) to produce intermediate 11. Finally, intermediate 11 in DMF was treated sequentially with stearic acid, then with silver carbonate, then with sodium iodide at 80°C, and then with methanol containing HCl to produce the final prodrug TB4 comprising stearic acid. (12). ( Figure 1 ). The synthesis shown in this example produces a TB4 prodrug with the following chemical structure:

[0554]

[0555] Example 2: Chemical synthesis of protecting group intermediates on the way to TB4 prodrugs.

[0556] In order to synthesize the protecting group intermediate, the following scheme was used. Briefly, at 15 ° C, to a solution of TB4 (13.0 g, 30.5 mmol, 1.00 equivalent) in DCM (1.50 L) was added Boc2O (8.00 g, 36.7 mmol, 8.42 mL, 1.20 equivalent) and DMAP (746 mg, 6.11 mmol, 0.20 equivalent). After addition, the reaction mixture was stirred at 30 ° C for 12 hours. TLC (dichloromethane: methanol = 10: 1) showed that TB4 (R f =0.2) was consumed and a major new spot was formed (Rf=0.5). LCMS confirmed that the expected mass was detected (RT=0.850 minutes). The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO2, dichloromethane: methanol = 50: 1-30: 1, R f =0.5) to obtain compound 2 (13.0 g, 24.7 mmol, yield 80.9%) as a white solid. Figure 2 shown.

[0557] Example 3: Chemical synthesis of protecting group intermediates on the way to TB4 prodrugs.

[0558] In another embodiment, other protecting group intermediates are synthesized in the following manner. Briefly, TFA (16.6 g, 145 mmol, 10.8 mL, 20.0 equivalents) was added to a solution of compound 3 (4.50 g, 7.28 mmol, 1.00 equivalents) in DCM (225 mL) at 25 ° C. After addition, the reaction mixture was stirred for another 4 hours at 25 ° C. LCMS confirmed that the reaction was complete and that the desired mass (RT=0.874 minutes) was detected. The reaction mixture was adjusted to pH=7-8 with saturated NaHCO solution and washed with DCM (150 mL). * 2) extraction. The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered and concentrated to give compound 4 (3.50 g, 6.76 mmol, 92.81% yield) as a yellow solid. Figure 3 shown.

[0559] Example 4: Chemical synthesis of TB4 prodrugs including stearic acid.

[0560] The chemical synthesis of TB4 prodrug including stearic acid was synthesized using the following scheme. First, at 15 ° C, to a solution of TB4 (13.0 g, 30.5 mmol, 1.00 equivalent) in DCM (1.50 L), Boc2O (8.00 g, 36.7 mmol, 8.42 mL, 1.20 equivalent) and DMAP (746 mg, 6.11 mmol, 0.20 equivalent) were added. After addition, the reaction mixture was stirred at 30 ° C for 12 hours. TLC (dichloromethane: methanol = 10: 1) showed that TB4 (R f =0.2) was consumed and a major new spot was formed (Rf=0.5). LCMS showed that the expected mass was detected (RT=0.850 minutes). The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (SiO2, dichloromethane: methanol = 50: 1-30: 1, R f=0.5) was purified to obtain compound 2 (13.0 g, 24.7 mmol, 80.9% yield) as a white solid. Then, LiHMDS (1 M, 37.6 mL, 2.20 equiv) was added to a solution of compound 2 (9.00 g, 17.1 mmol, 1.00 equiv) in DCM (1000 mL) at -70 ° C under N2 for 1 hour. DCM (50 mL) containing compound 2a (9.36 g, 72.5 mmol, 6.45 mL, 4.24 equiv) was added to the mixture at -70 ° C. The mixture was stirred at -70 ° C for 6 hours. The mixture was then stirred at 15 ° C for another 6 hours. LCMS showed that compound 2 (RT = 1.038 minutes) remained 18.4%, and 33.1% of the expected mass was detected (RT = 1.195 minutes). The mixture was poured into saturated NH4Cl solution (500 mL) and washed with DCM (200 mL). * 2) extraction. The combined organic layers were washed with brine (500 mL), dried over Na2SO4 and concentrated, and confirmed by LCMS. The crude product was purified by reversed-phase MPLC (ACN / H2O, TFA conditions), and after removing ACN, and using ethyl acetate (300 mL) * 3) aqueous phase was extracted. The combined organic layer was washed with brine (200mL), dried over Na2SO4, and concentrated under reduced pressure to obtain compound 3 (6.20g, 10.0mmol, 29.2% yield) as a yellow solid, which was confirmed by LCMS. Then, TFA (16.6g, 145mmol, 10.8mL, 20.0 equivalents) was added to a solution of compound 3 (4.50g, 7.28mmol, 1.00 equivalents) in DCM (225mL) at 25°C. After addition, the reaction mixture was stirred for another 4 hours at 25°C. LCMS showed that the reaction was complete and the desired mass was detected (RT=0.874 minutes). The reaction mixture was adjusted to pH=7-8 with saturated NaHCO3 solution and washed with DCM (150mL). *4 (3.50g, 6.76mmol, 92.81% yield) was added to the 4-nitro-1-oxo-2-nitropropane (2-nitro-1-oxo-2-nitropropane) mixture of 4-nitro-1-oxo-2-nitropropane (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) (2-nitro-1-oxo-2-nitropropane) (3-nitro-1-oxo-2-nitropropane) ( The crude product is merged with ET34822-17, to be purified by reverse MPLC (MeOH / H2O / TFA conditions), and then concentrated to obtain a crude product. The crude product is passed through column chromatography (SiO2, petroleum ether: ethyl acetate=1:1 to ethyl acetate: methanol=10:1), which is detected by TLC (ethyl acetate: methanol=10:1, R f =0.3). TB4 including stearic acid (1.3 g, 1.59 mmol, yield 21.2%, purity 93.9%) was obtained as a yellow gel. 1 H NMR, FNMR, LCMS and HPLC confirmed the obtained compounds and the synthesis process. Figure 7 The synthesis shown in this example produces a TB4 prodrug with the following chemical structure:

[0561]

[0562] Example 5: Synthesis and characterization of LNP-TB4 liposomes.

[0563] In another experiment, the liposome (being expressed as LNP-TB4) that comprises TB4 prodrug is synthetic in the following manner.In brief, in the first step, in ethanol (20mg / ml), prepare the lipid stock solution of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), CHOL and DSPE-PEG respectively.In acetonitrile (20mg / ml), prepare TB4 prodrug (TB4+ stearic acid) stock solution, because it is insoluble in ethanol.The lipid mixture of POPC, CHOL, TB4+ stearic acid and DSPE-PEG is mixed with the mol ratio of 51:29:16.5:3.5, and use ethanol dilution subsequently, to obtain the total lipid concentration of 10mg / ml.Use the heating block annex in the microfluidizer to heat this lipid mixture under 55-60 degrees centigrade. In the embodiment of the present invention, the microfluidic box of embodiment 1 is carried out filtration.Similarly, the aqueous phase that contains 1mM PBS buffer solution is also preheated under 55-60 degrees centigrade, then passes through microfluidic box with the flow velocity of 5:1 (aqueous phase: organic phase, lipid mixture).Use the dialysis membrane that is 12KDa (Sigma Aldrich company (Sigma Aldrich)) of the cut-off value size for DI water to remove solvent, continues at least 24 hours.Dialysis water is changed at least 5 times during the time period of 24 hours, to remove solvent to greatest extent.After removing solvent, use Amicon centrifugal filter unit (cut-off value size is 10KDa during 3000g) as required that LNP-TB4 is concentrated.

[0564] The characterization of LNP-TB4 liposomes was determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, two (2) ml of LNP-TB4 liposomes (liposome concentration 0.5-1 mg / ml) were placed in a 4-sided clear plastic cuvette and analyzed directly at 25°C. Figure 8 The results shown indicate that the nanoparticles have a Zav size of approximately 87 nm with a PDI of approximately 0.265.

[0565] In addition, the zeta potential of LNP-TB4 liposomes in aqueous dispersion was measured using a Malvern zeta seizer instrument (Malvern Instruments, Inc., Westborough, MA, USA). Briefly, approximately one (1) ml of liposomes (at a concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell suitable for a Zetasizer. The measurement was performed at 25°C. The results showed that the zeta potential of LNP-TB4 was measured to be approximately -15.1 mV ( Figure 9 ).

[0566] Example 6: Synthesis and characterization of LNP-TB4-ID3 liposomes.

[0567] In another experiment, comprise that the liposome (being expressed as LNP-TB4) of TB4 prodrug is allotted and synthesized in the following manner with ID3.In brief, in the first step, in ethanol (20mg / ml), prepare the lipid stock solution of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), CHOL and DSPE-PEG respectively.In acetonitrile (20mg / ml), prepare the stock solution of TB4 prodrug (TB4+ stearic acid) and ID3 prodrug then, because it is insoluble in ethanol.Then the lipid mixture of POPC, CHOL, TB4+ stearic acid, ID3 and DSPE-PEG is mixed with the mol ratio of 53:31:6:6:4, and use ethanol dilution subsequently, to obtain the total lipid concentration of 10mg / ml.Use the heating block annex in the microfluidizer to heat this lipid mixture under 50 degrees centigrade. In the embodiment of the present invention, the microfluidic cell of embodiment 1 is carried out filtration.Similarly, the aqueous phase that contains 1mM PBS buffer solution is also preheated at 50 degrees centigrade, then passes microfluidic box with the flow velocity of 4.5:1 (aqueous phase: organic phase, lipid mixture).Use the dialysis membrane that is 12KDa (Sigma Aldrich (Sigma Aldrich)) of the cut-off value size for DI water to remove solvent, continues at least 24 hours.Dialysis water is changed at least 5 times during the time period of 24 hours, to remove solvent to greatest extent.After removing solvent, use Amicon centrifugal filter unit (cut-off value size is 10KDa during 3000g) as required that LNP-TB4-ID3 is concentrated.

[0568] Characterization of LNP-TB4-ID3 liposomes was determined using a Malvern Zetasizer (Malvern Instruments, Inc., Westborough, MA, USA). Briefly, two (2) ml of LNP-TB4-ID3 liposomes (liposome concentration 0.5-1 mg / ml) were placed in a 4-sided clear plastic cuvette and analyzed directly at 25°C. Figure 10 The results shown show that the nanoparticles have a Zav size of approximately 87 nm with a PDI of approximately 0.075.

[0569] In addition, the zeta potential of LNP-TB4-ID3 liposomes in aqueous dispersion was measured using a Malvern zeta seizer instrument (Malvern Instruments, Inc., Westborough, MA, USA). Briefly, approximately one (1) ml of liposomes (at a concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell suitable for a Zetasizer. The measurement was performed at 25°C. The results showed that the zeta potential of LNP-TB4-ID3 was measured to be approximately -11.5 mV ( Figure 11 ).

[0570] Additionally, Table IV shows a summary of additional co-formulated LNP-TB4.

[0571] Example 7: Synthesis and characterization of SLNP-TB4 solid lipid nanoparticles.

[0572] In another experiment, the solid lipid nanoparticles (SLNP) comprising the TB4 prodrug (expressed as SLNP-TB4) were synthesized in the following manner. In short, the SLNP comprising the TB4 prodrug was prepared using different types of emulsifiers, such as Moliwol488 (polyvinyl alcohol), Pluronic F 127 and Kolliphor RH 40. In the first step, a lipid stock solution of POPC, CHOL, DSPE-PEG was prepared in ethanol (20 mg / ml) respectively. Then, a TB4 prodrug stock solution was prepared in acetonitrile (20 mg / ml). The lipid mixture of POPC, CHOL, TB4 and DSPE-PEG was mixed in a mol ratio of 51:29:15:5, and then diluted with ethanol to obtain a lipid concentration of 10 mg / ml. The heating block attachment in the microfluidizer was used to heat this lipid mixture at 55 degrees Celsius. Similarly, the aqueous phase containing 2% w / v Moliwol 488 (or 2% w / v Pluronic F127 / Kolliphor RH 40) solution is also preheated at 55 degrees Celsius, then passed through the microfluidic box with a flow rate of 5: 1 (aqueous phase: organic phase, lipid mixture). Use the dialysis membrane of 12KDa (Sigma Aldrich) for the cutoff value of DI water to remove solvent, continue at least 24 hours. During the time period of 24 hours, change dialysis water at least 5 times, to remove solvent to greatest extent. After removing solvent, SLNP is passed through 0.2 micron filter membrane (cellulose acetate). Use Amicon centrifugal filter device (cutoff value size is 10KDa when 3000g) SLNP-TB4 is concentrated as needed.

[0573] Characterization of SLNP-TB4 liposomes was determined using a Malvern Zetasizer (Malvern Instruments, Inc., Westborough, MA, USA). Briefly, two (2) ml of SLNP-TB4 (SLNP concentration 0.5-1 mg / ml) were placed in a 4-sided clear plastic cuvette and analyzed directly at 25°C. Figure 12 The results shown show that the Zav size of the nanoparticles is about 90 nm with a PDI of about 0.074.

[0574] In addition, the zeta potential of SLNP-TB4 solid lipid nanoparticles in aqueous dispersion was measured using a Malvern zeta seizer instrument (Malvern Instruments, Inc., Westborough, MA, USA). Briefly, approximately one (1) ml of SLNP (at a concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell suitable for a Zetasizer. The measurement was performed at 25°C. The results showed that the zeta potential of SLNP-TB4 was measured to be approximately -11.9 mV ( Figure 13 ).

[0575] Example 8: Synthesis and characterization of SLNP-TB4-ID3 solid lipid nanoparticles.

[0576] In another experiment, solid lipid nanoparticles (SLNPs) (expressed as SLNP-TB4-ID3) comprising the TB4 prodrug co-allocated with ID3 were synthesized in the following manner. In short, the SLNPs comprising the TB4 prodrug were prepared using different types of emulsifiers, such as Moliwol 488 (polyvinyl alcohol), Pluronic F 127, and Kolliphor RH 40. In the first step, lipid stock solutions of POPC, CHOL, and DSPE-PEG were prepared in ethanol (20 mg / ml) respectively. Then, TB4 and ID3 prodrug stock solutions were prepared in acetonitrile (20 mg / ml). The lipid mixture of POPC, CHOL, TB4, ID3, and DSPE-PEG was mixed in a mol ratio of 52:29:7:7:5, then diluted with ethanol to obtain a lipid concentration of 10 mg / ml. This lipid mixture was heated in a microfluidizer using a heating block attachment at 55 degrees Celsius. Similarly, the aqueous phase containing 2% w / v Moliwol 488 (or 2% w / v Pluronic F127 / Kolliphor RH 40) solution is also preheated at 55 degrees Celsius, then passed through the microfluidic box with a flow rate of 5: 1 (aqueous phase: organic phase, lipid mixture). Use the dialysis membrane of 12KDa (Sigma Aldrich) for the cutoff value of DI water to remove solvent, continue at least 24 hours. During the time period of 24 hours, change dialysis water at least 5 times, to remove solvent to greatest extent. After removing solvent, SLNP is passed through 0.2 micron filter membrane (cellulose acetate). Use Amicon centrifugal filter device (cutoff value size is 10KDa when 3000g) SLNP-TB4-ID3 is concentrated as needed.

[0577] Characterization of SLNP-TB4-ID3 was determined using a Malvern Zetasizer (Malvern Instruments, Inc., Westborough, MA, USA). Briefly, two (2) ml of SLNP-TB4-ID3 (SLNP concentration 0.5-1 mg / ml) were placed in a 4-sided clear plastic cuvette and analyzed directly at 25°C. Figure 14 The results shown indicate that the nanoparticles have a Zav size of approximately 104.3 nm with a PDI of approximately 0.119.

[0578] In addition, the zeta potential of SLNP-TB4-ID3 solid lipid nanoparticles in aqueous dispersion was measured using a Malvern zeta seizer instrument (Malvern Instruments, Inc., Westborough, MA, USA). Briefly, approximately one (1) ml of SLNP (at a concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell suitable for a Zetasizer. The measurement was performed at 25°C. The results showed that the zeta potential of SLNP-TB4-ID3 was measured to be approximately -10.3 mV ( Figure 15 ).

[0579] Additionally, Table V shows a summary of additional co-formulated SLNP-TB4.

[0580] Example 9: In vivo tumor inhibition by SLNP-TB4 using B16F10 cells.

[0581] In this experiment, SLNP-TB4 was evaluated using the following protocol. Mouse melanoma B16F10 cells (cell number (0.2 × 10 6 )) were inoculated subcutaneously in the right hind flank region of C57BL / 6 mice. Animals were treated with the following twice weekly by intravenous injection: vehicle control; 3 mg / kg of LNP-MTO (mitoxantrone dihydrochloride in liposome form); 3 mg / kg of LNP-AR5 (AR5 stearic acid in liposome form); 3 mg / kg of a combination of LNP-AR5 and LNP-TB4 (TB4 stearic acid in liposome form); and 3 mg / kg of LNP-MTO and SLNP-TB4 (TB4 stearic acid in solid lipid nanoparticle form). Tumor volume was measured three (3) times in two dimensions using a caliper and calculated using the following formula: V=(L x W x W) X 0.5, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on tumor size data on day 15.

[0582] The results showed that the combination of SLNP-TB4 and LNP-MTO provided significant antitumor activity. The TGI value was calculated to be 44.12% (all p < 0.05). Figure 16 ).

[0583] Example 10: In vivo tumor inhibition by LNP-TB4 in various combinations using B16F10 cells.

[0584] In this experiment, LNP-TB4 was evaluated using the following protocol. Mouse melanoma B16F10 cells (cell number (0.2 × 10 6 )) were inoculated subcutaneously in the right hind flank region of C57BL / 6 mice. Animals were treated twice weekly with the following by intravenous injection: vehicle control; 3 mg / kg of LNP-MTO (mitoxantrone dihydrochloride in liposomal form); 3 mg / kg of a combination of LNP-TB4 (TB4 stearic acid in liposomal form) and LNP-TR6 (TR6 chemical in liposomal form); 3 mg / kg of a combination of additional LNP-TB4 and LNP-AR5 (AR5 stearic acid in liposomal form); 3 mg / kg of a combination of additional LNP-TB4 and LNP-ID3 (ID3 stearic acid in liposomal form); 3 mg / kg of a combination of additional LNP-TB4 and LNP-PD3 (PD3 cholesterol); 3 mg / kg of a combination of additional LNP-TB4, LNP-MTO, and LNP-ID3; and 3 mg / kg of a combination of additional LNP-TB4, LNP-MTO, and LNP-AR5. Tumor volume was measured three (3) times in two dimensions using calipers and calculated using the following formula: V = (L x W x W) x 0.5, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on the tumor size data on day 16.

[0585] The results showed that treatment with 3 mg / kg of LNP-MTO as a single agent produced antitumor activity. The TGI value was calculated to be 32.6% (p < 0.05). In addition, the combination of LNP-TB4 + LNP-MTO + LNP-ID3 and LNP-AR5 + LNP-MTO + LNP-TB4 produced antitumor activity. The TGI values ​​were calculated to be 32.86% (p < 0.05) and 37.86% (p < 0.05), respectively. Figure 17 ).

[0586] Example 11: In vitro validation of the mechanism of action of LNP-TB4 and SLNP-TB4.

[0587] In this experiment, the mechanism of action of LNP-TB4 and SLNP-TB4 was evaluated in vitro using the following protocol. The following assays were performed to confirm that TB4 in liposome form (LNP) and in solid lipid nanoparticle form (SLNP) can have biological effects in vitro. Briefly, HEK-Blue TM TGF-β cells and QUANTI Blue TM (InvivoGen, San Diego, CA) assay. HEK-Blue was stimulated with TGF-β TM TGF-β cells induce activation of the TGF-β / Smad signaling pathway, leading to the formation of the Smad3 / Smad4 complex. The complex enters the nucleus and binds to the SBE site, thereby inducing the production of SEAP. The amount of SEAP secreted in the supernatant can be easily assessed using QUANTI-Blue. In the presence of 10 ng / ml of TGF-β, cells were incubated with different concentrations of TB4, LNP-TB4, and SLNP-TB4. After twenty-four (24) hours of incubation with the corresponding compound, the cells were quantified using QUANTI-Blue. TM The assay measures the level of SEAP optimal density (OD) and normalizes the data to the control group (cells treated with TGF-β alone) to estimate the percentage (%) of TGF-β inhibition.

[0588] The results showed that treatment of cells with LNP-TB4 and SLNP-TB4 inhibited TGF-β. (See Figure 18 ).

[0589] Example 12: Human clinical trials for treating human cancer using formulated and / or co-formulated liposomes including TB prodrugs Bed test.

[0590] According to the present invention, formulated and / or co-formulated liposomes containing TB prodrugs are used which specifically accumulate in tumor cells and are used to treat certain tumors and other immune disorders and / or other diseases. In conjunction with each of these indications, two clinical approaches have been successfully achieved.

[0591] 1.) Adjuvant Therapy: In adjuvant therapy, patients are treated with formulated and / or co-formulated liposomes containing TB prodrugs in combination with chemotherapeutic agents, drugs, or biologics, or a combination thereof. Primary cancer targets are treated under standard regimens by adding formulated and / or co-formulated liposomes containing TB prodrugs. The regimens are designed to address efficacy, as assessed in the following examples, including but not limited to reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, overall survival, improved patient well-being, disease stabilization, and the ability to reduce the typical doses of standard chemotherapy and other biologics. These dose reductions allow for additional and / or prolonged therapy by reducing the dose-related toxicity of the chemotherapeutic or biologic agent.

[0592] II.) Monotherapy: In conjunction with the use of formulated and / or co-formulated liposomes containing TB prodrugs as monotherapy for tumors, formulated and / or co-formulated liposomes containing TB prodrugs are administered to patients in the absence of chemotherapeutic agents, drugs, or biologics. In one embodiment, monotherapy is performed clinically in advanced cancer patients with extensive metastatic disease. The regimen design addresses efficacy, as assessed in the following examples, including but not limited to reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, overall survival, improved patient health, stable disease, and the ability to reduce the commonly used doses of standard chemotherapies and other biologics.

[0593] dose

[0594] The dosage regimen can be adjusted to provide the optimal desired response. For example, a single formulated and / or co-formulated liposome containing a TB prodrug can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for the mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the dosage unit forms of the present invention are determined by and directly depend on: (a) the unique characteristics of the formulated and / or co-formulated liposome containing a TB prodrug, (b) the individual mechanisms of the combined compounds (if any), (c) the specific therapeutic or prophylactic effect to be achieved, and (d) the limitations inherent in the art of using such compounds to treat individual allergies.

[0595] Clinical Development Plan (CDP)

[0596] CDP has developed and developed treatments using formulated and / or co-formulated liposomes containing TB prodrugs in combination with adjuvant therapy or monotherapy. The trial initially demonstrated safety and subsequently confirmed efficacy with repeated doses. The trial is open-label and compares standard chemotherapy and / or current standard of care plus formulated and / or co-formulated liposomes containing TB prodrugs. As will be appreciated, a non-limiting criterion that may be utilized in conjunction with patient inclusion is the expression of TGFβ in the tumor, as determined by standard assays known in the art.

[0597] It is believed that the formulated and / or co-formulated liposomes or any of the embodiments disclosed herein can have satisfactory pharmacological properties and promising biopharmaceutical properties, such as toxicological properties, metabolic and pharmacokinetic properties, solubility and permeability. It should be understood that determining suitable biopharmaceutical properties is within the knowledge of those skilled in the art, for example, determining cytotoxicity in cells or inhibiting certain targets or pathways to determine potential toxicity.

[0598] The present invention is not limited in scope to the embodiments disclosed herein, which are intended as simple illustrations of various aspects of the invention, and any functionally equivalent embodiments are within the scope of the invention. Various modifications to the models, methods, and lifecycle methodologies of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments may be practiced without departing from the true scope and spirit of the invention.

[0599] Table I: Examples of lipids .

[0600]

[0601]

[0602] Table II: Examples of helper lipids .

[0603]

[0604] Table III: Examples of phospholipids / fatty acids .

[0605]

[0606] Table IV: Summary of LNP-TB4 co-formulations.

[0607] Nano formulations Diameter in nm PDI Zeta potential (mV) LNP-TB4-PD3 91.4 0.148 -12.1 LNP-TB4-ID3 87.29 0.075 -11.5 LNP-TB4-TR5 101.4 0.136 -11.1

[0608] Table V: Summary of SLNP-TB4 formulations and co-formulations.

[0609]

Claims

1. A TB prodrug composition comprising: (i) drug component; (ii) a lipid portion, wherein the lipid portion comprises stearic acid; as well as (iii) connection unit LU, wherein the drug moiety comprises a TGFβ antagonist, and wherein the LU conjugates the drug moiety to the lipid moiety, and wherein the TB prodrug has the following chemical structure:

2. A nanocarrier comprising a TB prodrug composition, wherein the nanocarrier releases an active ALK5 inhibitor upon cleavage of the linker unit LU, and wherein the TB prodrug has the following chemical structure:

3. The nanocarrier of claim 2, further comprising a helper lipid, wherein the helper lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol, cholesterol hemisuccinate, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-3-trimethylammonium propane chloride.

4. The nanocarrier of claim 2, wherein the nanocarrier is further co-formulated with one or more immunomodulatory agents, wherein the immunomodulatory agent is selected from the group consisting of: an immunogenic cell death-inducing chemotherapeutic agent, a toll receptor agonist, a STING agonist, a CTLA-4 inhibitor, an IDO inhibitor, a PD-1 / PD-L1 inhibitor, and a CD1D agonist.

5. The nanocarrier of claim 2, wherein the nanocarrier is further co-formulated with an ICD-inducing chemotherapeutic agent, wherein the ICD-inducing chemotherapeutic agent is selected from the group consisting of doxorubicin DOX, mitoxantrone MTO, oxaliplatin OXA, cyclophosphamide CP, bortezomib, carfilzomib, or paclitaxel.

6. The nanocarrier of claim 2, wherein the nanocarrier is further co-formulated with a toll receptor agonist or a lipid prodrug thereof, wherein the toll receptor agonist is selected from the group consisting of: resiquimod R848, gademod, 852A, DSR 6434, tiramid, CU-T12-9, monophosphoryl lipid A, 3D (6-acyl)- SMU127, Pam3CSK4 or 7. The nanocarrier of claim 2, wherein the nanocarrier is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid prodrug thereof, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of AUNP12, CA-170, or BMS-986189.

8. The nanocarrier of claim 2, wherein the nanocarrier comprises a liposome.

9. The nanocarrier of claim 8, wherein the liposomes have a Zav size of 87 nm and a PDI of 0.

075.

10. The nanocarrier of claim 2, wherein the nanocarrier comprises solid lipid nanoparticles (SLNPs).

11. The nanocarrier of claim 10, wherein the SLNPs have a Zav size of 90 nm and a PDI of 0.

074.

12. A composition comprising a nanocarrier for treating a subject suffering from cancer, wherein the nanocarrier comprises a TB prodrug composition having the following chemical structure: or a pharmaceutically acceptable salt thereof.

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