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

Abstract

To provide formulated and / or co-formulated liposomal compositions containing TFG β antagonist prodrugs useful in the treatment of cancer, and methods thereof.SOLUTION: Disclosed herein are formulated and / or co-formulated liposomes (LNPs) and solid lipid nanoparticles (SLNPs) comprising TB prodrugs, and methods of making said LNPs and SLNPs. TB pro-drug compositions comprise a drug moiety, a lipidic moiety, and a linking unit, and inhibit ALK5. TB prodrugs can be formulated and / or co-formulated into liposomes or solid lipid nanoparticles to provide a method of treating cancer, immunological disorders, and other diseases by utilizing a targeted drug delivery vehicle.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 995,887, filed February 19, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable.

[0003] FIELD OF THE INVENTION The invention described herein relates to prodrug compositions that inhibit signal transduction induced by transforming growth factor beta 1, 2, or 3 ("TGFβ") proteins after the active inhibitor is released from the prodrug, and nanoformulations containing such prodrugs. In particular, the invention relates to prodrug compositions formulated within nanocarriers (e.g., liposomes) and used as vehicles for cancer treatment in humans. The invention also relates to co-formulations of such prodrugs with other immunomodulators or prodrugs. The invention further relates to the treatment of cancer and other immunological disorders and diseases. [Background technology]

[0004] Background of the Invention Cancer is the second leading cause of death worldwide, after coronary artery disease. Millions of people die from cancer each year; in the United States alone, cancer causes well over 500,000 deaths annually, with 1,688,780 new cases of cancer diagnosed in 2017 (American Cancer Society). While deaths from heart disease have declined significantly, cancer deaths in general are on the rise. Early in the next century, cancer is predicted to become the leading cause of death unless medical advances change current trends.

[0005] Some cancers stand out for their high mortality rates. In particular, lung cancer (18.4% of all cancer deaths), breast cancer (6.6% of all cancer deaths), colorectal cancer (9.2% of all cancer deaths), liver cancer (8.2% of all cancer deaths), and gastric cancer (8.2% of all cancer deaths) are the leading causes of cancer death worldwide for men and women of all ages (GLOBOCAN 2018). These and virtually all other cancers share a common fatal characteristic in that they metastasize to sites distal to the primary tumor, almost exclusively resulting in fatal metastatic disease. Furthermore, even cancer patients who survive their primary cancer early on report a common experience of their lives being dramatically altered. Many cancer patients experience intense anxiety caused by the awareness of the potential for recurrence or treatment failure. Many cancer patients also experience physical debilitation after treatment. Furthermore, many cancer patients experience disease recurrence.

[0006] While cancer treatment has improved over the past few decades, increasing survival rates, new therapeutic strategies utilizing multiple treatment modalities are still needed due to the heterogeneity of cancer. This is particularly true for the treatment of solid tumors in critical anatomical locations (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma), which may be limited to standard radiation therapy and / or chemotherapy. Nevertheless, adverse effects of these treatments include chemoresistance and radioresistance, which promote locoregional recurrence, distant metastases, and second primary tumors, in addition to severe side effects that reduce patients' quality of life.

[0007] TGF-β refers to a subset of polypeptides in the transforming growth factor beta superfamily of cytokines. TGF-β is a secreted protein that performs many cellular functions, including regulating cell growth, proliferation, differentiation, and apoptosis. In humans, TGF-β1 is encoded by the TGFB1 gene. Functionally, TGF-β acts synergistically with TGF-α in inducing transformation. TGF-β1 also acts as a negative autocrine growth factor. Dysregulation of TGF-β activation and signaling can lead to apoptosis. Many cells synthesize TGF-β, and nearly all cells possess specific receptors for these cytokines. TGF-β1, TGF-β2, and TGF-β3 all function through the same receptor signaling pathway. TGF-β plays an important role in regulating the immune system and exhibits diverse activities on different cell types or at different developmental stages. Most immune cells (or white blood cells) secrete TGF-β. See LETTERIO, et. al., Regulation of Immune Responses by TGF-beta, Annu. Rev. Immunol. 16: pp. 137-161 (1998). Some T cells (e.g., regulatory T cells) are thought to release TGF-beta, which inhibits the action of other T cells. For example, interleukin-1- and interleukin-2-dependent proliferation of activated T cells and activation of quiescent helper and cytotoxic T cells are prevented by the activity of TGF-beta1. GILBERT, et. al., Transforming growth factor-beta 1 induces antigen-specific unresponsiveness in naive T cells, Immunol. Invest. 26(4): pp. 459-472 (1997). & WAHL, et. al., TGF-beta: a mobile purveyor of immune privilege, Immunol. Rev. 213: pp. 213-227 (2006). Similarly, TGF-beta can inhibit the secretion and activity of many other cytokines, including interferon-gamma, tumor necrosis factor-alpha (TNF-α), and various interleukins. TGF-beta can also downregulate the activity of immune cells by reducing the expression levels of cytokine receptors, such as the IL-2 receptor. In addition, TGF-beta has similar effects on B cells, although its effects vary depending on the differentiation state of the cells. TGF-beta inhibits B cell proliferation, stimulates apoptosis, and plays a role in regulating 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 TGFβ on macrophages and monocytes are primarily inhibitory; this cytokine inhibits the proliferation of these cells and reduces their production of reactive oxygen species (e.g., superoxide (O2)). - ) and nitrogen (e.g., nitric oxide (NO)) intermediates. However, as with other cell types, TGF-β may also have adverse effects on cells of myeloid origin. See WAHL, et. al., supra. TGF-β has also been shown to reduce the availability of MHC II on astrocytes and dendritic cells, thereby reducing the activation of appropriate helper T cell populations. 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): pp. 311-319 (2001).

[0008] TGFβ elicits an intracellular signal by first binding to TGFβ receptor II (TGFβRII), which then recruits a second receptor, TGFβ receptor I (TGFβRI), also known as activin-like kinase 5 (ALK5). Upon recruitment to the TGFβ receptor:ligand complex, TGFβRII phosphorylates and activates ALK5, thereby mediating downstream signaling and resulting in transcriptional regulation. It has been suggested that ALK5 deletion or mutation in tumors appears to be a common form of pathway alteration. However, studies targeting inhibition of 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): pp. 2431-2447 (2016).

[0009] Currently, several small molecule kinase inhibitors that selectively inhibit ALK5 have been identified and advanced to the clinical stage for testing as anticancer drugs. Galunisertib (LY2157229) and bactosertib (TEW-7197) are cutting-edge therapeutic candidates in clinical trials, demonstrating anticancer efficacy in humans as monotherapy and in combination with approved drugs, validating this mechanism as a clinically meaningful approach. However, administration is limited by the cardiotoxicity induced by these molecules, and combination with other therapies increases the risk of systemic off-target toxicity.

[0010] A prodrug is a pharmaceutical or compound that is metabolized (i.e., converted in the body) into a pharmacologically active drug after administration. Instead of directly administering a drug, a corresponding prodrug is used as an alternative to improve how the drug is absorbed, distributed, metabolized, and / or excreted. Prodrugs are often designed to improve bioavailability when the drug itself is poorly absorbed, for example, from the gastrointestinal tract. Prodrugs can be used to improve how a drug selectively interacts with cells or processes that are not its intended target. This reduces the harmful or unintended effects of the drug, which is particularly important in treatments such as chemotherapy, which can have severe unintended and undesirable side effects. Therefore, prodrugs can be considered drugs containing special non-toxic protecting groups that are used temporarily to alter or eliminate undesirable properties of the parent molecule.

[0011] Finally, nanocarriers are nanomaterials used as a means of transporting other substances, such as drugs. Many different types of nanocarriers exist. Examples include polymer conjugates, polymer nanoparticles, lipid-based carriers, and dendrimers, to name a few. The various types of nanomaterials used in nanocarriers enable the delivery of hydrophobic and hydrophilic drugs throughout the body. Because the human body contains primarily water, the ability to effectively deliver hydrophobic drugs to humans is a major therapeutic benefit of nanocarriers. Nanocarriers hold promise in the drug delivery process because they enable site-specific targeting of drugs, allowing drugs to be delivered to certain organs and cells rather than to other organs and cells. Site specificity is a major therapeutic benefit because it prevents drugs from being delivered to the wrong location. Additionally, nanocarriers are particularly promising for use in chemotherapy because they can help reduce the harmful, broad-scale toxicity of chemotherapy to rapidly growing healthy cells throughout the body. Because chemotherapy drugs can be highly toxic to human cells, it is important that they be delivered to tumors without being released into other parts of the body.

[0012] From the above, it is readily apparent to those skilled in the art that new treatment paradigms are needed in the treatment of cancer and other immunological diseases. By using novel prodrugs in conjunction with modern nanocarrier modalities, new disease treatments can achieve the overall goals of more effective treatment(s), reduced side effects, and greater therapeutic utility in the treatment of cancer, particularly solid tumor cancer.

[0013] In view of the current deficiencies associated with cancer treatment, it is an object of the present invention to provide new and improved methods of treating cancer(s), immunological disorders, and other diseases utilizing prodrugs encapsulated within nanocarriers. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] LETTERIO, et. al., Regulation of Immune Responses by TGF-beta, Annu. Rev. Immunol. 16: pp 137-161 (1998) [Non-patent document 2] GILBERT, et. al., Transforming growth factor-beta 1 induces antigen-specific unresponsiveness in naive T cells, Immunol. Invest. 26(4): pp. 459-472 (1997) [Non-patent document 3] WAHL, et. al., TGF-beta: a mobile purveyor of immune privilege, Immunol. Rev. 213: pp. 213-227 (2006) Summary of the Invention [Means for solving the problem]

[0015] Summary of the Invention The present invention provides ALK5 inhibitor prodrug ("TB prodrug") compositions comprising an ALK5 inhibitor, a lipid, and a biologically cleavable linker. In certain embodiments, nanocarriers comprising the TB prodrug are formulated for use as a delivery modality to treat human diseases, such as cancer, including solid tumor cancer, and other immunological disorders. In certain embodiments, the nanocarriers comprise a lipid bilayer that can be incorporated into a drug delivery vehicle (i.e., a liposome). In a further preferred embodiment, the liposomes comprise cholesterol hemisuccinate ("CHEMS"). In a further preferred embodiment, the liposomes of the present invention comprise stearic acid.

[0016] In further embodiments, the TB prodrugs of the present disclosure include TB4 prodrugs.

[0017] In a further embodiment, 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 present invention into a nanocarrier of the present invention; and (iii) administering the nanocarrier to a patient.

[0018] In another embodiment, the present invention comprises a method of delivering an ALK5 inhibitor together with one or more additional immunomodulatory agents to a tumor, the method comprising: (i) synthesizing a TB prodrug; (ii) co-forming the TB prodrug of the present invention into a nanocarrier with one or more additional immunomodulatory agents of the present invention; and (iii) administering the nanocarrier to a patient.

[0019] In another embodiment, the immunomodulatory agent comprises 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.

[0020] In another embodiment, the present disclosure teaches methods for synthesizing TB prodrugs.

[0021] In another embodiment, the present disclosure teaches methods for synthesizing TB4 prodrugs.

[0022] In another embodiment, the present disclosure teaches methods of formulating TB prodrugs into nanocarriers, including but not limited to liposomes.

[0023] In another embodiment, the present disclosure teaches methods of formulating TB4 prodrugs into nanocarriers, including but not limited to liposomes.

[0024] In another embodiment, the present disclosure teaches methods of using the nanocarriers of the present disclosure to treat cancer(s), immunological disorders, and other diseases in humans. The present invention provides, for example, the following items. (Item 1) (i) a drug moiety; (ii) a lipid moiety, and (iii) Consolidated Unit (“LU”) wherein the drug moiety comprises a TGFβ antagonist, and the LU conjugates the drug moiety to the lipid moiety. (Item 2) 2. The TB prodrug composition of claim 1, wherein the drug moiety comprises the chemical structure set forth as TB4. (Item 3) 2. The TB prodrug composition of claim 1, wherein the lipid moiety comprises stearic acid. (Item 4) 2. The TB prodrug composition of claim 1, wherein the lipid moiety comprises stearic acid. (Item 5) Item 1, a TB prodrug composition comprising the following chemical structure: [ka] (Item 6) A nanocarrier comprising a TB prodrug, which releases an active ALK5 inhibitor after cleavage of the LU. (Item 7) 7. The nanocarrier of item 6, wherein the LU is a hydromethylcarbamate linker. (Item 8) 7. The nanocarrier of item 6, further comprising a helper lipid, wherein the helper lipid is listed in Table II. (Item 9) 7. The nanocarrier of item 6, wherein the TB prodrug comprises TB4. (Item 10) 7. The nanocarrier of item 6, wherein the TB prodrug comprises TB4 and further comprises stearic acid. (Item 11) Item 11. The nanocarrier of item 10, wherein the TB4 prodrug has the following chemical structure: [ka] (Item 12) 12. The nanocarrier of item 11, further co-formulated with one or more immunomodulatory agents or lipid-prodrugs thereof, 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, a CD1D agonist, and / or a prodrug thereof. (Item 13) 12. The nanocarrier of item 11, further co-formulated with an ICD-inducing chemotherapeutic agent, wherein the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, or paclitaxel. (Item 14) 12. The nanocarrier of item 11, 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), gardquimod, 852A, DSR6434, telluritolimod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD®. (Item 15) 12. The nanocarrier of item 11, 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. (Item 16) Item 12. The nanocarrier according to item 11, comprising a liposome. (Item 17) 17. The liposome according to item 16, wherein the liposome is LNP-TB4. (Item 18) 12. The nanocarrier of item 11, comprising a solid lipid nanoparticle (SLNP). (Item 19) 19. The SLNP of item 18, wherein the SLNP is SLNP-TB4. (Item 20) 1. A method of treating a subject suffering from or diagnosed with cancer, comprising: (i) administering to a subject in need of such treatment an effective amount of a nanocarrier, wherein the nanocarrier comprises a TB prodrug; (ii) a pharmaceutically acceptable salt thereof; A method comprising: [Brief explanation of the drawings]

[0025] [Figure 1] Chemical synthesis for TB4 prodrugs.

[0026] [Figure 2]Chemical synthesis for protecting group intermediates en route to the final TB4 prodrug.

[0027] [Figure 3] Chemical synthesis for protecting group intermediates en route to the final TB4 prodrug.

[0028] [Figure 4] Synthetic scheme for ALK5 inhibitor prodrugs using a carboxylic acid functional group.

[0029] [Figure 5] Synthesis scheme of ALK5 inhibitor prodrugs using alcohol functional groups.

[0030] [Figure 6] Scheme for the synthesis of ALK5 inhibitor prodrugs using secondary amine, amide, or aniline functional groups.

[0031] [Figure 7] Chemical synthesis for TB4 prodrugs containing stearic acid.

[0032] [Figure 8] Characterization of LNP-TB4 liposomes.

[0033] [Figure 9] Characterization of LNP-TB4 liposomes (zeta potential).

[0034] [Figure 10] Characterization of LNP-TB4-ID3 liposomes.

[0035] [Figure 11] Characterization of LNP-TB4-ID3 liposomes (zeta potential).

[0036] [Figure 12] Characterization of SLNP-TB4 solid lipid nanoparticles.

[0037] [Figure 13] Characterization of SLNP-TB4 solid lipid nanoparticles (zeta potential).

[0038] [Figure 14] Characterization of SLNP-TB4-ID3 solid lipid nanoparticles.

[0039] [Figure 15] Characterization of SLNP-TB4-ID3 solid lipid nanoparticles (zeta potential).

[0040] [Figure 16] Tumor inhibition of SLNP-TB4 in combination with LNP-MTO using B16F10 cells in vivo.

[0041] [Figure 17] Tumor inhibition of LNP-TB4 in multiple combinations using B16F10 cells in vivo.

[0042] [Figure 18] In vitro validation of the mechanism of action of LNP-TB4 and SLNP-TB4. DETAILED DESCRIPTION OF THE INVENTION

[0043] Detailed Description of the Invention Section Overview I.) Definition II.) Prodrugs III.) Chemical compounds IV.) Lipids V.) Consolidated Units (“LUs”) VI.) Nanocarriers VII.) Liposomes VIII.) Pharmaceutical Formulations IX.) Combination Therapy X.) Methods for delivering liposomes containing prodrugs to cells XI.) Methods of Treating Cancer(s) and Other Immunological Disorder(s) XII.) Kits / Manufactured Products I.) Definition

[0044] Unless otherwise defined, all terms of the art, notation, and other scientific or technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains, unless the context clearly indicates otherwise. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0045] When a trademark is used herein, reference to that trademark also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trademark product, unless the context dictates otherwise.

[0046] 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 of ±20% or ±10% in one example, ±5% in another example, ±1% in another example, and ±0.1% from the specified amount, as such variations are suitable for practicing the disclosed methods.

[0047] As used herein, the term "and / or," when used in the context of a list of entities, refers to the entities present either alone or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes not only A, B, C, and D individually, but also any and all combinations and subcombinations of A, B, C, and D.

[0048] As used herein, numerical ranges recited by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes, but is not limited to, 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5).

[0049] As used herein, the phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and, in addition, those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0050] The terms "advanced cancer," "locally advanced cancer," "progressive disease," and "locally advanced disease" refer to cancer that has spread through the relevant tissue capsule and are meant to include stage C disease in the American Urological Association (AUA) system, stages C1-C2 disease in the Whitmore-Jewett system, and stages T3-T4 and N+ disease in the TNM (tumor, node, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes than patients with clinically localized (organ-confined) cancer.

[0051] As used herein, the term "alkyl" refers to any alkyl group selected from C1 to C6, inclusive, including, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. 20"Alkyl" can refer to a linear (i.e., "straight chain"), branched, or cyclic, saturated or at least partially, and in some cases unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chain. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl, or propyl, is attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms (i.e., C1-C8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to C1-C8 straight chain alkyl. In other embodiments, "alkyl" refers, in particular, to C1-C8 straight chain alkyl. i~8 Refers to branched chain alkyl.

[0052] An alkyl group can be optionally substituted with one or more alkyl group substituents, which can be the same or different ("substituted alkyl"). The term "alkyl group substituent" includes, but is not limited to, alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. In some embodiments, one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms can be optionally inserted along the alkyl chain, and the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl"), or aryl.

[0053] Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group have been replaced with another atom or functional group, including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0054] The term "aryl" is used herein to refer to an aromatic substituent, which may be a single aromatic ring or multiple aromatic rings that are fused together, covalently linked, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group may 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. The aromatic ring(s) may include, among others, phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, and benzophenone. In certain embodiments, the term "aryl" refers to cyclic aromatics containing about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, including 5- and 6-membered aromatic and heteroaromatic rings. Aryl groups can be optionally substituted with one or more aryl group substituents, which can be the same or different ("substituted aryl"); "aryl group substituents" include alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R'', where R' and R'' can each independently be hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl. Illustrative 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.

[0055] "Heteroaryl," as used herein, refers to an aryl group that contains 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, etc.

[0056] The terms "anti-cancer drug," "chemotherapeutic agent," and "anti-cancer prodrug" refer to drugs (i.e., chemical compounds) or prodrugs that are known to or suspected to be capable of treating cancer (i.e., killing cancer cells, inhibiting the growth of cancer cells, or treating symptoms associated with cancer). In some embodiments, the term "chemotherapeutic agent," as used herein, refers to a non-PS molecule that is used to treat cancer and / or has cytotoxic capabilities. More traditional or conventional chemotherapeutic agents can be described by mechanism of action or by class of chemical compound, 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), inhibitors of topoisomerase I or II (e.g., irinotecan or etoposide), kinase inhibitors (e.g., bortezomib), nucleotide analogs or precursors thereof (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).

[0057] "Aralkyl" refers to the group -alkyl-aryl, wherein the alkyl and / or aryl portions are optionally substituted.

[0058] "Alkylene" refers to a straight-chain or branched divalent aliphatic hydrocarbon group having 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Alkylene groups can be straight-chain, branched, or cyclic. Alkylene groups can also be optionally unsaturated and / or substituted with one or more "alkyl group substituents." Optionally inserted along the alkylene group are one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl"), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH2-), ethylene (-CH2-CH2-), propylene (-(CH2)3-), cyclohexylene (-CH6-), 10 -), -CH=CH-CH=CH-, -CH=CH-CH2-, -(CH2) q Examples include -N(R)-(CH)- (where each q is an integer from 0 to about 20, e.g., 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 lower alkyl), methylenedioxyl (-O-CH-O-), and ethylenedioxyl (-O-(CH)-O-). The alkylene group can have from about 2 to about 3 carbon atoms and can further have from 6 to 20 carbons.

[0059] The term "arylene" refers to a divalent aromatic group, such as a divalent phenyl or naphthyl group. An arylene group can be optionally substituted with one or more aryl group substituents and / or can contain one or more heteroatoms.

[0060] 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" can refer to the group -N(R)2, where each R is H, alkyl, or substituted alkyl, and 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 at least one R is aryl or substituted aryl, such as aniline (i.e., -NHC6H5).

[0061] "Bulk" (also known as drug substance) means a drug substance or drug product that has not been filled into a final container for distribution. Final formulated bulk generally refers to a drug product that has been formulated and stored or held prior to filling. Drug substances may be stored or held as "bulk" or "concentrated bulk" prior to formulation into a drug product.

[0062] The terms "carboxylate" and "carboxylic acid" refer to -C(=O)O, respectively. - The term "carboxyl" can also refer to the group -C(=O)OH.

[0063] The terms "conjugate" and "conjugated," as used herein, can refer to the attachment (e.g., covalent bonding) of two or more components (e.g., chemical compounds, polymers, biomolecules, particles, etc.) to one another. In some embodiments, a conjugate can include monovalent moieties derived from two different chemical compounds covalently linked via a divalent linker moiety (e.g., an optionally substituted alkylene or arylene). In some embodiments, the linker can contain one or more biodegradable bonds, such that one or more bonds of the linker can be broken when the prodrug is exposed to certain physiological environments or enzymes (e.g., esterases).

[0064] The term "compound" refers to and includes the chemical compound (e.g., a prodrug) per se, as well as the following, whether expressly stated or not, unless the context makes it clear that the following is excluded: amorphous and crystalline forms of the compound, including polymorphic forms, where these forms may be part of a mixture or may be isolated; free acid and free base forms of the compound, which are typically those 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 and mixtures of optical isomers, including racemic and non-racemic mixtures, where an isomer may be in isolated form or may be a mixture with one or more other isomers; isomers of the compound, which refers to the isomers of the compound, including enantiomers and diastereomers, chiral isomers and non-chiral isomers, and topes, including deuterium- and tritium-containing compounds, including compounds containing radioisotopes, including therapeutically and diagnostically effective radioisotopes; multimeric forms of the compounds, including dimeric, trimeric, etc. forms; salts of the compounds, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts, including salts with organic and inorganic counterions, including zwitterionic forms, where if the 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, disolvates, etc., including organic solvates and inorganic solvates, where said inorganic solvates include hydrates, where if the compound is associated with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some cases, reference herein to a compound of the invention includes an explicit reference to one or of the above forms, e.g., salts and / or solvates, but this reference is for emphasis only and should not be construed as excluding any form other than the one specified above.

[0065] "Drug product" generally refers to a final formulation (i.e., liposomes containing an ALK5 inhibitor prodrug) containing an active drug ingredient, in association with, but not necessarily, an inactive ingredient. The term also includes final dosage forms that do not contain an active ingredient but are intended to be used as a placebo.

[0066] The term "disulfide" can refer to a -SS- group.

[0067] The term "empty vesicles" refers to unloaded lipid vesicles as such.

[0068] The term "ester," as used herein, means a chemical compound derived from an acid (organic or inorganic) in which at least one -OH hydroxy group is replaced by an -O-alkyl (alkoxy) or -O-aryl (aryloxy) group.

[0069] The term "esterase," as used herein, is a hydrolytic enzyme that breaks down esters into acids and alcohols.

[0070] "Excipient" means an inert substance used as a carrier for an active ingredient in a drug, such as a vaccine. Excipients are also sometimes used to increase the bulk of formulations containing highly potent active ingredients, allowing for convenient and accurate dosing. Examples of excipients include, but are not limited to, anti-adherents, binders, coatings, disintegrants, fillers, diluents, flavors, colorants, lubricants, and preservatives.

[0071] The terms "halo," "halide," or "halogen," as used herein, refer to fluoro, chloro, bromo, and iodo groups.

[0072] The terms "hydroxyl" and "hydroxy" refer to the group --OH.

[0073] The terms "inhibit" or "inhibition of," as used herein, means to reduce by a measurable amount or to prevent completely.

[0074] The terms "individual" or "patient" can be used interchangeably when used in the context of this disclosure.

[0075] As used herein, the term "ligand" generally refers to a species, e.g., a molecule or ion, that interacts with, e.g., binds to, another species in some way. See MARTELL, AE, and HANCOCK, RP, Metal Complexes in Aqueous Solutions, Plenum: New York (1996), which is incorporated herein by reference in its entirety.

[0076] The term "lipid," as used herein, refers to a class of naturally occurring (organic) compounds that are insoluble in polar solvents. In the context of this disclosure, lipids refer to conventional lipids, phospholipids, cholesterol, lipids chemically functionalized for attachment of PEG and ligands, and the like.

[0077] The term "lipid bilayer" or "LB" refers to any bilayer of oriented amphiphilic lipid molecules with their hydrocarbon tails pointing inward to form a continuous non-polar phase.

[0078] The terms "liposome" or "lipid vesicle" or "vesicle" are used interchangeably to refer to aqueous compartments enclosed by a lipid bilayer, as conventionally defined (see STRYER (1981) Biochemistry, 2d Edition, W.H. Freeman & Co., p. 213).

[0079] 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 invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0080] The term "mercapto" or "thiol" refers to a -SH group.

[0081] The terms "metastatic cancer" and "metastatic disease" are meant to mean spread to regional lymph nodes or distant sites and include stage D disease in the AUA system and stage TxNxM+ in the TNM system.

[0082] The terms "nanocarrier," "nanoparticle," and "nanoparticle drug carrier" are used interchangeably and refer to nanostructures having an aqueous, solid, or polymeric inner core. In certain embodiments, nanocarriers comprise a lipid bilayer enveloping (or surrounding or covering) a porous particle core. In certain embodiments, nanocarriers are liposomes, lipid nanoparticles ("LNPs"), or solid lipid nanoparticles ("SLNPs").

[0083] The terms "nanoscale particle," "nanomaterial," "nanocarrier," and "nanoparticle" refer to a structure having at least one region with a dimension (e.g., length, width, diameter, etc.) of 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 dimensions are between about 20 nm and about 250 nm (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm).

[0084] The term "nanovesicle" refers to a "lipid vesicle" (or a population of vesicles having an average diameter) having a diameter ranging from about 20 nm, or from about 30 nm, or from about 40 nm, or from about 50 nm, to about 500 nm, or to about 400 nm, or to about 300 nm, or to about 200 nm, or to about 150 nm, or to about 100 nm, or to about 80 nm. In certain embodiments, the nanovesicles have a diameter ranging from about 40 nm to about 80 nm, or from about 50 nm to about 70 nm.

[0085] "Pharmaceutically acceptable" refers to a composition that is non-toxic, inert, and / or physiologically compatible with humans or other mammals.

[0086] "Pharmaceutical formulation" refers to the process of combining various chemicals with pure drug substances to produce a final drug product.

[0087] The term "phosphonate" refers to the group -P(=O)(OR), where each R can independently be H, alkyl, aralkyl, aryl, or a negative charge (i.e., there are substantially no R groups attached to the oxygen atom, resulting in the presence of 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.

[0088] The term "phosphate" refers to the group -OP(=O)(OR')2, where R' is H or a negative charge.

[0089] The term "prodrug" refers to a pharmaceutical agent or compound that is metabolized into a pharmacologically active drug after administration. For purposes of this disclosure, the prodrugs of the present invention comprise three components: (i) a drug moiety, (ii) a lipid moiety, and (iii) a linking unit ("LU").

[0090] The term "TB prodrug" refers to a prodrug of the present invention, wherein the drug moiety comprises an ALK5 inhibitor.

[0091] The term "pyrolipid" refers to a conjugate of a lipid with a porphyrin, a porphyrin derivative, or a porphyrin analog. In some embodiments, a pyrolipid can 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.

[0092] As used herein, the terms "specific," "specifically binds," and "specifically binds to" refer to the selective binding of the nanocarriers of the present invention to the target TGFβ1 or related family member.

[0093] The term "supported lipid bilayer" refers to a lipid bilayer that encapsulates a porous particle core. This definition is provided because, as described herein, the lipid bilayer is located on the surface and supported by the porous particle core. In certain embodiments, the lipid bilayer can have a thickness ranging from about 6 nm to about 7 nm, comprising a 3-4 nm thick hydrophobic core, plus a layer of hydrated hydrophilic head groups (about 0.9 nm each), plus two partially hydrated regions, each about 0.3 nm. In various embodiments, the lipid bilayer surrounding the liposome comprises a continuous or substantially continuous bilayer, which effectively encases and seals the ALK5 inhibitor.

[0094] The term "thioalkyl" can refer to the 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 the group -SR, where R is aralkyl and aryl, respectively.

[0095] As used herein, "treating" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as extending survival, reducing morbidity, and / or reducing side effects that are a by-product of alternative treatment modalities; as will be readily recognized in the art, complete eradication of the disease, while preferred, is not a requirement of the act of treatment.

[0096] The term "therapeutically effective amount" refers to that amount of active prodrug, nanoencapsulated prodrug, or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal, individual, or human.

[0097] The term "unsupported lipid bilayer" refers to the uncoated lipid bilayer in a lipid vesicle or liposome. II.) Prodrugs

[0098] As shown in this disclosure, and for 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 an LU of the present disclosure (see the section entitled "Linking Unit"). For purposes of the present disclosure, the formation of TB prodrugs can utilize several strategies. (See, e.g., Figures 4, 5, and 6.)

[0099] Thus, in some embodiments, the prodrug is a drug-lipid moiety that comprises an ALK5 inhibitor of the present disclosure.

[0100] In one embodiment, the prodrug comprises the following chemical structure according to Formula I: [ka] wherein, in an exemplary embodiment of Formula I, R1=C 11 ~C 21 is a saturated alkyl; R2=H, CH3].

[0101] In a further embodiment, the prodrug comprises the following chemical structure shown in Formula II: [ka] wherein, in an exemplary embodiment of Formula II, A= [ka] and R1=C 11 ~C 21 is a saturated alkyl; R2=H, CH3].

[0102] In a further embodiment, the prodrug comprises the following chemical structure shown in Formula III: [ka] wherein, in an exemplary embodiment of Formula III, A= [ka] and R1=C 11 ~C 21 is a saturated alkyl; R2=H, CH3].

[0103] Thus, in one embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of Formula I.

[0104] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of Formula II.

[0105] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of Formula III.

[0106] In one embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor as depicted in FIG.

[0107] In one embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor as depicted in FIG.

[0108] In one embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor as depicted in FIG.

[0109] In a further embodiment, the TB prodrug is a drug-lipid moiety comprising a lipid of the present disclosure.

[0110] In a further embodiment, the TB prodrug is a drug-lipid moiety, wherein the lipid is a CHEMS.

[0111] In a further embodiment, the TB prodrug is a drug-lipid moiety, wherein the lipid is stearic acid.

[0112] In a further embodiment, the TB prodrug is a drug-lipid moiety comprising an LU of the present disclosure.

[0113] In a further embodiment, the TB prodrug is a drug-lipid moiety, where LU is a hydromethylcarbamate linker.

[0114] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises the chemical composition(s) denoted TB4.

[0115] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the present invention, where the ALK5 inhibitor comprises TB4 and has the following chemical structure: [ka]

[0116] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the present invention, where the ALK5 inhibitor comprises TB4 and further comprises a lipid of the present disclosure having the following chemical formula: [ka]

[0117] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the invention, where the ALK5 inhibitor comprises TB4 and further comprises CHEMS.

[0118] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the invention, where the ALK5 inhibitor comprises TB4 and further comprises stearic acid.

[0119] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the invention, where the ALK5 inhibitor comprises TB4 and further comprises CHEMS, and LU is a hydromethylcarbamate linker.

[0120] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the invention, where the ALK5 inhibitor comprises TB4 and further comprises stearic acid, and LU is a hydromethylcarbamate linker.

[0121] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the invention, where the ALK5 inhibitor comprises TB4 and further comprises a stearic acid having the following structure: [ka]

[0122] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of the invention, where the ALK5 inhibitor comprises TB4 and further comprises a stearic acid having the following structure: [ka]

[0123] In additional embodiments of the present disclosure, the present subject matter provides an ALK5 inhibitor prodrug comprising a therapeutic parent drug conjugated to a lipid. In some embodiments, the prodrug comprises (a) a monovalent drug moiety, (b) a monovalent lipid moiety, and (c) a linking unit that degrades in vivo, such as a bivalent linker moiety containing a disulfide bond, where the monovalent drug moiety and the monovalent lipid moiety are linked via the linker (e.g., covalently linked). The monovalent drug moiety and the monovalent lipid moiety 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 containing a hydroxyl, thiol, amino, or carboxylic acid group.

[0124] In a further embodiment of the present disclosure, the present subject matter provides an ALK5 inhibitor prodrug comprising a therapeutic parent drug conjugated to a lipid. In some embodiments, the prodrug comprises (a) a bivalent drug moiety, (b) a bivalent lipid moiety, and (c) a bivalent linker moiety comprising an in vivo degradable linkage, wherein the bivalent drug moiety and the bivalent lipid moiety are linked via a linker (e.g., covalently linked). The bivalent drug moiety and the bivalent lipid moiety can be bivalent derivatives of a chemical compound and a lipid, respectively. For example, the bivalent derivatives can be deprotonated derivatives of a chemical compound or lipid comprising a hydroxyl, thiol, amino, or carboxylic acid group.

[0125] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without changing the function and purpose of the inventions disclosed herein, and such changes and modifications are intended to be within the scope of this disclosure. III.) Drug Moiety

[0126] Another aspect of the present invention provides novel TB prodrug compound(s) comprising an ALK5 inhibitor, having the following formula designated TB4:

[0127] Those skilled in the art will appreciate that compounds are useful as ALK5 signal transduction inhibitors (e.g., inhibiting ALK5 and other family members). By way of brief background, ALK5, also known as TGFβ receptor I (TGFβRI), is a membrane-bound receptor belonging to the cytokine superfamily that acts on protein kinase receptors in the plasma membrane to induce a plethora of biological signals that regulate cell growth and death, differentiation, immune response, angiogenesis, and inflammation. Dysregulation of this pathway contributes to a wide range of pathologies, including cancer. TGFβ is an important tumor-regulating suppressor in epithelial cells, where it inhibits early proliferation and induces apoptosis. See FABREGAT, et. al., TGF-beta Signaling in Cancer Treatment, Curr. Pharm. Des. 20(17): pp. 2934-2947 (2014). Research has shown that the development of therapeutic compounds that target or block TGFβ production may be useful in the treatment of cancer. See HAQUE, et. al., Transforming growth factor-β: A Therapeutic Target for Cancer, Hum. Vaccin. Immunother., 13(8): pp. 1741-1750 (2017).

[0128] Based on the above, the present disclosure describes a class of TGFβ inhibitors.

[0129] In one embodiment, the drug moiety of the present disclosure comprises a compound having the following chemical structure (denoted TB4): [ka]

[0130] In one embodiment, the drug moieties of the present disclosure include protecting group intermediates en route to the final TB4 prodrug depicted in FIG.

[0131] In further embodiments, the drug moieties of the present disclosure include protecting group intermediates en route to the final TB4 prodrug depicted in FIG.

[0132] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without changing the function and purpose of the inventions disclosed herein, and such changes and modifications are intended to be within the scope of this disclosure. IV.) Lipids

[0133] Generally speaking, and for purposes of this disclosure, the term "lipid" is used in its broadest sense and includes several sub-categories of lipids, including, but not limited to, phospholipids / fatty acids. As recognized by those skilled in the art, phospholipids refer to a class of lipids that are the primary components of all cell membranes. Phospholipids are capable of forming lipid bilayers due to their amphipathic characteristics. The structure of a phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group that can be modified with simple organic molecules such as choline, ethanolamine, or serine. These two components are usually linked together by a glycerol molecule. A representative list of phospholipid / fatty acid(s) of the present invention is set forth in Table III.

[0134] As a brief background, at the most fundamental level, liposome properties depend on subtle physicochemical interactions among the various lipid species in their composition. Individual lipids can be combined to form a wide variety of superstructures, including bilayers, whose properties can be tailored to modulate drug release and membrane stability. In a simplified bilayer model, acyl chain length defines bilayer thickness and phase transition temperature (Tm), acyl chain saturation controls bilayer fluidity, and headgroup interactions affect inter- and intra-lipid forces. Liposome behavior can be tuned by incorporating synthetic lipids, such as lipid prodrugs, fusogenic lipids, and functionalizable lipids, into the bilayer. See Kohli, et al., J. Control Release, 0: pp. 274-287 (September 28, 2014).

[0135] In one embodiment of the present disclosure, the TB prodrug comprises a monovalent lipid moiety.

[0136] In one embodiment, the TB prodrug comprises a divalent lipid moiety.

[0137] In one embodiment, the lipid comprises cholesterol, which has the following chemical structure: [ka]

[0138] In one embodiment, the lipid comprises DPPG, which has the following chemical structure: [ka]

[0139] In one embodiment, the lipid comprises DMPG, which has the following chemical structure: [ka]

[0140] In one embodiment, the lipid comprises Lyso PC, which has the following chemical structure: [ka]

[0141] In one embodiment, the lipid comprises (Δ9-Cis)PG, which has the following chemical structure: [ka]

[0142] In one embodiment, the lipid comprises Soy Lyso PC, which has the following chemical structure: [ka]

[0143] In one embodiment, the lipid comprises PG, which has the following chemical structure: [ka]

[0144] In one embodiment, the lipid comprises C16 PEG2000 ceramide (Ceramde), which has the following chemical structure: [ka]

[0145] In one embodiment, the lipid comprises cholesterol hemisuccinate (“CHEMS”), which has the following chemical structure: [ka]

[0146] For reference, a complete list of the chemical formulas and abbreviation(s) of the lipids disclosed herein is provided in Table I.

[0147] In additional embodiments, the lipid comprises a phospholipid / fatty acid disclosed herein and set forth in Table III.

[0148] In a further embodiment, the lipid comprises stearic acid.

[0149] In addition, the TB prodrug and / or liposome(s) of the present disclosure may contain one or more helper lipids, also referred to herein as "helper lipid components." The helper lipid component is preferably selected from the group including phospholipids and steroids. Phospholipids are preferably diesters and monoesters of phosphoric acid. Preferred members of phospholipids are phosphoglycerides and sphingolipids. Steroids, as used herein, 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.

[0150] Without being bound by any theory, it should be noted that due to the specific molar percentage of helper lipid(s) contained in the lipid composition according to the present invention, which may be either PEG-free helper lipids or in particular PEG-containing helper lipids, surprising effects may be realized, more particularly when the content of any of this type of helper lipid is encompassed within the concentration ranges specified herein.

[0151] In a further aspect of the present 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. In a preferred embodiment, the lipid composition comprises any helper lipid or combination of helper lipids and any ALK5 inhibitor described herein (e.g., TB4). In a further embodiment, the composition according to the present invention containing nucleic acid(s) forms a lipoplex. In a preferred embodiment, the term lipoplex, as used herein, refers to a composition composed of a neutral or anionic lipid, a neutral helper lipid, and an ALK5 inhibitor of the present invention. 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., JR. Soc. Interface. 9, pp. 548-561 (2012), and MOCHIZUKI, et. See, e.g., W. et al., Biochimica et. Biophysica Acta, 1828, pp. 412-418 (2013).

[0152] In a preferred embodiment, the helper lipids of the present invention comprise the helper lipids set forth in Table II.

[0153] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is CHEMS and the drug moiety is TB4.

[0154] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is CHEMS, the drug moiety is TB4, and further comprises LU, wherein LU is a hydromethylcarbamate linker.

[0155] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is CHEMS, the drug moiety is TB4, and further comprises LU, which is a hydromethylcarbamate linker, and further comprises a helper lipid component, which comprises a helper lipid of Table II.

[0156] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is CHEMS, the drug moiety is TB4, and CHEMS is monovalent.

[0157] In one embodiment, the TB prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid and the drug moiety is TB4.

[0158] In one embodiment, the TB prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the drug moiety is TB4, and the stearic acid is monovalent.

[0159] In one embodiment, the TB prodrug comprises a lipid of the invention, wherein the lipid is stearic acid, the drug moiety is TB4, and further comprises LU, wherein LU is a hydromethylcarbamate linker.

[0160] In one embodiment, the TB prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the chemical composition is TB4, and further comprises LU, wherein LU is a hydromethylcarbamate linker, and further comprises a helper lipid component, wherein the helper lipid component comprises a helper lipid of Table II.

[0161] In one embodiment, the TB prodrug comprises a lipid of the present invention, where the lipid is stearic acid, the drug moiety is TB4, and the TB prodrug has the following chemical structure: [ka]

[0162] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without changing the function and purpose of the inventions disclosed herein, and such changes and modifications are intended to be within the scope of this disclosure. V.) Consolidated Units (“LUs”)

[0163] In some embodiments, the presently disclosed subject matter provides prodrugs that include drug-lipid conjugates that include biodegradable linkages, such as esters, thioesters, and other linkers known in the art.

[0164] Exemplary embodiments of ester chemistry are described herein. [ka]

[0165] In some embodiments, the prodrug is a drug-lipid conjugate, where the drug-lipid conjugate is cleaved by an esterase.

[0166] In one embodiment, the prodrugs of the present invention contain LU via a secondary amine, amide, or aniline using the following scheme: [ka] An exemplary synthesis is as follows: [ka] Cleavage of prodrug structures containing secondary amines, amides, or anilines is achieved via esterase hydrolysis of the secondary amine, amide, or aniline prodrug under the following exemplary synthesis: [ka] [In the formula, R1-NH-R2 can be any molecule with a secondary amine, amide, or aniline].

[0167] In one embodiment, the secondary amide nitrogen of the TB4 drug moiety is conjugated to CHEMS via a hydromethylcarbamate linker.

[0168] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without changing the function and purpose of the inventions disclosed herein, and such changes and modifications are intended to be within the scope of this disclosure. VI.) Nanocarriers

[0169] Generally speaking, and for purposes of this disclosure, nanocarriers(s) are within the scope of the present invention. Nanocarriers are nanomaterials used as transport modules for other substances, such as drugs. Commonly used nanocarriers include micelles, polymers, carbon-based materials, liposomes, and other substances. Due to their small size, nanocarriers can deliver drugs to otherwise inaccessible sites throughout the body. Nanocarriers may include polymer conjugates, polymer nanoparticles, lipid-based carriers, dendrimers, carbon nanotubes, and gold nanoparticles. Lipid-based carriers include both liposomes, solid lipid nanoparticles, and micelles. In certain embodiments, the nanocarrier is a liposome, lipid nanoparticle ("LNP"), or solid lipid nanoparticle ("SLNP").

[0170] In addition, nanocarriers are useful in the drug delivery process because they can deliver drugs to site-specific targets, delivering drugs to certain organs or cells rather than elsewhere. Site specificity has great therapeutic benefits because it prevents the drug being delivered from being delivered to the wrong location. In addition, nanocarriers are promising for use in chemotherapy because they can help reduce the harmful, broader-scale toxicity of chemotherapy to rapidly growing healthy cells throughout the body. Because chemotherapy drugs can be highly toxic to human cells, it is important that chemotherapy drugs be delivered to the tumor without being released into other parts of the body.

[0171] Generally speaking, there are four ways in which nanocarriers can deliver drugs, including passive targeting, active targeting, pH specificity, and temperature specificity.

[0172] Passive targeting refers to the ability of nanocarriers to travel through the tumor vasculature, be captured, and accumulate in the tumor. This accumulation is caused by enhanced permeability and retention effects. The leaky vasculature of tumors is a vascular network formed in tumors, which contains many small pores. These pores not only allow nanocarriers to enter, but also contain many bends that can capture nanocarriers. As more nanocarriers are captured, the drug accumulates at the tumor site. This accumulation allows a large dose of drug to be delivered directly to the tumor site.

[0173] Active targeting involves incorporating targeting modules, such as ligands or antibodies, onto the surface of the nanocarrier that are specific for a particular type of cell in the body. Nanocarriers generally have a high surface area to volume ratio and can incorporate multiple ligands on their surface.

[0174] In addition, certain nanocarriers release their drugs only within a specific pH range. pH specificity also allows nanocarriers to deliver drugs directly to tumor sites. This is because tumors are generally more acidic than normal human cells, with a pH of approximately 6.8. Normal tissue has a pH of approximately 7.4. Therefore, nanocarriers that release drugs only within a specific pH range can be used to release drugs only within the acidic tumor environment. A highly acidic environment causes the nanocarrier to degrade, releasing the drug. Generally, these nanocarriers do not release drugs in neutral or basic environments, effectively targeting the acidic environment of tumors while leaving normal somatic cells untouched. This pH sensitivity can also be achieved in micellar systems by adding copolymer chains to the micelles, which are determined to be pH-independent. See WU, et. al., Biomaterials, 34(4): 1213-1222 (2012). These micelle-polymer complexes also help prevent cancer cells from developing multidrug resistance: the low pH environment causes the micelle polymer to release rapidly, releasing most of the drug at once, rather than gradually as with other drug treatments.

[0175] Additionally, some nanocarriers have been shown to deliver drugs more effectively at certain temperatures. Because tumor temperatures are generally higher than the rest of the body, approximately 40°C, this temperature gradient helps act as a safeguard for tumor-specific site delivery. See REZAEI, et. al., Polymer, 53(16): 3485-3497 (2012).

[0176] 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 and hydrophilic molecules, exhibit minimal or no toxicity, and can increase drug duration by extending drug half-life and controlling drug release. Lipid nanoparticles can contain chemical modifications to avoid detection by the immune system (gangliosides or polyethylene glycol (PEG)) or to improve drug solubility. In addition, lipid nanoparticles can be formulated into pH-sensitive formulations to promote drug release in acidic environments, and can also be associated with small molecules or antibodies that recognize tumor cells or their receptors (e.g., folic acid (FoA)). Nanodrugs can also be used in combination with other therapeutic strategies to improve patient response. See Garcia-Pinel, et al., Nanomaterials 9(639) (2019).

[0177] In various embodiments, the silicasome drug carriers described herein comprise porous silica (or other material) nanoparticles (e.g., silica bodies having a surface and defining a plurality of pores suitable for receiving molecules therein) coated with a lipid bilayer. The fact that the nanoparticles are referred to as silica nanoparticles does not preclude materials other than silica from being incorporated within the silica nanoparticles. In some embodiments, the silica nanoparticles may be substantially spherical, with a plurality of pore openings on the surface that provide access to the pores. However, in various embodiments, the silica nanoparticles can have shapes other than substantially spherical. Thus, for example, in certain embodiments, the silica nanoparticles may be substantially ovoid, rod-shaped, substantially regular polygonal, irregular polygonal, etc.

[0178] Generally, silica nanoparticles include a silica body defining an exterior surface and sidewalls within the pores between pore openings. The pores can extend throughout the silica body to another pore opening, or the pores can extend only partially throughout the silica body, having a base defined by the silica body.

[0179] In some embodiments, the silica body is mesoporous. In other embodiments, the silica body is microporous. As used herein, "mesoporous" means having pores with diameters between about 2 nm and about 50 nm, while "microporous" means having pores with diameters smaller 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, e.g., therapeutic compounds, e.g., anti-cancer compounds, to attach or bind to the interior surfaces of the pores and be released from the silica body when used for therapeutic purposes. In some embodiments, the pores are substantially cylindrical.

[0180] In certain embodiments, the nanoparticles comprise a plurality of pores having a diameter between about 1 nm and about 10 nm, or between about 2 nm and about 8 nm. In certain embodiments, the nanoparticles comprise a plurality of pores having a diameter between about 1 nm and about 6 nm, or between about 2 nm and about 5 nm. Other embodiments include particles having a pore diameter of less than 2.5 nm.

[0181] In other embodiments, the pore size is between 1.5 and 2.5 nm. Silica nanoparticles with other pore sizes can be prepared, for example, by using different surfactants or swelling agents during the preparation of the silica nanoparticles. In various embodiments, nanoparticles can include particles as large as about 1000 nm (e.g., mean or median diameter (or another characteristic dimension)). In various embodiments, however, nanoparticles are typically less than 500 nm or less than about 300 nm, because particles larger than 300 nm generally may not be effective at entering living cells or vascular fenestrations. In certain embodiments, nanoparticles range in size from about 40 nm, or from about 50 nm, or from about 60 nm to about 100 nm, or to about 90 nm, or to about 80 nm, or to about 70 nm. In certain embodiments, nanoparticles range in size from about 60 nm to about 70 nm. Some embodiments include nanoparticles having average maximum dimensions between about 50 nm and about 1000 nm. Other embodiments include nanoparticles having average maximum dimensions between about 50 nm and about 500 nm. Other embodiments include nanoparticles having average maximum dimensions between about 50 nm and about 200 nm.

[0182] In some embodiments, the average maximum dimension is greater than about 20 nm, greater than about 30 nm, greater than about 40 nm, or greater than about 50 nm. Other embodiments include nanoparticles having average maximum dimensions less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 75 nm. As used herein, nanoparticle size refers to the average or median size of the primary particles as measured by transmission electron microscopy (TEM) or similar visualization techniques known in the art. Further examples of mesoporous silica nanoparticles include, but are not limited to, MCM-41, MCM-48, and SBA-15. See KATIYARE, et al., J. Chromotog. 1122(1-2): 13-20 (2006).

[0183] Methods for preparing porous silica nanoparticles are well known to those skilled in the art. In a specific embodiment, mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate (TEOS) with a template made from micellar rods. The result is a collection of nano-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).

[0184] In certain 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 certain embodiments, tetraethyl orthosilicate can also be used with an additional polymer monomer as a template. In certain embodiments, 3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS.

[0185] In certain embodiments, mesoporous silica nanoparticles are cores are synthesized by a modification of the sol / gel procedure described by MENG et. al. (2015) ACS Nemo, 9(4): 3540-3557.

[0186] Although the methods described herein have been demonstrated with respect to porous silica nanoparticles (e.g., mesoporous silica), those skilled in the art will recognize that similar methods can be used with other porous nanoparticles. Many other mesoporous materials that can be used in drug delivery nanoparticles are known to those skilled in the art. For example, in certain embodiments, mesoporous carbon nanoparticles could be utilized.

[0187] 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.).

[0188] Similarly, in certain embodiments, mesoporous polymer particles can be utilized. The synthesis of highly ordered mesoporous polymers and carbon frameworks from organic-organic assemblies of triblock copolymers and soluble low molecular weight phenolic resin precursors (resoles) via an evaporation-induced self-assembly strategy was reported by MENG, et al. (2006) Chem. Mat. 6(18): 4447-4464.

[0189] The nanoparticles described herein are exemplary and non-limiting, and numerous other lipid bilayer-coated nanoparticles will be available to those skilled in the art using the teachings provided herein.

[0190] In one embodiment, the present invention teaches a nanocarrier comprising a TB prodrug.

[0191] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises CHEMS.

[0192] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid.

[0193] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises CHEMS, and the liposome further comprises a TB prodrug.

[0194] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises CHEMS, and the liposome further comprises TB4.

[0195] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises an ALK5 inhibitor.

[0196] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TB4.

[0197] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TB4 (denoted LNP-TB4).

[0198] In a further embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TB4, wherein the liposome is co-formulated with ID3 (denoted LNP-TB4-ID3).

[0199] In a preferred embodiment, the lipid particles comprise solid lipid nanoparticles (SLNPs) that comprise liposomes containing a TB4 prodrug.

[0200] In one embodiment, the present invention teaches a nanocarrier comprising a solid lipid nanoparticle (“SLNP”), wherein the solid lipid nanoparticle comprises stearic acid, and the solid lipid nanoparticle further comprises TB4 (denoted as SLNP-TB4).

[0201] In a further embodiment, the present invention teaches a nanocarrier comprising solid lipid nanoparticles (“SLNPs”), wherein the solid lipid nanoparticles comprise stearic acid, and the solid lipid nanoparticles further comprise TB4, wherein the SLNPs are co-formulated with ID3 (denoted as SLNP-TB4-ID3).

[0202] In a further preferred embodiment, the solid lipid nanoparticles of the present invention comprise a composition having the following ratios: [Table 1] wherein lipid 1 comprises a TB4 prodrug, the lipid moiety comprises stearic acid, the helper lipid is a helper lipid described in Table II, and the stabilizer is polyvinyl alcohol (e.g., Moliwol 488), poloxamer (e.g., Pluronic F127), Tween® 80, PEG 400, and Kolliphor RH 40, and 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, ID1 inhibitors (e.g., ID3-STEA, ID3-CHEM, AR5-STEA, TR3-STEA, ID1-CHOL, etc.), MPLA, and tellutolimod.

[0203] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without changing the function and purpose of the inventions disclosed herein, and such changes and modifications are intended to be within the scope of this disclosure.

[0204] Additionally, the scope of the present disclosure teaches three possible treatment modalities using the formulated prodrugs of the present invention. See PCT Patent Application Publication WO2018 / 213631.

[0205] The first treatment modality involves combining a TB prodrug with another therapy (e.g., another formulated prodrug that inhibits ALK5 and other family members, a chemotherapeutic agent (e.g., an ICD-inducing chemotherapy agent), etc.) into a single liposome that allows for systemic (or local) biodistribution and drug delivery to the tumor site. The dual delivery approach achieved synergistic enhancement of adaptive and innate immunity, significantly improving animal survival. In certain embodiments, the nanocarrier comprises a vesicle (i.e., a lipid bilayer encapsulating a fluid).

[0206] A second treatment modality involves the local delivery to the tumor or peritumoral area of ​​drugs that inhibit ALK5 and other family members in combination with lipids (e.g., liposomes) containing inhibitors of ALK5 signaling.

[0207] The third treatment modality involves vaccination with dying cancer cells (e.g., KPC cells), where ALK5 inhibition is induced ex vivo.It has been discovered that such vaccination can generate a systemic immune response that can prevent tumor growth at distant sites, and can be adopted into non-immunized animals.Those skilled in the art can recognize and implement the methods of treatment modalities provided herein. VII.) Liposomes

[0208] In one aspect, the subject matter of the present disclosure is based on an approach to provide TB prodrugs of the present disclosure (see the section entitled "Prodrugs") that are suitable for incorporation into nanocarriers comprising a lipid coating layer to enhance the delivery of the corresponding prodrug and provide a combination therapy that includes the prodrug. Advantages of using the prodrugs of the present disclosure include facilitating controlled formulation into LNPs (e.g., liposomes) of the present disclosure. This allows the prodrug to remain in an inactive form during systemic circulation, allowing the liposome to release the active agent, for example, after phagocytosis by cells in a tumor.

[0209] 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 with lipid moieties that can form vesicular (e.g., liposomal) structures in aqueous solution or form components of the lipid bilayers that comprise liposomes. The liposomes can be used directly and provided as components in combined formulations (e.g., in combination with another drug moiety or therapeutic modality disclosed herein).

[0210] In certain embodiments, liposomes formulated with TB prodrugs comprise lipids, PHGP, vitamin E, cholesterol, and / or fatty acids.

[0211] In one embodiment, the liposome comprises cholesterol.

[0212] In one embodiment, the liposome comprises DSPC.

[0213] In one embodiment, the liposome comprises HSPCs.

[0214] In one embodiment, the liposome is DSPE-PEG 2000 Includes:

[0215] In one embodiment, the liposome comprises DPPG.

[0216] In one embodiment, the liposome comprises DMPG.

[0217] In one embodiment, the liposome is Lyso PC.

[0218] In one embodiment, the liposome is (Δ9-Cis)PG.

[0219] In one embodiment, the liposome comprises Soy Lyso PC.

[0220] In one embodiment, the liposome comprises PG.

[0221] In one embodiment, the liposome comprises PA-PEG3-mannose.

[0222] In one embodiment, the liposome comprises C16 PEG2000 ceramide.

[0223] In one embodiment, the liposome comprises MPLA.

[0224] In one embodiment, the liposome comprises CHEMS.

[0225] In one embodiment, the liposomes comprise stearic acid.

[0226] In one embodiment, the liposomes comprise a phospholipid listed in Table III.

[0227] In one embodiment, the liposome comprises TB4, further comprises CHEMS, and further comprises LU, wherein said LU is a hydromethylcarbamate linker.

[0228] In one embodiment, the liposome comprises TB4, further comprises stearic acid, and further comprises LU, wherein said LU is a hydromethylcarbamate linker.

[0229] In one embodiment, the liposome comprises TB4, further comprises CHEMS, and further comprises LU, wherein said LU is a hydromethylcarbamate linker, and further comprises a helper lipid listed in Table II.

[0230] In one embodiment, the liposome comprises TB4, further comprises stearic acid, and further comprises LU, wherein said LU is a hydromethylcarbamate linker, and further comprises a helper lipid listed in Table II.

[0231] In one embodiment, the liposomes of the present disclosure comprise a TB prodrug co-formulated with one or more additional immunomodulatory agents, including but 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.

[0232] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with an ICD-inducing chemotherapy drug.

[0233] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with an ICD-inducing chemotherapy drug selected from the list of doxorubicin (DOX), mitoxantrone (MTO), oxaliplatin (OXA), cyclophosphamide (CP), bortezomib, carfilzomib, or paclitaxel.

[0234] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a Toll receptor TLR agonist / prodrug.

[0235] In a further preferred embodiment, the Toll receptor TLR agonist / prodrug is selected from the group consisting of TR3, TR4, TR5, and TR6.

[0236] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a Toll receptor (TLR) agonist / prodrug selected from the list of resiquimod (R848), gardquimod, 852A, DSR6434, telluritolimod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD® or its prodrug.

[0237] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a PD-1 inhibitor / prodrug.

[0238] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a PD-1 inhibitor / prodrug selected from the list of AUNP12, CA-170, or BMS-986189 or prodrugs thereof.

[0239] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with an IDO-1 inhibitor / prodrug.

[0240] In a preferred embodiment, the liposomes contain 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 (BMS986205), MK-7162, LY-3381916, KHK-2455, HTI-1090, DN-1406131, or BGB-5777.

[0241] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with doxorubicin (DOX).

[0242] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with mitoxantrone (MTO).

[0243] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with doxorubicin (DOX) and a PD-1 prodrug.

[0244] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with mitoxantrone (MTO) and a PD-1 prodrug.

[0245] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with doxorubicin (DOX) and an IDO-1 prodrug.

[0246] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with mitoxantrone (MTO) and an IDO-1 prodrug.

[0247] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with doxorubicin (DOX) and a TLR agonist / prodrug.

[0248] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with mitoxantrone (MTO) and a TLR agonist / prodrug.

[0249] In a preferred embodiment, the liposomes contain doxorubicin (DOX) and a TB prodrug co-formulated with a PD-1 prodrug and a TLR agonist / prodrug.

[0250] In a preferred embodiment, the liposomes contain mitoxantrone (MTO) and a TB prodrug co-formulated with a PD-1 prodrug and a TLR agonist / prodrug.

[0251] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a TLR agonist / prodrug.

[0252] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with an IDO antagonist / prodrug.

[0253] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a CD1D agonist / prodrug.

[0254] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a TLR agonist / prodrug and a PD-1 prodrug.

[0255] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a TLR agonist / prodrug and an IDO-1 prodrug.

[0256] In a preferred embodiment, the liposomes contain a TB4 prodrug co-formulated with doxorubicin (DOX).

[0257] In a preferred embodiment, the liposomes contain a TB4 prodrug co-formulated with mitoxantrone (MTO).

[0258] In a preferred embodiment, the liposomes contain a TB4 prodrug co-formulated with doxorubicin (DOX) and / or and IDO prodrug and / or TLR agonist / prodrug.

[0259] In a preferred embodiment, the liposomes contain TB4 prodrugs co-formulated with mitoxantrone (MTO) and / or and IDO prodrugs and / or TLR agonists / prodrugs.

[0260] Those skilled in the art will recognize and understand that solubility is one of the most common problems that those skilled in the art face in the drug development process.The chemical conjugation of drugs / anticancer drugs through lipid molecules (i.e., lipid-based prodrugs) provides a platform for solving the problem of formulating drugs into aqueous suspensions.The main advantage of using lipid conjugation (lipid-based prodrugs) to deliver drug(s) is its ability to improve pharmacokinetics / half-life and targeted delivery.

[0261] By appropriately selecting the lipid molecules, lipid-based prodrug(s) can be integrated / formulated into liposomal formulations using techniques known in the art, which have many advantages over conventional drug delivery systems (KOHLI, et. al., J. Control Release, 0: pp 274-287 (September 28, 2014) and GARCIA-PINEL, et. al., Nanomaterials 9:638 (2019). The advantages of combining lipid-prodrugs with liposomes are twofold: (i) lipid-prodrug-containing liposomes not only increase the solubility of the drug / prodrug itself, but also (ii) have the ability to encapsulate multiple drugs (both hydrophilic and lipophilic) (see the section titled Nanocarriers).

[0262] For the purposes of this disclosure, the primary advantages of liposomal formulations are: i) The liposomal formulation is biocompatible / biodegradable and has no general toxicity; ii) flexibility and manipulation of size and surface charge depending on the desired purpose. The liposome formulation(s), for purposes of this disclosure, can have a size range of 40-150 nm in diameter and a surface charge ranging from -40 to +40 mV; and iii) The liposomes of the present invention have either single or multiple lipid-prodrugs as the lipid portion of the liposome(s). In addition, multiple drugs with different solubility profiles (hydrophilicity or lipophilicity) (e.g., acting by different mechanisms of action) can be formulated into these liposomes (either in the lipid bilayer or in the hydrophilic core).

[0263] As will be appreciated by those skilled in the art, all methods for making liposomes involve four basic steps: (i) drying the lipid from the organic solvent; (ii) dispersing lipids in an aqueous solution; (iii) purifying the resulting liposomes; and (iv) Analyzing the final product. See AKBARZADEH, et. al., Nanoscale Research Letters, 8:102 (2013).

[0264] Another aspect of the present invention discloses liposome encapsulation technology (LET), which is a delivery technology used to deliver drugs. LET is a method of producing submicroscopic foams called liposomes that encapsulate a variety of materials. These "liposomes" form a barrier around their contents that is resistant to oral and gastric enzymes, alkaline solutions, digestive fluids, bile salts, and intestinal flora produced in the human body, as well as free radicals. Thus, the liposomal contents are protected from oxidation and degradation. This protective phospholipid shield or barrier remains intact until the liposomal contents are delivered to the correct target gland, organ, or system, where they are utilized (see the section entitled "Nanocarriers").

[0265] In one embodiment, the liposome(s) of the present disclosure are synthesized using different ratios of TB prodrug, lipid, and / or lipid-prodrug. As disclosed herein, the TB prodrug may include a helper lipid disclosed herein (see, e.g., Table II).

[0266] In one embodiment, the liposome(s) of the present disclosure are synthesized using different ratios of TB prodrug, lipid, and / or lipid-prodrug. As disclosed herein, the TB prodrug may further comprise DSPE-PEG.

[0267] In a preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 2-1] [Table 2-2]

[0268] In a further preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 3]

[0269] In a further preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 4] Here, lipid 1 contains the TB4 prodrug and the lipid moiety contains CHEMS. In a further preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 5] Here, lipid 1 contains the TB4 prodrug and the lipid portion contains stearic acid.

[0270] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without changing the function and purpose of the inventions disclosed herein, and such changes and modifications are intended to be within the scope of this disclosure. VIII.) Pharmaceutical Formulations

[0271] As used herein, the term "drug" is synonymous with "pharmaceutical product." In certain embodiments, the liposomes of the present disclosure are processed into an encapsulated dosage form and given to a patient for the treatment of a disease.

[0272] Generally speaking, pharmaceutical formulation is the process of combining various chemicals into pure drug substances to produce the final drug product. Formulation studies involve developing preparations of the drug that are stable and tolerated by patients. For drugs taken orally, this usually involves incorporating the drug into tablets or capsules. It is important to understand that dosage forms contain a variety of other substances apart from the drug itself, and studies must be conducted to ensure that the drug is compatible with these other substances.

[0273] Excipients are inert substances used as carriers for the active ingredient of a drug product, in this case, liposomes containing a TB prodrug. In addition, excipients can be used to aid in the process by which the drug product is manufactured. The active ingredient is then dissolved or mixed with the excipient. Excipients are also sometimes used to increase the bulk of formulations containing highly potent active ingredients, allowing for convenient and accurate dosing. Once an active ingredient is purified, it cannot remain in purified form for a long time. Often, the active ingredient denatures, separates from the solution, or sticks to the sides of the container.

[0274] Excipients are added to stabilize the active ingredient to ensure that the active ingredient remains active and stable for a long enough period of time so that the product's shelf life makes it competitive with other products and safe for the end user. Examples of excipients include, but are not limited to, anti-adherents, binders, coatings, disintegrants, fillers, diluents, flavors, colorants, lubricants, and preservatives. The final formulation contains the active ingredient and excipients, which are then encapsulated into a pharmaceutical dosage form.

[0275] Pre-formulation involves characterizing the physical, chemical, and mechanical properties of a drug to select which other ingredients should be used in the preparation. Formulation studies then consider factors such as stability, particle size, polymorphism, pH, and solubility, as these can all affect bioavailability and therefore drug activity. The drug must be combined with inert additives in a manner that ensures the amount of drug present is consistent in each dosage unit (e.g., each vial). The dosage should have a uniform appearance.

[0276] These studies are unlikely to be completed by the time clinical trials begin. This means that simple formulations are developed early for use in Phase I clinical trials. These typically consist of vials, hand-filled capsules containing small amounts of drug and diluents. Evidence of the long-term stability of these formulations is not necessary because they are used (tested) within only a few days. However, the time between the time the final formulation is packaged and its delivery to patients can be months or even years, so long-term stability is crucial in supply chain management. What is called drug loading (i.e., the ratio of active drug to the total dose content) must be considered. Low drug loading can cause homogeneity problems. High drug loading can result in flow problems or require large capsules if the compound has a low bulk density. By the time Phase III clinical trials are reached, the drug formulation should have been developed to closely resemble the formulation that will ultimately be used in the market.

[0277] Knowledge of stability is essential up to this stage, and conditions must be developed to ensure the drug is stable in the formulation. If the drug proves unstable, the results of clinical trials will be invalid, since it will be impossible to know what the actual dose was. Stability studies are conducted to test whether temperature, humidity, oxidation, or photolysis (ultraviolet or visible light) have any effects, and the formulation is analyzed to determine whether any degradation products are formed. It is also important to check for any undesirable interactions between the formulation and the container. If plastic containers are used, tests are conducted to determine whether any of the ingredients are adsorbed onto the plastic and whether any plasticizers, lubricants, pigments, or stabilizers are leaching from the plastic into the formulation. The adhesive for the container label also needs to be tested to ensure that it does not leach from the plastic container into the formulation. Drug formulation practices can avoid some of the problems associated with oral administration. Drugs are usually taken orally as tablets or capsules. The drug (active substance) itself needs to dissolve in aqueous solution at a controlled rate. Factors such as particle size and crystalline morphology can significantly affect dissolution. Rapid dissolution is not always ideal. For example, a slower dissolution rate can extend the duration of action or avoid initial high plasma levels.

[0278] In some embodiments, nanocarriers (e.g., liposomes containing a TB prodrug) and / or liposomes co-formulated with an immunomodulatory agent are administered alone or in a mixture with a physiologically acceptable carrier (e.g., saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. For example, when used as an injection, nanocarriers can be formulated as a sterile suspension, dispersion, or emulsion using a pharmaceutically acceptable carrier. In certain embodiments, normal 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 such as albumin, lipoproteins, globulins, etc., to enhance stability. In compositions that include a saline or other salt-containing carrier, the carrier is preferably added after nanocarrier formation. Thus, after nanocarriers are formed and loaded with the appropriate drug(s), they can be diluted in a pharmaceutically acceptable carrier, such as normal saline. Similarly, the TB prodrug liposomes can be incorporated into a carrier that facilitates suspension (eg, emulsification, dilution, etc.) of the nanomaterial.

[0279] Pharmaceutical compositions can be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions, suspensions, dispersions, emulsions, etc. can be packaged for use or filtered under aseptic conditions. In certain embodiments, drug delivery nanocarriers (e.g., LB-coated nanoparticles) are freeze-dried, and the freeze-dried preparation is mixed with a sterile aqueous solution before administration. The composition can also contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, osmotic adjusters, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0280] Additionally, in certain embodiments, pharmaceutical formulations may contain lipid-protective agents that protect lipids against free radical and lipid peroxidation damage during storage. Lipophilic free-radical quenchers, such as alpha-tocopherol, and water-soluble iron-specific chelators, such as ferrioxamine, are suitable and contemplated herein. The concentration of nanocarriers (e.g., liposomes containing TB prodrugs) in pharmaceutical formulations can vary widely, e.g., less than approximately 0.05%, typically at least approximately 2-5%, up to 10-50%, or even up to 40%, or even up to 30% by weight, and is selected primarily based on fluid volume, viscosity, and the like, depending on the particular administration method selected. For example, concentrations may be increased to reduce the fluid load associated with treatment. This may be particularly desirable in patients with atherosclerosis-related congestive heart failure or severe hypertension. Alternatively, nanocarriers composed of irritating lipids may be diluted to low concentrations to reduce inflammation at the administration site. The amount of nanocarrier administered will depend on the particular drug used, the condition being treated, and the judgment of the clinician, but will generally be between about 0.01 mg and about 50 mg per kilogram of body weight, preferably between about 0.1 mg and about 5 mg per kg of body weight.

[0281] Those skilled in the art will recognize that the exact dosage will vary depending on the particular TB prodrug and any co-formulated immunomodulatory agent, as well as the desired medical effect, and patient factors such as age, sex, overall condition, etc. Taking these factors into consideration and using them, those skilled in the art will be able to readily establish effective therapeutic concentrations without undue experimentation.

[0282] For administration to humans (or non-human mammals) in the curative, ameliorative, retardative, or preventative treatment of the diseases described herein, the prescribing physician will ultimately determine the appropriate drug dosage for a given human (or non-human) subject, which can be expected to vary according to the individual's age, weight, and response, as well as the nature and severity of the patient's disease. In certain embodiments, the dosage of drug provided by the nanocarrier may be approximately the same as that used for the free drug. However, as noted above, the nanocarriers described herein can significantly reduce the toxicity and significantly increase the therapeutic window of the drug(s) administered therewith. Thus, in some cases, dosages greater than those prescribed for the free drug(s) are utilized.

[0283] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without changing the function and purpose of the inventions disclosed herein, and such changes and modifications are intended to be within the scope of this disclosure. IX.) Combination Therapy

[0284] As those skilled in the art will recognize and understand, the growth and survival of cancer cell can be influenced by multiple signal transduction pathways.Therefore, in order to treat such a condition, it is useful to combine different enzyme / protein / receptor inhibitors, which have different target preferences and modulate the activity of target.By targeting one or more signal transduction pathways (or one or more biological molecules involved in a given signal transduction pathway), it can reduce the likelihood of drug resistance occurring in cell population and / or reduce the toxicity of treatment.

[0285] Therefore, the liposomes containing 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 the treatment of diseases, such as cancer or infectious diseases. Examples of diseases and indications that can be treated using combination therapy include those described in this disclosure. Examples of cancer include, but are not limited to, solid tumors and liquid tumors, such as blood cancers. Examples of infectious diseases include viral infections, bacterial infections, fungal infections, or parasitic infections.

[0286] For example, liposomes containing the TB prodrugs of the present disclosure 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 (alpha, beta, gamma, delta), CSFIR, KIT, FLK-II, KDR / FLK-1, FLK-4, flt- 1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, TAM kinase (Axl, Mer, Tyro3), FLT3, VEGFR / Flt2 , Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf.

[0287] In further embodiments, liposomes containing the TB prodrugs of the present disclosure may be combined with one or more of the following inhibitors for the treatment of cancer or infectious diseases: Non-limiting examples of inhibitors that can be combined with compounds of the present disclosure for the treatment of cancer and infectious diseases 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-delta inhibitors (e.g., INCB50797 and INCB50465), PI3K-gamma inhibitors, e.g., PI3K-gamma selective inhibitors, Pim inhibitors (e.g., INCB53914), CSF1R inhibitors, TAM receptor tyrosine kinase inhibitors, and the like. These include histone deacetylase inhibitors (Tyro-3, Axl, and Mer), adenosine receptor antagonists (e.g., A2a / A2b receptor antagonists), HPK1 inhibitors, histone deacetylase inhibitors (HDACs), such as HDAC8 inhibitors, angiogenesis inhibitors, interleukin receptor inhibitors, bromo- 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 combinations thereof.

[0288] Additionally, liposomes containing the TB prodrugs of the present disclosure can further be used in combination with other methods of treating cancer, for example, by chemotherapy, radiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, or surgery.

[0289] Examples of immunotherapies include cytokine treatments (e.g., interferon, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccines, monoclonal antibodies, adoptive T cell transfer, Toll receptor agonists, STING agonists, oncolytic virotherapy, and immunomodulatory small molecules, including thalidomide or JAK1 / 2 inhibitors.

[0290] Liposomes containing TB prodrugs can be used in combination with one or more anti-cancer drugs, such as chemotherapeutic drugs, including abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezombi, bortezomib, intravenous busulfan, oral busulfan, calcitonin, capecitabine, and carboplatin. cisplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, Rulotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alpha-2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenylpropionate phenpropionate), nelarabine, nofetumomab, olaparib, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, ruxolitinib, rucaparib, sorafenib, streptozocin, sucralose These include any of the following: nitinib, sunitinib 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.

[0291] Other anti-cancer agent(s) include antibody therapeutics such as trastuzumab (Herceptin), antibodies against costimulatory molecules such as CTLA-4 (e.g., ipilimumab), antibodies against 4-1BB (e.g., urelumab, utomilumab), antibodies against PD-1 and PD-L1 / L2, or antibodies against cytokines (e.g., IL-10, TGF-beta, etc.).

[0292] Examples of antibodies against PD-1 and / or PD-L1 / L2 that can be combined with compounds of the present disclosure for the treatment of cancer or infectious diseases, such as viral, bacterial, fungal, and parasitic infections, include, but are not limited to, nivolumab, pembrolizumab, MPDL3280A, MEDI-4736, and SHR-1210.

[0293] In addition, liposomes containing the TB prodrugs of the present disclosure can be used in combination with one or more immune checkpoint inhibitors for the treatment of diseases such as cancer or infectious diseases. Exemplary immune checkpoint inhibitors include inhibitors of immune checkpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, 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.

[0294] In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137. In further embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, and VISTA. In further embodiments, the liposomes comprising the TB prodrugs provided herein can be used in combination with one or more agents selected from a KIR inhibitor, a TIGIT inhibitor, a LAIR1 inhibitor, a CD160 inhibitor, a 2B4 inhibitor, and a TGF-beta inhibitor. X.) Methods for delivering nanocarriers containing TB prodrugs to ALK5-expressing cells

[0295] As is known in the art, a wide variety of compositions and methods for using prodrugs and / or nanocarriers to kill tumor cells are known in the art. In the context of cancer, a typical method involves administering to a tumor-bearing mammal a biologically effective amount of a TB prodrug of the present disclosure, and / or a nanocarrier of the present disclosure comprising a TB prodrug.

[0296] An exemplary embodiment is a method of delivering a therapeutic agent to a cell expressing ALK5, comprising conjugating a drug moiety of the present disclosure to a lipid of the present disclosure via a linking unit to form a TB prodrug, and exposing the cell to the TB prodrug.

[0297] In one embodiment, a TB prodrug comprises a drug moiety of Formula I and a CHEMS conjugated via an LU that comprises a hydromethylcarbamate linker.

[0298] In one embodiment, the TB prodrug comprises a drug moiety of Formula I and stearic acid conjugated via an LU that comprises a hydromethylcarbamate linker.

[0299] In one embodiment, a TB prodrug comprises a drug moiety of Formula II and a CHEMS conjugated via an LU that comprises a hydromethylcarbamate linker.

[0300] In one embodiment, the TB prodrug comprises a drug moiety of Formula II and stearic acid conjugated via an LU that comprises a hydromethylcarbamate linker.

[0301] In one embodiment, a TB prodrug comprises a drug moiety of formula III and a CHEMS conjugated via an LU that comprises a hydromethylcarbamate linker.

[0302] In one embodiment, the TB prodrug comprises a drug moiety of Formula III and stearic acid conjugated via an LU that comprises a hydromethylcarbamate linker.

[0303] In one embodiment, the TB prodrug comprises a TB4 prodrug and the lipid moiety comprises a CHEMS conjugated via an LU that comprises a hydromethylcarbamate linker.

[0304] In one embodiment, the TB prodrug comprises a TB4 prodrug and the lipid moiety comprises stearic acid conjugated via a LU that comprises a hydromethylcarbamate linker.

[0305] Another exemplary embodiment is a method of treating an individual suspected of having metastatic cancer, comprising 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 linking unit, thereby exposing the cells to the TB prodrug.

[0306] In one embodiment, a TB prodrug comprises a drug moiety of Formula I and a CHEMS conjugated via an LU that comprises a hydromethylcarbamate linker.

[0307] In one embodiment, the TB prodrug comprises a drug moiety of Formula I and stearic acid conjugated via an LU that comprises a hydromethylcarbamate linker.

[0308] In one embodiment, a TB prodrug comprises a drug moiety of Formula II and a CHEMS conjugated via an LU that comprises a hydromethylcarbamate linker.

[0309] In one embodiment, the TB prodrug comprises a drug moiety of Formula II and stearic acid conjugated via an LU that comprises a hydromethylcarbamate linker.

[0310] In one embodiment, a TB prodrug comprises a drug moiety of formula III and a CHEMS conjugated via an LU that comprises a hydromethylcarbamate linker.

[0311] In one embodiment, the TB prodrug comprises a drug moiety of Formula III and stearic acid conjugated via an LU that comprises a hydromethylcarbamate linker.

[0312] In one embodiment, the TB prodrug comprises a TB4 prodrug and the lipid moiety comprises a CHEMS conjugated via an LU that comprises a hydromethylcarbamate linker.

[0313] In one embodiment, the TB prodrug comprises a TB4 prodrug and the lipid moiety comprises stearic acid conjugated via a LU that comprises a hydromethylcarbamate linker.

[0314] The TB prodrugs, liposomes, and co-formulated liposomes of the present disclosure inhibit TGFβ protein activity / protein interactions and are therefore useful for treating diseases and disorders associated with TGFβ activity and diseases and disorders associated with kinase inhibition. In further embodiments of the present disclosure, the TB prodrugs, liposomes, or pharmaceutically acceptable salts or stereoisomers thereof are useful for therapeutic administration to enhance, stimulate, and / or increase immunity in cancer, chronic infection, or sepsis, including enhancing responses to vaccinations.

[0315] In a further embodiment, the present disclosure provides a method for inhibiting T cell function of ALK5. The method includes administering to an individual or patient a TB prodrug, liposome, SLNP, and / or any of the formulas described herein (e.g., TB4 and / or TB4 prodrug), or a TB prodrug, liposome, SLNP, and nanoencapsulated ALK5 inhibitor prodrug listed in any of the claims and described herein, or a pharmaceutically acceptable salt or stereoisomer thereof. The TB prodrug, liposome, SLNP, and nanoencapsulated ALK5 inhibitor prodrug of the present disclosure can be used alone, in combination with other drugs or therapies, or as an adjuvant or neoadjuvant to treat diseases or disorders, including cancer and other diseases. Any of the TB prodrugs, liposomes, and nanoencapsulated TB prodrugs of the present disclosure, including any of the embodiments thereof, can be used for the uses and methods described herein.

[0316] Additionally, the TB prodrugs, liposomes, SLNPs, and nanoencapsulated TB prodrugs of the present disclosure inhibit ALK5 and / or T cell function, thereby blocking the TGFβ pathway.

[0317] In further embodiments, the present disclosure provides for the in vivo treatment of individuals or patients using TB prodrugs, liposomes, and nanoencapsulated TB prodrugs or salts or stereoisomers thereof to inhibit the growth of cancerous tumors.

[0318] The TB prodrugs, liposomal, and nanoencapsulated TB prodrugs, or any of the formulas described herein (e.g., TB4 prodrugs), or any of the claims and described herein, or salts or stereoisomers thereof, can be used to inhibit the growth of cancerous tumors.

[0319] Alternatively, the TB prodrugs, liposomal, SLNP, and nanoencapsulated TB prodrugs of the present disclosure, or any of the formulas described herein, or any of the claims listed and described herein (e.g., TB4 prodrugs), or salts or stereoisomers thereof, can be used in conjunction with other drugs or standard cancer treatments, as described in this disclosure.

[0320] In a further embodiment, the present disclosure provides a method for inhibiting tumor cell growth in vitro, comprising contacting tumor cells in vitro with a TB prodrug, liposomal, and nanoencapsulated TB prodrug of the present disclosure, or any of the formulas described herein (e.g., TB4 prodrug), or a TB prodrug, liposomal, SLNP, and nanoencapsulated TB prodrug listed in any of the claims and described herein, or a salt or stereoisomer thereof.

[0321] In a further embodiment, the present disclosure provides a method for inhibiting the growth of tumor cells in a patient, the method comprising contacting tumor cells with a TB prodrug, liposomal, and nanoencapsulated TB prodrug of the present disclosure, or any of the formulas described herein (e.g., TB4 prodrug), or a TB prodrug, liposomal, SLNP, and nanoencapsulated TB prodrug listed in any of the claims and described herein, or a salt or stereoisomer thereof. XI.) Methods of Treating Cancer and Other Immunological Disorders

[0322] Another embodiment of the present disclosure is a method for treating cancer. The method comprises administering to a patient a therapeutically effective amount of a liposome containing a TB prodrug (i.e., a TB4 prodrug), a compound listed in any of the claims and described herein, or a salt thereof. Examples of cancers include cancers whose growth can be inhibited using the ALK5 inhibitors of the present disclosure and the TB prodrugs of the present disclosure, and cancers that typically respond to immunotherapy.

[0323] In some embodiments, the disclosure provides a method of 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 TB prodrug (i.e., a TB4 prodrug), a compound or composition recited in any of the claims and described herein, or a salt thereof.

[0324] In one embodiment, the method(s) comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0325] In a further embodiment, the method(s) comprise administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0326] Non-limiting examples of cancers treatable using liposomes comprising TB prodrugs, TB prodrugs and co-formulated liposomes of the present disclosure include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma of soft tissue, urethral cancer, and penile cancer. Cancer includes but is not limited to, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumor, lymphocytic lymphoma, bladder cancer, kidney or urethral cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, including asbestos-induced cancer, and the combination of above-mentioned cancer.Compound of the present disclosure is also useful for treating metastatic cancer, particularly the metastatic cancer that expresses ALK5.

[0327] In some embodiments, cancers treatable with the liposomes or TB prodrugs of the present disclosure include melanoma (e.g., metastatic malignant melanoma), kidney 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), head and neck squamous cell carcinoma, urothelial cancer (e.g., bladder), and microsatellite instability-high (MSI) cancer. high In addition, the present disclosure includes refractory or recurrent malignancies whose growth can be inhibited using the liposomes or TB prodrugs or co-formulated liposomes of the present disclosure.

[0328] In additional embodiments, cancers treatable 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., lymphoma, leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular NHL), Hodgkin's lymphoma, or multiple myeloma), and combinations of the foregoing cancers.

[0329] In further embodiments, cancers treatable using formulated and / or co-formulated liposomes or TB prodrugs of the present disclosure include, but are not limited to, cholangiocarcinoma, bile duct carcinoma, 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 cavity cancer, mouth cancer, throat cancer, laryngeal cancer, lip cancer, mesothelioma, neck cancer, nasal cavity cancer, eye cancer, intraocular melanoma, pelvic cancer, rectal cancer, renal cell carcinoma, salivary gland cancer, paranasal sinus cancer, spinal cancer, tongue cancer, tubular carcinoma, urethral cancer, and ureteral cancer.

[0330] Additionally, in some embodiments, the formulated and / or co-formulated liposomes of the present disclosure, or TB prodrugs, can be used to treat sickle cell disease and sickle cell anemia.

[0331] Furthermore, in some embodiments, diseases and indications treatable using the formulated and / or co-formulated liposomes of the present disclosure, or TB prodrugs, include, but are not limited to, hematological cancers, sarcoma, lung cancer, gastrointestinal cancer, genitourinary cancer, liver cancer, bone cancer, nervous system cancer, gynecological cancer, and skin cancer.

[0332] 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's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma, myeloproliferative disorders (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocytosis (ET)), myelodysplastic syndromes (MDS), T-cell acute lymphoblastic lymphoma (T-ALL), and multiple myeloma (MM).

[0333] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, hamartoma, and teratoma.

[0334] Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatous hamartoma, and mesothelioma.

[0335] 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 tumor, vipoma), small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), colon cancer (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), and colorectal cancer.

[0336] Exemplary genitourinary cancers include kidney cancer (adenocarcinoma, Wilms' 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, adenomatous tumor, lipoma).

[0337] Exemplary liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0338] Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor.

[0339] Exemplary nervous system cancers include cranial cancers (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), meningeal cancers (meningioma, meningiosarcoma, gliomatosis), brain cancers (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pinealoma), glioblastoma, glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), and spinal cancers (neurofibroma, meningioma, glioma, sarcoma), as well as neuroblastoma and Lhermitte-Duclos disease.

[0340] Exemplary gynecological cancers include uterine cancer (endometrial carcinoma), cervical cancer (cervical carcinoma, pre-tumor cervical dysplasia), ovarian cancer (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulvar cancer (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), and fallopian tube cancer (carcinoma).

[0341] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, molar dysplastic nevus, lipoma, angioma, dermatofibroma, and keloid. In some embodiments, diseases and indications treatable 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 carcinoma.

[0342] Additionally, TGFβ, ALK5, and / or kinase pathway blockade using the formulated and / or co-formulated liposomes of the present disclosure, or TB prodrugs, can also be used to treat infectious diseases, such as viral, bacterial, fungal, and parasitic infections.

[0343] The present disclosure provides a method for treating an infectious disease, e.g., a viral infection, comprising administering to a patient a therapeutically effective amount of any of the formulated and / or co-formulated liposomes recited in any of the claims and described herein, or a TB prodrug or formula (i.e., a TB4 prodrug), or salt thereof, described herein.

[0344] In one embodiment, the method(s) comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0345] In a further embodiment, the method(s) comprise administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0346] Examples of viruses that cause infectious diseases treatable by the methods of the present disclosure include, but are not limited to, human immunodeficiency virus, human papillomavirus, influenza, hepatitis A, B, C, or D virus, adenovirus, poxvirus, herpes simplex virus, human cytomegalovirus, severe acute respiratory syndrome virus, Ebola virus, and measles virus. In some embodiments, viruses causing infections treatable by the methods of the present disclosure include, but are not limited to, hepatitis (types A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), 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 contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0347] Additionally, the present disclosure provides a method for treating a bacterial infection, comprising administering to a patient a therapeutically effective amount of any of the formulated and / or co-formulated liposomes or TB prodrugs or formulas described herein (i.e., TB4 prodrugs), or salts thereof, as recited in any of the claims and described herein.

[0348] Examples of infectious disease-causing pathogens treatable by the methods of the present disclosure include, but are not limited to, chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and lyme disease bacteria.

[0349] Additionally, the present disclosure provides a method for treating a fungal infection, comprising administering to a patient a therapeutically effective amount of any of the formulated and / or co-formulated liposomes or TB prodrugs or formulas described herein (i.e., TB4 prodrugs) recited in any of the claims, or salts thereof.

[0350] In one embodiment, the method(s) comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0351] In a further embodiment, the method(s) comprise administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0352] Examples of pathogenic fungi causing infections treatable by the methods of the present disclosure include, but are not limited to, Candida (such as albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (such as fumigatus and Niger), Mucorales (such as Mucor, absidia, and rhizophus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0353] Additionally, the present disclosure provides a method for treating a parasitic infection comprising administering to a patient a therapeutically effective amount of any of the formulated and / or co-formulated liposomes or TB prodrugs or formulas described herein (i.e., TB4 prodrugs), or salts thereof, as recited in any of the claims and described herein.

[0354] In one embodiment, the method(s) comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0355] In a further embodiment, the method(s) comprise administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof.

[0356] Examples of pathogenic parasites that cause infections treatable by the methods of the present disclosure include, but are not limited to, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0357] In a further set of embodiments within the scope of the present disclosure, formulated and / or co-formulated nanocarriers, liposomes, SLNPs or TB prodrugs, or any of the formulas described herein (i.e., TB4 prodrugs), are useful for preventing or reducing the risk of developing any of the diseases mentioned in this disclosure, for example, in individuals who may be predisposed to the disease, condition or disorder but who have not yet experienced or exhibited any pathology or symptomatic symptoms of the disease.

[0358] In one embodiment, the method(s) comprises administering to the patient a therapeutically effective amount of LNP-TB4 or a salt thereof.

[0359] In a further embodiment, the method(s) comprise administering to the patient a therapeutically effective amount of SLNP-TB4 or a salt thereof. XII.) Kits / Manufactured Products

[0360] Kits for use in the laboratory, prognostic, preventive, diagnostic, and therapeutic applications described herein are within the scope of the present invention. Such kits can include a carrier, package, or container configured to receive one or more containers, such as vials, tubes, etc., each containing one of the separate elements used in the method, along with a label or insert containing instructions for use, e.g., for use as described herein. For example, the container(s) can contain formulated and / or co-formulated nanocarriers that are detectably labeled or can be detectably labeled and / or are loaded with a TB prodrug of the present disclosure. The kit can include a container containing a drug unit. The kit can include all or a portion of the formulated and / or co-formulated nanocarriers and / or TB prodrug.

[0361] Kits of the present invention typically include the container described above and one or more other containers associated therewith that contain materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, shipping containers, packages, containers, vials, and / or tube labels listing the contents and / or instructions for use, and package inserts containing instructions for use.

[0362] A label can be present on or associated with the container to indicate that the composition is used for a particular therapeutic or non-therapeutic application, such as prognostic, preventative, diagnostic, or laboratory application, and can indicate directions for either in vivo or in vitro use, such as those described herein. Directions for use and other information can also be included on insert(s) or label(s) included with or on the kit. The label can be on or associated with the container. The label can be on the container when letters, numbers, or other symbols forming the label are molded or etched into the container itself. The label can also be associated with the container when present in a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for the diagnosis, treatment, prevention, or prognosis of a condition, such as cancer or other immunological disorder.

[0363] The terms "kit" and "article of manufacture" can be used synonymously.

[0364] In another embodiment of the present invention, an article(s) of manufacture containing a composition, e.g., formulated and / or co-formulated nanocarriers, and / or TB prodrugs, is within the scope of this disclosure. The article of manufacture typically includes 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 formulated and / or co-formulated nanocarriers loaded with a TB prodrug.

[0365] The container may alternatively hold a composition effective for treating, diagnosing, prognosing or preventing a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition may be a TB prodrug and / or formulated and / or co-formulated nanocarriers loaded with a TB prodrug disclosed herein.

[0366] The article of manufacture may further comprise 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 standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and / or package inserts containing instructions for indications and / or use.

[0367] In one embodiment, the kit or article of manufacture comprises LNP-TB4 and / or a therapeutically effective amount of LNP-TB4.

[0368] In one embodiment, the kit or article of manufacture comprises SLNP-TB4 and / or a therapeutically effective amount of SLNP-TB4. Illustrative Embodiments

[0369] The provided embodiments include the following: 1) (i) a drug moiety; (ii) a lipid moiety, and (iii) Consolidated Unit (“LU”) wherein the drug moiety comprises a TGFβ antagonist, and the LU conjugates the drug moiety to a lipid moiety.

[0370] 2) The TB prodrug of claim 1, further comprising the chemical structure set forth in Formula I.

[0371] 3) The TB prodrug of claim 1, further comprising the chemical structure set forth in Formula II.

[0372] 4) The TB prodrug of claim 1, further comprising the chemical structure set forth in Formula III.

[0373] 5) The TB prodrug of claim 1, wherein the drug moiety comprises the chemical structure set forth as TB4.

[0374] 6) The TB prodrug of claim 1, wherein LU is a hydromethylcarbamate linker.

[0375] 7) The TB prodrug of claim 1, wherein the lipid moiety comprises a lipid set forth in Table I.

[0376] 8) The TB prodrug of claim 1, wherein the lipid moiety comprises a lipid set forth in Table III.

[0377] 9) The TB prodrug of claim 1, wherein the lipid moiety comprises CHEMS.

[0378] 10) The TB prodrug of claim 1, wherein the lipid moiety comprises stearic acid.

[0379] 11) The TB prodrug of claim 1, wherein the drug moiety comprises the chemical structure described as TB4, the lipid moiety comprises stearic acid, and the compound has the following chemical structure: [ka]

[0380] 12) (i) a drug moiety containing TB4 (ii) a lipid moiety containing CHEMS, and (iii) LUs containing hydromethylcarbamate linkers 1. A TB prodrug composition comprising:

[0381] 13)(i) a drug moiety containing TB4 (ii) a lipid portion comprising stearic acid, and (iii) LUs containing hydromethylcarbamate linkers 1. A TB prodrug composition comprising:

[0382] 14) The TB prodrug composition of claim 13, having the following chemical structure: [ka]

[0383] 15) A nanocarrier comprising a TB prodrug, which releases an active ALK5 inhibitor after cleavage of the LU.

[0384] 16) The nanocarrier of claim 15, wherein the LU is a hydromethylcarbamate linker.

[0385] 17) The nanocarrier of claim 15, further comprising a helper lipid, wherein the helper lipid is listed in Table II.

[0386] 18) The nanocarrier of claim 15, wherein the TB prodrug comprises TB4.

[0387] 19) The nanocarrier of claim 15, which is a liposome.

[0388] 20) The liposome of claim 19, wherein the TB prodrug comprises TB4 and is designated LNP-TB4.

[0389] 21) The liposome of claim 19, further co-formulated with one or more immunomodulatory agents or lipid-prodrugs thereof, wherein the immunomodulatory agent is selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, toll receptor agonists, STING agonists, CTLA-4 inhibitors, IDO inhibitors, PD-1 / PD-L1 inhibitors, CD1D agonists and / or prodrugs thereof.

[0390] 22) The liposome of claim 19, 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.

[0391] 23) The liposome of claim 19, further comprising DOX.

[0392] 24) The liposome of claim 19, further comprising MTO.

[0393] 25) The liposome of claim 22, further comprising DOX.

[0394] 26) The liposome of claim 22, further comprising MTO.

[0395] 27) The liposome of claim 19, 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), gardquimod, 852A, DSR6434, telluritolimod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD®.

[0396] 28) The liposome of claim 19, 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.

[0397] 29) A kit comprising the liposome according to any one of claims 15 to 28.

[0398] 30) The nanocarrier of claim 15, which is a solid lipid nanoparticle (SLNP).

[0399] 31) The SLNP of claim 30, wherein the TB prodrug comprises TB4 and is designated SLNP-TB4.

[0400] 32) The SLNP of claim 30, further co-formulated with one or more immunomodulatory agents or lipid-prodrugs thereof, wherein the immunomodulatory agent is selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, Toll receptor agonists, STING agonists, CTLA-4 inhibitors, IDO inhibitors, PD-1 / PD-L1 inhibitors, CD1D agonists and / or prodrugs thereof.

[0401] 33) The SLNP of claim 30, 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.

[0402] 34) The SLNP of claim 30, further comprising DOX.

[0403] 35) The SLNP of claim 30, further comprising MTO.

[0404] 36) The SLNP of claim 33, further comprising DOX.

[0405] 37) The SLNP of claim 33, further comprising MTO.

[0406] 38) The SLNP of claim 30, wherein the liposome is further co-formulated with a toll receptor agonist or a lipid-prodrug thereof, and the toll receptor agonist is selected from the group consisting of resiquimod (R848), gardquimod, 852A, DSR6434, telluritolimod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD®.

[0407] 39) The SLNP of claim 30, wherein the liposome is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid-prodrug thereof, and the PD-1 / PD-L1 antagonist is selected from the group consisting of AUNP12, CA-170, or BMS-986189.

[0408] 40) A kit comprising the SLNP of any one of claims 30 to 39.

[0409] 41) A method of treating a subject suffering from or diagnosed with cancer, comprising: (i) administering to a subject in need of such treatment an effective amount of a nanocarrier, wherein the nanocarrier comprises a TB prodrug; (ii) a pharmaceutically acceptable salt thereof; A method comprising:

[0410] 42) The method of claim 41, wherein the TB prodrug comprises a TB4 prodrug.

[0411] 43) The method of claim 41, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an ICD-inducing chemotherapy drug.

[0412] 44) The method of claim 41, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an immunomodulatory agent.

[0413] 45) The method of claim 41, wherein the nanocarrier is a liposome.

[0414] 46) The method of claim 45, wherein the liposome is LNP-TB4.

[0415] 47) The method of claim 41, wherein the nanocarrier is a solid lipid nanoparticle.

[0416] 48) The method of claim 47, wherein the liposome is SLNP-TB4.

[0417] 49) A method of treating a subject suffering from or diagnosed with cancer, comprising: (iii) administering to a subject in need of such treatment an effective amount of a nanocarrier, wherein the nanocarrier comprises a TB prodrug; and (iv) a pharmaceutically acceptable salt thereof; A method comprising:

[0418] 50) The method of claim 49, wherein the TB prodrug comprises a TB4 prodrug.

[0419] 51) The method of claim 49, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an ICD-inducing chemotherapy drug.

[0420] 52) The method of claim 49, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an immunomodulatory agent.

[0421] 53) The method of claim 49, wherein the nanocarrier is a solid lipid nanoparticle ("SLNP").

[0422] 54) The method of claim 53, wherein the SLNP is SLNP-TB4.

[0423] 55) The method of claim 49, wherein the nanocarrier is a liposome.

[0424] 56) The method of claim 55, wherein the liposome is LNP-TB4.

[0425] 57) A TB4 prodrug having the following chemical structure: [ka]

[0426] 58) A liposome comprising the TB4 prodrug of claim 57.

[0427] 59) A liposome comprising the TB4 prodrug of claim 57 and further comprising a helper lipid.

[0428] 60) The liposome of claim 59, wherein the helper lipid is listed in Table II.

[0429] 61) A solid lipid nanoparticle (SLNP) comprising the TB4 prodrug of claim 57.

[0430] 62) A TB4 prodrug having the following chemical structure: [ka]

[0431] 63) A liposome comprising the TB4 prodrug of claim 62.

[0432] 64) A liposome comprising the TB4 prodrug of claim 62 and further comprising a helper lipid.

[0433] 65) The liposome of claim 64, wherein the helper lipid is listed in Table II.

[0434] 66) The liposome of claim 62, designated LNP-TB4.

[0435] 67) A solid lipid nanoparticle (SLNP) comprising the TB4 prodrug of claim 62.

[0436] 68) The SLNP of claim 67, designated SLNP-TB4.

[0437] 69) The liposome of claim 63 co-formulated with AR5.

[0438] 70) The liposome of claim 63 co-formulated with TR6.

[0439] 71) The liposome of claim 63 co-formulated with ID3.

[0440] 72) The liposome of claim 63 co-formulated with PD3.

[0441] 73) The liposome of claim 63 co-formulated with MTO.

[0442] 74) The liposome of claim 63 co-formulated with MTO and ID3.

[0443] 75) The liposome of claim 63 co-formulated with MTO and AR5.

[0444] 76) The SLNP of claim 68 co-formulated with MTO.

[0445] 77) The SLNP of claim 68 co-formulated with AR5.

[0446] 78) The SLNP of claim 68 co-formulated with ID3.

[0447] 79) The SLNP of claim 68 co-formulated with PD3.

[0448] 80) The SLNP of claim 68 co-formulated with MTO and ID3.

[0449] 81) The SLNP of claim 68 co-formulated with MTO and AR5. [Example]

[0450] Various aspects of the present invention are further described and illustrated by the following several examples, none of which are intended to limit the scope of the invention.

[0451] Example 1 Chemical synthesis of TB4 prodrugs containing stearic acid

[0452] The chemical synthesis of TB4 prodrugs containing stearic acid is performed using the following protocol. First, compound (1) is treated with compound (2) and KHMDS to give intermediate 3. Intermediate 3 is then treated sequentially with DMF dimethyl acetal and then hydrazine hydrate to give intermediate 4. Intermediate 4 is then treated with trityl chloride to give intermediate 5. Intermediate 5 is then treated with reagent (6) and palladium triphenylphosphine, followed by hydrolysis with sodium hydroxide to give intermediate 7. Intermediate 7 is then treated with reagent (8) and EDCI / HOBt to give intermediate 9. Intermediate 9 is then treated with lithium hexamethyldisilazide, followed by treatment with chloromethyl chloroformate (10) to give intermediate 11. Finally, intermediate 11 is treated sequentially with stearic acid, then silver carbonate, then sodium iodide in DMF at 80°C, followed by treatment with HCl in methanol to give the final stearic acid-containing prodrug TB4 (12) (Figure 1). The synthesis described in this example yields the TB4 prodrug with the following chemical structure: [ka]

[0453] Example 2 Chemical synthesis for protecting group intermediates en route to TB4 prodrugs

[0454] The following protocol was used to synthesize the protected intermediate. Briefly, to a solution of TB4 (13.0 g, 30.5 mmol, 1.00 equiv.) in DCM (1.50 L) was added BocO (8.00 g, 36.7 mmol, 8.42 mL, 1.20 equiv.) and DMAP (746 mg, 6.11 mmol, 0.20 equiv.) at 15 °C. After the addition, the reaction mixture was stirred at 30 °C for 12 h. TLC (dichloromethane:methanol = 10:1) showed that TB4 (R f =0.2) is consumed and one large new spot (R f=0.5). LCMS confirmed that the desired mass (RT=0.850 min) was detected. The reaction mixture was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (SiO, dichloromethane:methanol=50:1 to 30:1, R f =0.5), compound 2 (13.0 g, 24.7 mmol, 80.9% yield) was obtained as a white solid. The resulting compound is depicted in Figure 2.

[0455] Example 3 Chemical synthesis for protecting group intermediates en route to TB4 prodrugs

[0456] In another embodiment, additional protected group intermediates were synthesized in the following manner. Briefly, to a solution of compound 3 (4.50 g, 7.28 mmol, 1.00 equiv.) in DCM (225 mL) was added TFA (16.6 g, 145 mmol, 10.8 mL, 20.0 equiv.) at 25 °C. After the addition, the reaction mixture was stirred at 25 °C for an additional 4 h. LCMS confirmed that the reaction was complete and the desired mass was detected (RT = 0.874 min). The reaction mixture was adjusted to pH = 7-8 with saturated NaHCO3 solution and extracted with DCM (150 mL × 2). 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. The resulting compound is depicted in Figure 3.

[0457] Example 4 Chemical synthesis of TB4 prodrugs containing stearic acid

[0458] The chemical synthesis of TB4 prodrug containing stearic acid was performed using the following protocol. First, to a solution of TB4 (13.0 g, 30.5 mmol, 1.00 equiv.) in DCM (1.50 L), BocO (8.00 g, 36.7 mmol, 8.42 mL, 1.20 equiv.) and DMAP (746 mg, 6.11 mmol, 0.20 equiv.) were added at 15 °C. After the addition, the reaction mixture was stirred at 30 °C for 12 h. TLC (dichloromethane:methanol = 10:1) showed that TB4 (R f =0.2) is consumed and one large new spot (R f =0.5). LCMS showed that the desired mass (RT=0.850 min) was detected. The reaction mixture was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (SiO, dichloromethane:methanol=50:1 to 30:1, R f=0.5), compound 2 (13.0 g, 24.7 mmol, 80.9% yield) was obtained as a white solid. Next, to a solution of compound 2 (9.00 g, 17.1 mmol, 1.00 equiv.) in DCM (1000 mL) was added LiHMDS (1 M, 37.6 mL, 2.20 equiv.) at -70 °C under N for 1 h. To the mixture was added compound 2a (9.36 g, 72.5 mmol, 6.45 mL, 4.24 equiv.) in DCM (50 mL) at -70 °C. The mixture was stirred at -70 °C for 6 h. The mixture was then stirred at 15 °C for an additional 6 h. LCMS showed that 18.4% of compound 2 (RT = 1.038 min) remained and 33.1% of the desired mass (RT = 1.195 min) was detected. The mixture was poured into saturated NH4Cl solution (500 mL) and extracted with DCM (200 mL × 2). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, concentrated, and confirmed by LCMS. The crude product was purified by reverse-phase MPLC (ACN / H2O, TFA condition). After removing ACN, the aqueous phase was extracted with ethyl acetate (300 mL × 3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, and concentrated under reduced pressure to give compound 3 (6.20 g, 10.0 mmol, 29.2% yield) as a yellow solid, which was confirmed by LCMS. Next, to a solution of compound 3 (4.50 g, 7.28 mmol, 1.00 equiv.) in DCM (225 mL), TFA (16.6 g, 145 mmol, 10.8 mL, 20.0 equiv.) was added at 25 °C. After the addition, the reaction mixture was stirred at 25 °C for an additional 4 h. LCMS showed that the reaction was complete and the desired mass was detected (RT = 0.874 min). The reaction mixture was adjusted to pH = 7-8 with saturated NaHCO3 solution and extracted with DCM (150 mL × 2). 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, which was used directly for the next step without further purification.Finally, to a solution of compound 4 (3.50 g, 6.76 mmol, 1.00 equiv.) and stearic acid (2.88 g, 10.1 mmol, 3.41 mL, 1.50 equiv.) in ACN (175 mL) was added DIEA (2.62 g, 20.3 mmol, 3.53 mL, 3.00 equiv.). After the addition, the reaction mixture was stirred at 80 °C for an additional 36 h. LCMS showed that compound 4 was not completely converted, and the desired mass (RT = 1.383 min) was detected. The mixture was concentrated under reduced pressure at 45 °C to give the crude product, which was confirmed by LCMS. The crude product was combined with ET34822-17 and purified by reverse-phase MPLC (MeOH / HO / TFA conditions) and then concentrated to give the crude product. The crude product was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=1:1 to ethyl acetate:methanol=10:1) and purified by TLC (ethyl acetate:methanol=10:1, R. f =0.3). TB4 containing stearic acid (1.3 g, 1.59 mmol, 21.2% yield, 93.9% purity) was obtained as a yellow gum. 1 The compound was confirmed by H NMR, FNMR, LCMS, and HPLC. The resulting compound and synthesis are described in Figure 7. The synthesis described in this example yields a TB4 prodrug with the following chemical structure: [ka]

[0459] Example 5 Synthesis and characterization of LNP-TB4 liposomes

[0460] In another experiment, liposomes containing the TB4 prodrug (denoted LNP-TB4) were synthesized in the following manner. Briefly, in the first step, lipid stock solutions of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), CHOL, and DSPE-PEG were prepared separately in ethanol (20 mg / ml). Because the TB4 prodrug (TB4 + stearic acid) was not soluble in ethanol, its stock solution was prepared in acetonitrile (20 mg / ml). The lipid mixture of POPC, CHOL, TB4 + stearic acid, and DSPE-PEG was mixed together in a molar ratio of 51:29:16.5:3.5 and then diluted with ethanol to obtain a total lipid concentration of 10 mg / ml. This lipid mixture was heated at 55-60°C using a heating block attachment in a microfluidizer. Similarly, an aqueous phase containing 1 mM PBS buffer was preheated to 55-60°C and then passed through the microfluidic cartridge at a flow rate of 5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed using a 12 kDa cutoff dialysis membrane (Sigma-Aldrich) against DI water for at least 24 hours. The dialysis water was exchanged at least five times over the 24-hour period to maximize solvent removal. After solvent removal, LNP-TB4 was optionally concentrated using an Amicon centrifugal filter device (10 kDa cutoff, 3000 g).

[0461] LNP-TB4 liposomes were characterized using a Malvern Zetasizer (Malvern The PDI was determined using a HPLC-MS / MS instrument (Microfluidics Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-TB4 liposomes (liposome concentrations were 0.5–1 mg / ml) were placed in a clear, four-sided plastic cuvette and analyzed directly at 25 °C. The results, shown in Figure 8, indicate that the Z-average size of the nanoparticles was approximately 87 nm and the PDI was approximately 0.265.

[0462] Additionally, the zeta potential of LNP-TB4 liposomes in aqueous dispersion was determined using a Malvern Zetasizer instrument (Malvern Instrumentation Co, Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell available with the Zetasizer. Measurements were performed at 25°C. The results show that the zeta potential measurement of LNP-TB4 was approximately -15.1 mV (Figure 9).

[0463] Example 6 Synthesis and characterization of LNP-TB4-ID3 liposomes

[0464] In another experiment, liposomes containing the TB4 prodrug (denoted LNP-TB4) were co-formulated with ID3 and synthesized in the following manner. Briefly, in the first step, lipid stock solutions of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), CHOL, and DSPE-PEG were separately prepared in ethanol (20 mg / ml). Next, since the TB4 prodrug (TB4 + stearic acid) and ID3 prodrug were not soluble in ethanol, their stock solutions were prepared in acetonitrile (20 mg / ml). Next, the lipid mixture of POPC, CHOL, TB4 + stearic acid, ID3, and DSPE-PEG was mixed together in a molar ratio of 53:31:6:6:4 and then diluted with ethanol to obtain a total lipid concentration of 10 mg / ml. This lipid mixture was heated at 50°C using a heating block attachment in a microfluidizer. Similarly, an aqueous phase containing 1 mM PBS buffer was preheated to 50°C and then passed through the microfluidic cartridge at a flow rate of 4.5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed using a 12 KDa cutoff dialysis membrane (Sigma-Aldrich) against DI water for at least 24 hours. The dialysis water was exchanged at least five times over the 24-hour period to maximize solvent removal. After solvent removal, LNP-TB4-ID3 was optionally concentrated using an Amicon centrifugal filter device (10 KDa cutoff, 3000 g).

[0465] The characteristics of LNP-TB4-ID3 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-TB4-ID3 liposomes (liposome concentrations were 0.5–1 mg / ml) were placed in a clear, four-sided plastic cuvette and analyzed directly at 25 °C. The results, shown in Figure 10, indicate that the Z-average size of the nanoparticles was approximately 87 nm and the PDI was approximately 0.075.

[0466] Additionally, the zeta potential of LNP-TB4-ID3 liposomes in aqueous dispersion was determined using a Malvern Zetasizer instrument (Malvern Instrumentation Co, Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell available for the Zetasizer. Measurements were performed at 25°C. The results show that the zeta potential measurement of LNP-TB4-ID3 was approximately -11.5 mV (Figure 11).

[0467] Additionally, a summary table of additional co-formulated LNP-TB4 is provided in Table IV.

[0468] Example 7 Synthesis and characterization of SLNP-TB4 solid lipid nanoparticles

[0469] In another experiment, solid lipid nanoparticles (SLNPs) containing the TB4 prodrug (denoted SLNP-TB4) were synthesized in the following manner. Briefly, SLNPs containing the TB4 prodrug were prepared using various emulsifier(s), such as Moliwol 488 (polyvinyl alcohol), Pluronic F127, and Kolliphor RH 40. In the first step, a lipid stock solution of POPC, CHOL, and DSPE-PEG was prepared in ethanol (20 mg / ml). Next, a stock solution of the TB4 prodrug was prepared in acetonitrile (20 mg / ml). A lipid mixture of POPC, CHOL, TB4, and DSPE-PEG in a molar ratio of 51:29:15:5 was mixed together and then diluted with ethanol to obtain a lipid concentration of 10 mg / ml. This lipid mixture was heated at 55°C using a heating block attachment in a microfluidizer. Similarly, an aqueous phase containing 2% w / v Moliwol 488 (or 2% w / v Pluronic F127 / Kolliphor RH 40) solution was preheated to 55°C and then passed through the microfluidic cartridge at a flow rate of 5:1 (aqueous phase:organic phase, lipid mixture). Solvent was removed using a 12 KDa cutoff dialysis membrane (Sigma-Aldrich) against DI water for at least 24 hours. The dialysis water was exchanged at least five times over the 24-hour period to maximize solvent removal. After solvent removal, the SLNPs were passed through a 0.2 micron filter membrane (cellulose acetate). SLNP-TB4 was optionally concentrated using an Amicon centrifugal filter device (10 KDa cutoff, at 3000 g).

[0470] SLNP-TB4 was characterized using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-TB4 (SLNP concentrations were 0.5–1 mg / ml) was placed in a clear, four-sided plastic cuvette and analyzed directly at 25 °C. The results, shown in Figure 12, indicate that the Z-average size of the nanoparticles was approximately 90 nm and the PDI was approximately 0.074.

[0471] Additionally, the zeta potential of SLNP-TB4 solid lipid nanoparticles in aqueous dispersion was determined using a Malvern Zetasizer instrument (Malvern Instrumentation Co, Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell available with the Zetasizer. Measurements were performed at 25°C. The results show that the measured zeta potential of SLNP-TB4 was approximately -11.9 mV (Figure 13).

[0472] Example 8 Synthesis and characterization of SLNP-TB4-ID3 solid lipid nanoparticles

[0473] In another experiment, solid lipid nanoparticles (SLNPs) containing the TB4 prodrug co-formulated with ID3 (denoted SLNP-TB4-ID3) were synthesized in the following manner. Briefly, SLNPs containing the TB4 prodrug were prepared using various emulsifier(s), such as Moliwol 488 (polyvinyl alcohol), Pluronic F127, and Kolliphor RH 40. In the first step, lipid stock solutions of POPC, CHOL, and DSPE-PEG were prepared in ethanol (20 mg / ml). Next, stock solutions of TB4 and ID3 prodrugs were prepared in acetonitrile (20 mg / ml). A lipid mixture of POPC, CHOL, TB4, ID3, and DSPE-PEG in a molar ratio of 52:29:7:7:5 was mixed together and then diluted with ethanol to obtain a lipid concentration of 10 mg / ml. This lipid mixture was heated at 55°C using a heating block attachment in a microfluidizer. Similarly, an aqueous phase containing 2% w / v Moliwol 488 (or 2% w / v Pluronic F127 / Kolliphor RH 40) solution was preheated to 55°C and then passed through the microfluidic cartridge at a flow rate of 5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed using a 12 KDa cutoff dialysis membrane (Sigma-Aldrich) against DI water for at least 24 hours. The dialysis water was exchanged at least five times over the 24-hour period to maximize solvent removal. After solvent removal, the SLNPs were passed through a 0.2 micron filter membrane (cellulose acetate). The SLNP-TB4-ID3 was optionally concentrated using an Amicon centrifugal filter device (10 KDa cutoff at 3000 g).

[0474] SLNP-TB4-ID3 was characterized using a Malvern Zetasizer (Malvern The particle size was determined using a 1000-well plate (PDI) assay (PDI: 0.119, P ...). Briefly, 2 ml of SLNP-TB4-ID3 (SLNP concentration was 0.5–1 mg / ml) was placed in a clear, four-sided plastic cuvette.

[0475] Additionally, the zeta potential of the SLNP-TB4-ID3 solid lipid nanoparticles in aqueous dispersion was measured using a Malvern Zetasizer instrument (Malvern Instrumentation) The zeta potential was determined using a ZetaSizer (Zeta Potential Cell) (Zeta Potential Cell, ZetaSizer, Inc., Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell available for the Zetasizer. Measurements were performed at 25°C. The results show that the zeta potential measurement of SLNP-TB4-ID3 was approximately -10.3 mV (Figure 15).

[0476] Additionally, a summary table of additional co-formulated SLNP-TB4 is provided in Table V.

[0477] Example 9 In vivo tumor inhibition of SLNP-TB4 using B16F10 cells

[0478] In this experiment, SLNP-TB4 was evaluated using the following protocol: Mouse melanoma cancer B16F10 cells (0.2 × 10 cells) 6C57BL / 6 mice were subcutaneously inoculated with 100 mg / kg LNP-MTO (mitoxantrone dihydrochloride in liposomal form), 3 mg / kg LNP-AR5 (AR5-stearate in liposomal form), 3 mg / kg LNP-AR5 and LNP-TB4 (TB4-stearate in liposomal form) combined at 3 mg / kg, and 3 mg / kg LNP-MTO and SLNP-TB4 (TB4-stearate in solid lipid nanoparticles) combined at 3 mg / kg by intravenous injection twice weekly. Tumor volume was measured in three dimensions using calipers, and the volume was calculated using the formula: V = (L × W × W) × 0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on the tumor size data on day 15.

[0479] The results show that the combination of SLNP-TB4 and LNP-MTO resulted in significant antitumor activity. The TGI was calculated to be 44.12% (all p<0.05) (Figure 16).

[0480] Example 10 In vivo tumor inhibition of LNP-TB4 in multiple combinations using B16F10 cells

[0481] In this experiment, LNP-TB4 was evaluated using the following protocol: Mouse melanoma cancer B16F10 cells (0.2 × 10 cells) 6)) was inoculated subcutaneously into the right hind flank region of C57BL / 6 mice. Animals were treated twice weekly by i.v. injection with vehicle control, 3 mg / kg LNP-MTO (mitoxantrone dihydrochloride in liposomal form), 3 mg / kg combination of LNP-TB4 (TB4-stearate in liposomal form) and LNP-TR6 (TR6-Chemes in liposomal form), 3 mg / kg further combination of LNP-TB4 and LNP-AR5 (AR5-stearate in liposomal form), 3 mg / kg further combination of LNP-TB4 and LNP-ID3 (ID3-stearate in liposomal form), 3 mg / kg further combination of LNP-TB4 and LNP-PD3 (PD3-cholesterol), 3 mg / kg further combination of LNP-TB4, LNP-MTO and LNP-ID3, and 3 mg / kg further combination of LNP-TB4, LNP-MTO and LNP-AR5. Tumor volumes were measured in three dimensions using calipers, and the volumes were calculated using the formula: V = (L × W × W) × 0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on tumor size data on day 16.

[0482] The results show that treatment with LNP-MTO at 3 mg / kg as a single agent produced antitumor activity. The TGI was calculated to be 32.6% (p<0.05). In addition, the combinations of LNP-TB4 + LNP-MTO + LNP-ID3 and LNP-AR5 + LNP-MTO + LNP-TB4 produced antitumor activity. The TGI was calculated to be 32.86% (p<0.05) and 37.86% (p<0.05), respectively (Figure 17).

[0483] Example 11 In vitro verification of the mechanism of action of LNP-TB4 and SLNP-TB4

[0484] In this experiment, the mechanism of action of LNP-TB4 and SLNP-TB4 was evaluated in vitro using the following protocol. To confirm that TB4 in liposomal form (LNP) and solid lipid nanoparticle form (SLNP) can have biological effects in vitro, the following assays were performed. Briefly, HEK-Blue™ TGF-β cells and QUANTI-Blue™ (InvivoGen, San Diego, CA) assays were used using standard methods. Stimulation of HEK-Blue™ TGF-β cells with TGF-β induces activation of the TGF-β / Smad signaling pathway, forming the Smad3 / Smad4 complex. The heterocomplex enters the nucleus and binds to the SBE site, inducing the production of SEAP. The amount of SEAP secreted into the supernatant can be easily assessed using QUANTI-Blue. Cells were incubated with various concentrations of TB4, LNP-TB4, and SLNP-TB4 in the presence of 10 ng / ml TGF-β. After 24 hours of incubation with each compound, percent (%) TGF-β inhibition was assessed by measuring the level of SEAP optimal density (OD) using the QUANTI-Blue™ assay and normalizing the data to the control group (cells treated with TGF-β alone).

[0485] The results showed that treatment of cells with LNP-TB4 and SLNP-TB4 led to the inhibition of TGF-β (see FIG. 18).

[0486] Example 12 Human clinical trials for the treatment of human cancers using formulated and / or co-formulated liposomes containing TB prodrugs

[0487] Formulated and / or co-formulated liposomes containing TB prodrugs that specifically accumulate in tumor cells and are used in the treatment of certain tumors as well as other immunological disorders and / or other diseases are used in accordance with the present invention. For each of these indications, two clinical approaches have been successfully pursued.

[0488] I.) Adjunctive Therapy: In adjunctive therapy, patients are treated with formulated and / or co-formulated liposomes containing TB prodrugs in combination with chemotherapeutic drugs or pharmaceuticals or biopharmaceuticals or combinations thereof. The primary cancer target is treated with the addition of formulated and / or co-formulated liposomes containing TB prodrugs under standard protocols. Protocol designs may include, but are not limited to, the following: reduction in tumor burden of primary or metastatic lesions, prolonged progression-free survival, overall survival, improved patient well-being, disease stabilization, and improved efficacy and safety in patients with standard chemotherapy and other biologic agents. This addresses efficacy as assessed by example, including the ability to reduce the usual dose of a chemotherapeutic or biologic agent. These dose reductions allow for additional treatment and / or extended treatment by reducing dose-related toxicity of the chemotherapeutic or biologic agent.

[0489] II.) Monotherapy: In connection with the use of formulated and / or co-formulated liposomes containing TB prodrugs in tumor monotherapy, the formulated and / or co-formulated liposomes containing TB prodrugs are administered to patients without chemotherapeutic or pharmaceutical or biological agents. In one embodiment, monotherapy is implemented clinically in terminal cancer patients with widespread metastatic disease. Protocol designs address efficacy as assessed by the following examples, including but not limited to: reduction in tumor burden of primary or metastatic lesions, prolonged progression-free survival, overall survival, improved patient health, disease stabilization, and the ability to reduce usual doses of standard chemotherapy and other biological agents. Dosage

[0490] Dosage regimens can be adjusted to provide the optimum desired response. For example, a single formulated and / or co-formulated liposome containing a TB prodrug can be administered, or 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. "Unit dosage form," as used herein, refers to a physically discrete unit suitable as a unitary dosage for the mammalian subject being treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specifications for the unit dosage 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 mechanism, if any, of the combined compounds, (c) the specific therapeutic or prophylactic effect to be achieved, and (d) the limitations inherent in the technology for compounding such compounds to treat the susceptibility of the individual. Clinical Development Plan (CDP)

[0491] The CDP follows and develops the treatment of using formulations and / or co-formulated liposomes containing TB prodrugs in the context of adjunctive therapy or monotherapy. The trial will first demonstrate safety, and then confirm the efficacy of repeated administration. The trial is open-label, comparing standard chemotherapy and / or current treatment standards with formulations and / or co-formulated liposomes containing TB prodrugs. As will be understood, one non-limiting criterion that can be used in connection with patient enrollment is the expression of TGFβ in tumors, as determined by standard detection methods known in the art.

[0492] It is believed that the formulated and / or co-formulated liposomes, or any of the embodiments disclosed herein, may have a satisfactory pharmacological profile and promising biopharmaceutical properties, such as toxicological profile, metabolic and pharmacokinetic properties, solubility, and permeability. It will be understood that determining appropriate biopharmaceutical properties is within the knowledge of one of ordinary skill in the art, such as determining cytotoxicity in cells or inhibiting certain targets or channels to determine potential toxicity.

[0493] The present invention is not limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any functional equivalents are within the scope of the invention. Various modifications of the models, methods, and lifecycle methodologies of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the above description and teachings and are likewise intended to be within the scope of the present invention. Such modifications and other embodiments can be made without departing from the true scope and spirit of the invention. Table I. Examples of lipids [Table I] Table II. Examples of helper lipids [Table II] Table III. Examples of phospholipids / fatty acids [Table III] Table IV. Summary of LNP-TB4 co-formulations [Table IV] Table V. Overview of SLNP-TB4 Formulations and Co-formulations [Table V]

Claims

[Claim 1] The invention described in the specification.

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