Formulated and / or co-formulated liposome compositions containing TGFB antagonist prodrugs useful in cancer treatment, and methods thereof.

Abstract

This specification discloses formulated and / or co-formulated liposomes, lipid nanoparticles (LNPs), and solid lipid nanoparticles (SLNPs) containing TB prodrugs, as well as methods for producing LNPs and SLNPs. The TB prodrug composition comprises a drug moiety that inhibits ALK5, a lipid moiety, and a linking unit. The TB prodrugs can be formulated and / or co-formulated into nanocarriers to provide methods for treating cancer, immunological disorders, and other diseases by utilizing targeted drug delivery vehicles.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a continuation-in-part application of U.S. Patent Application No. 18 / 445,323 filed on 10 July 2023, which is a continuation-in-part application of 17 / 300,063 (currently U.S. Patent No. 11,801,304) filed on 19 February 2021, which claims priority under U.S. Provisional Patent Application No. 62 / 995,887 filed on 19 February 2020, the contents of which are fully incorporated herein by reference.

[0002] Statement of rights to inventions made under federally funded research. Not applicable.

[0003] Field of Invention The present invention as described herein relates to prodrug compositions that inhibit signaling induced by transforming growth factor beta 1, 2, or 3 ("TGFβ") proteins after the release of an active inhibitor from a prodrug, and to nanoformulations comprising such prodrugs. In particular, the present invention relates to prodrug compositions formulated within nanocarriers (e.g., liposomes) and used as vehicles for the treatment of cancer in humans. The present invention also relates to coformulations of such prodrugs with other immunomodulators or prodrugs. Furthermore, the present invention relates to the treatment of cancer and other immunological disorders and diseases.

[0004] Background of the Invention Cancer is the second leading cause of death globally, after coronary artery disease. Millions of people die from cancer every year, and in the United States alone, cancer accounts for well over 500,000 deaths annually. In 2017, 1,688,780 new cases of cancer were diagnosed (American Cancer Society). While deaths from heart disease have declined significantly, deaths from cancer have generally been on the rise. It is predicted that by the early part of the next century, cancer will be the leading cause of death unless medical advancements reverse current trends.

[0005] Several 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 stomach 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 cancers, and virtually all other cancers, share a common fatal characteristic: they metastasize to sites distal to the primary tumor, and are almost without exception fatal metastatic diseases. Furthermore, even cancer patients who have overcome primary cancer early on have shown a common experience of dramatically altered lives. Many cancer patients experience intense anxiety caused by the perception of potential recurrence or treatment failure. Many cancer patients also experience physical weakness after treatment. Additionally, many cancer patients experience disease recurrence.

[0006] While cancer treatment has improved and survival rates have increased over the past few decades, the heterogeneity of cancer necessitates new treatment strategies utilizing multiple treatment modalities. This is particularly true in the treatment of solid tumors in anatomically important sites (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma), where treatment may be limited to standard radiotherapy and / or chemotherapy. Nevertheless, the adverse effects of these treatments include chemotherapy resistance and radioresistance, which, in addition to severe side effects that reduce the patient's quality of life, promote localized recurrence, distant metastasis, and secondary primary tumors.

[0007] TGFβ refers to a subset of polypeptides in the beta superfamily, which are cytokine transformation growth factors. These are secreted proteins that play a role in many cellular functions, including the regulation of cell growth, cell proliferation, cell differentiation, and apoptosis. In humans, TGFβ1 is encoded by the TGFB1 gene. Functionally, TGFβ acts synergistically with TGF-α in inducing transformation. It also acts as a negative autocrine growth factor. Dysregulation of TGFβ activation and signaling can lead to apoptosis. Many cells synthesize TGFβ, and almost all cells have specific receptors for these cytokines. TGF-β1, TGF-β2, and TGF-β3 all function via the same receptor signaling pathway. TGFβ plays a crucial role in regulating the immune system and exhibits different activity depending on the cell type or developmental stage of the cell. Most immune cells (or leukocytes) secrete TGFβ. See LETTERIO, et. al., Regulation of Immune Responses by TGF-beta, Annu. Rev. Immunol. 16: pp 137-161 (1998). It has been taught that some T cells (e.g., regulatory T cells) release TGF-β to inhibit the action of other T cells. For example, the proliferation of interleukin-1 and interleukin-2-dependent activated T cells, as well as the activation of quiescent helper T cells and cytotoxic T cells, are prevented by TGF-β1 activity. See GILBERT, et. al., Transforming growth factor-beta 1 induces antigen-specific unresponsiveness in naive T cells, Immunol. Invest. 26(4): pp. 459-472 (1997) and WAHL, et. al., TGF-beta: a mobile purveyor of immune privilege, Immunol. Rev. 213: pp. 213-227 (2006).Similarly, TGFβ can inhibit the secretion and activity of many other cytokines, including interferon-γ, tumor necrosis factor-alpha (TNF-α), and various interleukins. It can also downregulate the activity of immune cells by reducing the expression levels of cytokine receptors such as the IL-2 receptor. In addition, TGFβ has similar effects on B cells, and these effects vary depending on the cell's differentiation state. TGFβ inhibits B cell proliferation, stimulates apoptosis, and plays a role in regulating the expression of antibodies, transferrin, and MHC class II proteins in 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 repressive. This cytokine inhibits the proliferation of these cells, preventing them from producing reactive oxygen species (e.g., superoxide (O2)). - It can prevent the formation of intermediates containing )) and nitrogen (e.g., nitric oxide (NO)). However, as with other cell types, TGFβ can have the opposite effect on bone marrow-derived cells. See WAHL, et al. above. It has also been shown that TGFβ reduces the effectiveness of MHC II in astrocytes and dendritic cells, resulting in reduced activation of appropriate helper T cell populations. See TANG, et al., The Smad3 protein is involved in TGF-beta inhibition of class II transactivator and class II MHC expression, J. of Immun, 167(1): pp. 311-319 (2001).

[0008] TGFβ first elicits intracellular signaling by binding to TGFβ receptor II (TGFβRII), thereby recruiting TGFβ receptor I (TGFβRI), a second receptor also known as activin-like kinase 5 (ALK5). Upon recruitment to the TGFβ receptor:ligand complex, TGFβRII phosphorylates and activates ALK5, thereby mediated downstream signaling and leading to transcriptional regulation. ALK5 deletion or mutation in tumors appears to be a common form of pathway alteration. However, efforts 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 are being developed into clinical trials as anticancer agents. Garnicertiveb (LY2157229) and bactocertiveb (TEW-7197) are the most advanced treatment candidates in clinical trials, and their anticancer efficacy has been demonstrated in humans, both as monotherapy and in combination with approved drugs, verifying that this mechanism is clinically meaningful. However, the cardiotoxicity induced by these molecules limits the dosage, and their combination with other therapies carries the risk of increased systemic off-target toxicity.

[0010] A prodrug is a drug or compound that is metabolized (i.e., converted in the body) into a pharmacologically active drug after administration. Instead of administering the drug directly, a corresponding prodrug is used as an alternative to improve how the drug is absorbed, distributed, metabolized, and / or eliminated. Prodrugs are often designed to improve bioavailability when the drug itself is poorly absorbed, for example, from the gastrointestinal tract. Prodrugs can also 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 can be particularly important in treatments such as chemotherapy, where severe unintended and undesirable side effects may occur. Therefore, prodrugs can be considered drugs containing special non-toxic protecting groups that are transiently used 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. For example, some examples include polymer conjugates, polymer nanoparticles, lipid-based carriers, and dendrimers. The various types of nanomaterials used in nanocarriers enable the delivery of hydrophobic and hydrophilic drugs throughout the body. Since the human body is primarily water-based, the ability to effectively deliver hydrophobic drugs to humans is a significant therapeutic benefit of nanocarriers. Nanocarriers are promising in drug delivery processes because they deliver drugs to site-specific targets, ensuring that drugs are delivered to specific organs or cells rather than other organs or cells. Site specificity is a significant therapeutic benefit because it prevents the delivered drug from being delivered to the wrong location. In addition, nanocarriers are particularly 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. Since chemotherapy drugs can be extremely toxic to human cells, it is crucial that chemotherapy drugs are delivered to tumors without being released to other parts of the body.

[0012] From the above, it will be readily apparent to those skilled in the art that a new treatment paradigm is required 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), reduction of side effects, and higher therapeutic utility in the treatment of cancer, particularly solid tumor cancer. Considering the current deficiencies associated with cancer treatment, one object of the present invention is to provide a new and improved method for treating cancer(s), immunological disorders, and other diseases by utilizing a prodrug encapsulated within a nanocarrier.

PRIOR ART DOCUMENTS

NON-PATENT LITERATURE

[0013]

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Summary of the Invention

Means for Solving the Problems

[0014] Summary of the Invention The present invention provides an ALK5 inhibitor prodrug ("TB prodrug") composition comprising an ALK5 inhibitor, a lipid, and a biologically cleavable linker. In certain embodiments, the nanocarrier containing the TB prodrug is formulated for use as a delivery modality for treating human diseases such as cancer, including solid tumor cancers, and other immunological disorders. In certain embodiments, the nanocarrier comprises a lipid bilayer that can be incorporated into a drug delivery vehicle (i.e., a liposome). In a more preferred embodiment, the liposome comprises cholesterol hemisuccinate ("CHEMS"). In a more preferred embodiment, the liposomes of the present invention comprise stearic acid.

[0015] In further embodiments, the TB prodrugs of the present disclosure include TB4 prodrugs.

[0016] In further embodiments, the present invention includes a method for 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.

[0017] In another embodiment, the present invention includes a method for delivering an ALK5 inhibitor to a tumor together with one or more additional immunomodulators, comprising: (i) synthesizing a TB prodrug; (ii) co-formulating the TB prodrug of the present invention with one or more additional immunomodulators of the present invention into a nanocarrier; and (iii) administering the nanocarrier to a patient.

[0018] In another embodiment, the immunomodulator includes immunogenic cell death-inducing chemotherapeutic agents, PD-1 antagonists, Toll receptor agonists, STING agonists, IDO inhibitors, CTLA4 inhibitors, CD1D agonists, and / or prodrugs thereof.

[0019] In another embodiment, the present disclosure teaches a method for synthesizing TB prodrugs.

[0020] In another embodiment, the present disclosure teaches a method for synthesizing a TB4 prodrug.

[0021] In another embodiment, the Disclosure teaches a method for formulating a TB prodrug within a nanocarrier, including but not limited to liposomes.

[0022] In another embodiment, the disclosure teaches a method for formulating a TB4 prodrug within a nanocarrier, including but not limited to liposomes.

[0023] In another embodiment, the Disclosure teaches a method for treating human cancers, immunological disorders, and other diseases using the nanocarriers of the Disclosure. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows the chemical synthesis for the TB4 prodrug. [Figure 2] Figure 2 shows the chemical synthesis of protecting group intermediates leading to the final TB4 prodrug. [Figure 3] Figure 3 shows the chemical synthesis of protecting group intermediates leading to the final TB4 prodrug. [Figure 4] Figure 4 shows the synthesis scheme for ALK5 inhibitor prodrugs using carboxylic acid functional groups. [Figure 5] Figure 5 shows the synthesis scheme for an ALK5 inhibitor prodrug using an alcohol functional group. [Figure 6] Figure 6 shows the synthesis scheme for ALK5 inhibitor prodrugs using secondary amine, amide, or aniline functional groups. [Figure 7] Figure 7 shows the chemical synthesis of a TB4 prodrug containing stearic acid. [Figure 8] Figure 8 shows the characterization of the LNP-TB4 liposome. [Figure 9] Figure 9 shows the characterization (zeta potential) of LNP-TB4 liposomes. [Figure 10] Figure 10 shows the characterization of the LNP-TB4-ID3 liposome. [Figure 11] Figure 11 shows the characterization (zeta potential) of the LNP-TB4-ID3 liposome. [Figure 12] Figure 12 shows the characterization of SLNP-TB4 solid lipid nanoparticles. [Figure 13] Figure 13 shows the characterization (zeta potential) of SLNP-TB4 solid lipid nanoparticles. [Figure 14] Figure 14 shows the characterization of SLNP-TB4-ID3 solid lipid nanoparticles. [Figure 15] Figure 15 shows the characterization (zeta potential) of SLNP-TB4-ID3 solid lipid nanoparticles. [Figure 16] Figure 16 shows in vivo tumor inhibition of the SLNP-TB4 and LNP-MTO combination using B16F10 cells. [Figure 17] Figure 17 shows tumor inhibition of LNP-TB4 in multiple combinations in vivo using B16F10 cells. [Figure 18] Figure 18 shows the in vitro verification of the mechanisms of action of LNP-TB4 and SLNP-TB4. [Figure 19] Figure 19 shows an alternative chemical synthesis for the TB4 prodrug (ALT-1) containing stearic acid. [Figure 20] Figure 20 shows alternative chemical synthesis for protecting group intermediates leading to the final TB4 prodrug (ALT-1). [Figure 21] Figure 21 shows alternative chemical synthesis for protecting group intermediates leading to the final TB4 prodrug (ALT-1). [Figure 22] Figure 22 shows alternative chemical synthesis methods leading to the final TB4 prodrug (ALT-1). [Figure 23] Figure 23 shows the in vitro verification of the mechanism of action of LNP-TB4(ALT-1) and SLNP-TB4(ALT-1). [Figure 24] Figure 24 shows tumor inhibition of SLNP-TB4 in combination in vivo using EMT-6 cells. [Figure 25] Figure 25 shows in vivo tumor inhibition of SLNP-TB4-IC1 using MC38 cells. [Figure 26] Figure 26 shows tumor inhibition of SLNP-TB4-IC1 at multiple dose levels in vivo using EMT-6 cells. [Figure 27] Figure 27 shows the characterization of SLNP-TB4(ALT-1) solid lipid nanoparticles. [Figure 28]Figure 28 shows the characterization (zeta potential) of SLNP-TB4(ALT-1) solid lipid nanoparticles. [Figure 29] Figure 29 shows the characterization of SLNP-IC1-TB4(ALT-1) solid lipid nanoparticles. [Figure 30] Figure 30 shows the characterization (zeta potential) of SLNP-IC1-TB4(ALT-1) solid lipid nanoparticles. [Modes for carrying out the invention]

[0025] Detailed description of the invention Section Summary I.) Definition II.) Prodrugs III.) Chemical compounds IV.) Lipids V.) Linked Unit ("LU") VI.) Nanocarriers VII.) Liposomes VIII.) Pharmaceutical Formulation IX.) Combination Therapy X.) Method for delivering liposomes containing prodrugs to cells XI.) Methods for treating cancer(s) and other immunological disorders(s). XII.) Kits / Manufactured Products

[0026] I.) Definition Unless otherwise defined, all terms, notations, and other scientific or technical terms used herein are intended to have meanings that are generally understood by those skilled in the art to which the invention relates, unless otherwise clearly indicated by the context. In some cases, terms that have a generally understood meaning are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from the generally understood meaning in the art.

[0027] Where a trademark is used herein, references to that trademark also refer to the product formulations, generic drugs, and the pharmaceutically active ingredients(s) of the trademarked product, unless otherwise specified by the context.

[0028] Where used herein, the term “about” means, when referring to a value or quantity of size (i.e., diameter), weight, concentration, or percentage, to include a variation of ±20% or ±10% in one example, ±5% in another example, ±1% in yet another example, and ±0.1% in yet another example from the specified quantity, because such variation is suitable for carrying out the disclosed method.

[0029] As used herein, the term "and / or" refers to entities that exist individually or in combination, when used in the context of a list of entities. Thus, for example, the phrase "A, B, C, and / or D" includes not only A, B, C, and D individually, but also any combination and partial combination of A, B, C, and D.

[0030] In this specification, the numerical range indicated by an endpoint includes all numbers and fractions contained within that range (for example, 1 to 5 includes, but is not limited to, 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5).

[0031] As used herein, the phrase “essentially from” limits the claims to the specified material or step, and in addition, any basic and novel feature(s) of the claimed subject matter.

[0032] The terms “advanced cancer,” “locally advanced cancer,” “progressive disease,” and “locally advanced disease” refer to cancer that has spread through the associated tissue capsule and include disease at stage C in the American Urological Association (AUA) system, disease at stages C1–C2 in the Whitmore-Jewett system, and disease at stages T3–T4 and N+ in the TNM (tumor, nodule, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, as these patients have substantially fewer favorable outcomes compared to patients with clinically localized (organ-limited) cancer.

[0033] As used herein, the term "alkyl" includes, 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, including C1-C1 groups at both ends. 20 A linear (i.e., "straight-chain"), branched, or cyclic hydrocarbon chain can be defined as saturated or at least partially, and in some cases as unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains. "Branched" refers to an alkyl group in which a lower alkyl group, e.g., methyl, ethyl, or propyl, is bonded 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" specifically refers to a C1-C8 straight-chain alkyl group. In other embodiments, "alkyl" specifically refers to a C i~8 This refers to branched-chain alkyl groups.

[0034] The alkyl group may optionally be substituted with one or more alkyl substituents, which may be the same or different ("substituted alkyl"). The term "alkyl substituent" includes, but is not limited to, alkyl, substituted alkyl, halo, arylamino, acyl, 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 may optionally be inserted along the alkyl chain, the nitrogen substituent being hydrogen, a lower alkyl (also referred to herein as "alkylaminoalkyl"), or an aryl.

[0035] Therefore, 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 are replaced by another atom or by a functional group including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate and mercapto.

[0036] The term “aryl” is used herein to refer to an aromatic substituent that may be a single aromatic ring, or may be fused together, covalently linked, or, but not limited to, multiple aromatic rings linked to a common group such as a methylene or ethylene moiety. The common linking group may also be a carbonyl group, as in benzophenone, or oxygen, as in diphenyl ether, or nitrogen, as in diphenylamine. The term “aryl” particularly encompasses heterocyclic aromatic compounds. Aromatic rings may include, among others, phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, and benzophenone. In certain embodiments, the term “aryl” means cyclic aromatic compounds containing about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5-membered and 6-membered aromatic rings and heteroaromatic rings. The aryl group may be optionally substituted with one or more aryl substituents, which may be the same or different ("substituted aryl"), where the "aryl 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'' may independently be hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl. Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, and carbazole.

[0037] As used herein, "heteroaryl" refers to an aryl group whose ring structure contains one or more non-carbon atoms (e.g., O, N, S, Se, etc.). Nitrogen-containing heteroaryl moieties include, but are not limited to, pyridine, imidazole, benzimidazole, pyrazole, pyrazine, triazine, and pyrimidine.

[0038] The terms “anti-cancer drug,” “chemotherapeutic agent,” and “anti-cancer prodrug” refer to a drug (i.e., a chemical compound) or prodrug that is known to or is thought to be able to treat cancer (i.e., kill cancer cells, inhibit the growth of cancer cells, or treat 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 activity. More traditional or conventional chemotherapeutic agents can be described by mechanism of action or by class of chemical compounds and may include, but are not limited to, alkylating agents (e.g., melphalan), anthracyclines (e.g., doxorubicin), cytoskeletal disruptors (e.g., paclitaxel), epothilons, histone deacetylase inhibitors (e.g., vorinostat), topoisomerase I or II inhibitors (e.g., irinotecan or etoposide), kinase inhibitors (e.g., bortezomib), nucleotide analogs or their precursors (e.g., methotrexate), peptide antibiotics (e.g., bleomycin), platinum-based drugs (e.g., cisplatin or oxaliplatin), retinoids (e.g., tretinoin), and vinka alkaloids (e.g., vinblastine).

[0039] "Aralkyl" refers to an alkyl-aryl group, and the alkyl and / or aryl parts are substituted as needed.

[0040] "Alkylene" refers to a linear or branched divalent aliphatic hydrocarbon group having 1 to about 20 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group may be linear, branched, or cyclic. The alkylene group may also be unsaturated as desired and / or substituted with one or more "alkyl substituents". Along the alkylene group, one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl") may be inserted as desired, the nitrogen substituent being alkyl as already described. Examples of alkylene groups include methylene (-CH2-), ethylene (-CH2-CH2-), propylene (-(CH2)3-), and cyclohexylene (-C6H 10 -), -CH=CH-CH=CH-, -CH=CH-CH2-, -(CH2) q Examples include -N(R)-(CH2)- (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 a lower alkyl group), methylenedioxyl (-O-CH2-O-), and ethylenedioxyl (-O-(CH2)2-O-). The alkylene group can have about 2 to about 3 carbon atoms, and can also have 6 to 20 carbon atoms.

[0041] The term "arylene" refers to a divalent aromatic group, such as a divalent phenyl or naphthyl group. The arylene group may be substituted with one or more aryl substituents as needed and / or may contain one or more heteroatoms.

[0042] The term "amino" refers to a -N(R)² group, where each R is independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, or substituted aralkyl. The terms "aminoalkyl" and "alkylamino" may refer to a -N(R)² group, where each R is H, alkyl, or substituted alkyl, and at least one R is alkyl or substituted alkyl. "Arylamine" and "aminoaryl" refer to a -N(R)² group, 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).

[0043] "Bulk" (also known as drug substance) refers to drug substance or drug product that has not been filled into final containers for distribution. Final formulation bulk generally refers to drug products that have been formulated and are stored or held before filling. Drug substance may be stored or held as "bulk" or "concentrated bulk" before being formulated into drug products.

[0044] The terms "carboxylate" and "carboxylic acid" are -C(=O)O, respectively. - The term "carboxyl" can refer to the -C(=O)OH group.

[0045] The terms “conjugate” and “conjugated,” as used herein, can refer to two or more components (e.g., chemical compounds, polymers, biomolecules, particles, etc.) being linked to one another (e.g., covalently). In some embodiments, the conjugate may include monovalent portions derived from two different chemical compounds covalently linked via a divalent linker portion (e.g., alkylene or arylene, as may be substituted). In some embodiments, the linker may contain one or more biodegradable bonds, so that one or more of the linker bonds can be broken when the prodrug is exposed to a particular physiological environment or enzyme (e.g., esterase).

[0046] The term “compound” refers to and includes chemical compounds (e.g., prodrugs) themselves, as well as, unless the context makes it clear whether explicitly stated or not, the following: amorphous and crystalline forms of a compound, including polymorphic forms, where these forms may be part of a mixture or be isolated; free acid and free base forms of a compound, these are typically the forms shown in the structures provided herein; isomers of a compound, where optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers, as well as mixtures of optical isomers, including racemic and non-racemic mixtures, where isomers may be in isolated forms or in mixtures with one or more other isomers; the same Topologies, including deuterium-containing compounds and tritium-containing compounds, and compounds containing radioisotopes, including radioisotopes that are therapeutically and diagnostically effective; polymeric forms of compounds, including dimers, trimers, and the like; salts of compounds, preferably pharmaceutically acceptable salts, including acid-addition salts and base-addition salts, and salts having organic and inorganic counterions, and zwitterionic forms, where the compound associates with two or more counterions, the two or more counterions may be the same or different; and solvates of compounds, including hemisolvates, monosolvates, disolvates, and the like, and organic solvent hydrates and inorganic solvates, where the inorganic solvates include hydrates, where the compound associates with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some cases, references to the compounds of the present invention herein include an explicit reference to one or of the above forms, e.g., salts and / or solvates, but such references are for emphasis only and should not be construed as excluding any forms other than those specified above.

[0047] The term "drug product" generally refers to the final formulation containing an active drug component associated with an inactive component, although this association is not strictly necessary (i.e., a liposome containing an ALK5 inhibitor prodrug). The term also includes final dosage forms that do not contain an active ingredient but are intended to be used as a placebo.

[0048] The term "disulfide" can refer to the -SS- group.

[0049] The term "empty vesicle" refers to an unloaded lipid vesicle, in itself.

[0050] As used herein, the term "ester" means a chemical compound derived from an acid (organic or inorganic) in which at least one -OH hydroxyl group is replaced by an -O-alkyl (alkoxy) or O-aryl (aryloxy) group.

[0051] As used herein, the term "esterase" refers to a hydrolytic enzyme that breaks down esters into acids and alcohols.

[0052] "Additives" refer to inert substances used as carriers for active ingredients in drugs, such as vaccines. Additives are also sometimes used to increase the volume of formulations containing very potent active ingredients, enabling convenient and precise dosage. Examples of additives include, but are not limited to, anti-adherents, binders, coatings, disintegrants, fillers, diluents, flavorings, colorants, lubricants, and preservatives.

[0053] The terms "halo," "halide," or "halogen" as used herein refer to fluoro, chloro, bromo, and iodine groups.

[0054] The terms "hydroxyl" and "hydroxy" refer to the -OH group.

[0055] The terms “inhibit” or “inhibit” as used herein mean reducing or completely preventing a measurable amount.

[0056] The terms “individual” and “patient” may be used interchangeably when used in the context of this disclosure.

[0057] As used herein, the term “ligand” generally refers to a species, such as a molecule or ion, that interacts with another species in several ways, for example, by binding. See Martell, AE, and Hancock, RP, Metal Complexes in Aqueous Solutions, Plenum: New York (1996), which is incorporated herein by reference in its entirety.

[0058] As used herein, the term "lipid" refers to a class of naturally occurring (organic) compounds that are insoluble in polar solvents. In the context of this disclosure, lipids include conventional lipids, phospholipids, cholesterol, and lipids chemically functionalized for PEG-ligand binding.

[0059] The term "lipid bilayer" or "LB" refers to any bilayer of oriented amphiphilic lipid molecules in which the hydrocarbon tails face inward, forming a continuous nonpolar phase.

[0060] The terms “liposome,” “lipid vesicle,” or “vesicle” are interchangeably used to refer to an aqueous compartment encapsulated by a lipid bilayer, as conventionally defined (see Stryer (1981) Biochemistry, 2d Edition, WH Freeman & Co., p. 213).

[0061] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cattle, horses, and humans. In one embodiment of the present invention, the mammal is a mouse. In another embodiment of the present invention, the mammal is a human.

[0062] The terms "mercapto" or "thiol" refer to the -SH group.

[0063] The terms "metastatic cancer" and "metastatic disease" refer to diseases that have spread to local lymph nodes or distal sites and include stage D diseases in the AUA system and stage T×N×M+ in the TNM system.

[0064] 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, the nanocarrier comprises a lipid bilayer enclosing (or surrounding or covering) a porous particle core. In certain embodiments, the nanocarrier is a liposome, lipid nanoparticle ("LNP"), or solid lipid nanoparticle ("SLNP").

[0065] The terms “nanoscale particles,” “nanomaterials,” “nanocarriers,” and “nanoparticles” refer to structures having at least one region with dimensions (e.g., length, width, diameter, etc.) less than about 1,000 nm. In some embodiments, the dimensions are 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 approximately 20 nm and approximately 250 nm (for example, approximately 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).

[0066] The term “nanopes” refers to “lipid vesicles” (or groups of vesicles with an average diameter) having diameters ranging from about 20 nm, or about 30 nm, or about 40 nm, or about 50 nm, up to about 500 nm, or about 400 nm, or about 300 nm, or about 200 nm, or about 150 nm, or about 100 nm, or about 80 nm. In certain embodiments, nanovesicles have diameters ranging from about 40 nm to about 80 nm, or from about 50 nm to about 70 nm.

[0067] "Pharmacologically acceptable" refers to a composition that is non-toxic, inert, and / or physiologically compatible with humans or other mammals.

[0068] "Pharmaceutical formulation" refers to the process of combining various chemical substances with pure drug substances to produce the final drug product.

[0069] The term "phosphonate" refers to a -P(=O)(OR)2 group, where each R can independently be H, alkyl, aralkyl, aryl, or negatively charged (i.e., there is virtually no R group bonded to the oxygen atom, resulting in the presence of a lone pair of electrons on the oxygen atom). In other words, each R may or may not be present, and if present, it can be selected from H, alkyl, aralkyl, or aryl.

[0070] The term "phosphate" refers to -OP(=O)(OR')2 groups, where R' is H or a negative charge.

[0071] The term "prodrug" means a pharmaceutical or compound that is metabolized into a pharmacologically active drug after administration. For the purposes of this disclosure, the prodrug of the present invention comprises three components: (i) a drug moiety, (ii) a lipid moiety, and (iii) a linking unit ("LU").

[0072] The term "TB prodrug" refers to the prodrug of the present invention, where the drug portion comprises an ALK5 inhibitor.

[0073] 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 may include a lipid conjugate in which a porphyrin or its derivative or analog is covalently bonded to a lipid side chain. See, for example, U.S. Patent Application Publication 2014 / 0127763.

[0074] As used herein, the terms “specific,” “specifically bind,” and “specifically bind to” refer to the selective binding of the nanocarrier of the present invention to the target TGFβ1 or related family members.

[0075] The term “supported lipid bilayer” refers to a lipid bilayer that encapsulates a porous particle core. This definition is given as such because the lipid bilayer is located on the surface and supported by the porous particle core, as described in this disclosure. In certain embodiments, the lipid bilayer may have a thickness ranging from about 6 nm to about 7 nm, including a hydrophobic core 3–4 nm thick, plus a layer of hydrated hydrophilic head groups (each about 0.9 nm thick), and plus two partially hydrated regions, each about 0.3 nm thick. In various embodiments, the lipid bilayer surrounding the liposome includes a continuous bilayer or substantially continuous bilayers, which effectively cover and seal the ALK5 inhibitor.

[0076] The term "thioalkyl" can refer to an -SR group, where R is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl. Similarly, the terms "thioaralkyl" and "thioaryl" refer to an -SR group, where R is aralkyl and aryl, respectively.

[0077] As used herein, “to treat” or “therapeutic” and grammatically related terms mean any improvement of any outcome of a disease, such as an extension of survival, a reduction in morbidity, and / or a reduction of side effects which are byproducts of an alternative therapeutic modality, and not a requirement of a treating act, although complete eradication of the disease is preferred, as is readily apparent in the art.

[0078] The term "therapeutic dose" refers to the amount of an active prodrug, nanoencapsulated prodrug, or pharmaceutical product that elicits a biological or medical response in a tissue, system, animal, individual, or human.

[0079] The term "unsupported lipid bilayer" refers to an uncoated lipid bilayer in lipid vesicles or liposomes.

[0080] II.) Prodrugs As shown in this disclosure and for the purposes of the present invention, a suitable prodrug is formed by conjugating the drug portion of the present invention (see the section titled "Drug Portion") to the lipid portion of the present invention (see the section titled "Lipids") via the LU (see the section titled "Concatenation Units") of this disclosure. For the purposes of this disclosure, several strategies can be used to form the TB prodrug (see, for example, Figures 4, 5, and 6).

[0081] Therefore, in some embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present disclosure.

[0082] In one embodiment, the prodrug has the following chemical structure represented by formula I: [ka] [In the formula, in an exemplary embodiment of formula I, R1=C 11 ~C 21is a saturated alkyl, R2 = H, CH3].

[0083] In a further embodiment, the prodrug has the following chemical structure represented by Formula II:

Chemical formula

Chemical formula

[0084] In a further embodiment, the prodrug has the following chemical structure represented by Formula III:

Chemical formula

Chemical formula

[0085] Thus, in one embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of Formula I.

[0086] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of Formula II.

[0087] In a further embodiment, the prodrug is a drug-lipid moiety comprising an ALK5 inhibitor of Formula III.

[0088] In one embodiment, the prodrug is a drug-lipid moiety containing an ALK5 inhibitor as shown in Figure 4.

[0089] In one embodiment, the prodrug is a drug-lipid moiety containing an ALK5 inhibitor as shown in Figure 5.

[0090] In one embodiment, the prodrug is a drug-lipid moiety containing an ALK5 inhibitor as shown in Figure 6.

[0091] In further embodiments, the TB prodrug is a drug-lipid moiety comprising the lipids of the present disclosure.

[0092] In a further embodiment, the TB prodrug is a drug-lipid moiety, where the lipid is CHEMS.

[0093] In a further embodiment, the TB prodrug is a drug-lipid moiety, where the lipid is stearic acid.

[0094] In further embodiments, the TB prodrug is a drug-lipid moiety comprising the LU of the present disclosure.

[0095] In further embodiments, the TB prodrug is a drug-lipid moiety, where LU is a hydromethylcarbamate linker.

[0096] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, where the ALK5 inhibitor comprises a chemical composition represented as TB4.

[0097] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, where the ALK5 inhibitor comprises TB4 and has the following chemical structure: [ka] It has.

[0098] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises the lipid of the present disclosure, and has the following chemical formula: [ka] It has.

[0099] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises the lipid of the present disclosure, and has the following chemical formula: [ka] It has the following properties [wherein Li = the lipid of the present invention (see Table I, Table II, or Table III)].

[0100] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises the lipid of the present disclosure, and has the following chemical formula: [ka] It has the following properties [wherein Li = the lipid of the present invention (see Table I, Table II, or Table III)].

[0101] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises the lipid of the present disclosure, and has the following chemical formula: [ka] It has the following properties [wherein Li = the lipid of the present invention (see Table I, Table II, or Table III)].

[0102] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises CHEMS.

[0103] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 and further comprises stearic acid.

[0104] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4, further comprising CHEMS, and LU is a hydromethylcarbamate linker.

[0105] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4, further comprising stearic acid, and LU is a hydromethylcarbamate linker.

[0106] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, where the ALK5 inhibitor comprises TB4 and further comprises stearic acid, and has the following structure: [ka] It has.

[0107] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, where the ALK5 inhibitor comprises TB4 and further comprises stearic acid, and has the following structure: [ka] It has.

[0108] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, wherein the ALK5 inhibitor comprises TB4 ALT-1 and further comprises stearic acid, and has the following structure: [ka] It has.

[0109] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, where the ALK5 inhibitor comprises TB4 ALT-2-pyrazole and further comprises stearic acid, and has the following structure: [ka] It has.

[0110] In further embodiments, the prodrug is a drug-lipid moiety comprising the ALK5 inhibitor of the present invention, where the ALK5 inhibitor comprises TB4 ALT-3-amide and further comprises stearic acid, and has the following structure: [ka] It has. In additional embodiments of the present disclosure, the subject provides ALK5 inhibitor prodrugs comprising a parent drug of a lipid-conjugated therapeutic agent. In some embodiments, the prodrug comprises (a) a monovalent drug moiety, (b) a monovalent lipid moiety, and (c) a divalent linker moiety comprising a linking unit that degrades in vivo, such as a disulfide bond, where the monovalent drug moiety and the monovalent lipid moiety are linked via a linker (e.g., covalently). The monovalent drug moiety and the monovalent lipid moiety may be monovalent derivatives of a chemical compound and a lipid, respectively. For example, the monovalent derivative may be a deprotonated derivative of a chemical compound or lipid containing a hydroxyl, thiol, amino, or carboxylic acid group.

[0111] In further embodiments of the present disclosure, the subject provides ALK5 inhibitor prodrugs comprising a parent drug of a lipid-conjugated therapeutic agent. In some embodiments, the prodrug comprises (a) a divalent drug moiety, (b) a divalent lipid moiety, and (c) a divalent linker moiety comprising a linkage that degrades in vivo, wherein the divalent drug moiety and the divalent lipid moiety are linked via a linker (e.g., by a covalent bond). The divalent drug moiety and the divalent lipid moiety may be divalent derivatives of a chemical compound and a lipid, respectively. For example, the divalent derivative may be a deprotonated derivative of a chemical compound or lipid containing a hydroxyl, thiol, amino, or carboxylic acid group.

[0112] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure.

[0113] PRO.) Drug Section Another aspect of the present invention provides a novel TB prodrug compound (or multiple compounds) comprising an ALK5 inhibitor having the formula represented as TB4.

[0114] Those skilled in the art will understand that the compound is useful as an ALK5 signaling inhibitor (e.g., inhibiting ALK5 and other family members). As brief background, ALK5, also known as TGFβ receptor I (TGFβRI), is a membrane-bound receptor belonging to a superfamily of cytokines that acts on protein kinase receptors in the cell membrane to induce numerous biological signals that regulate cell growth and death, differentiation, immune responses, angiogenesis, and inflammation. Dysregulation of this pathway contributes to a wide variety of pathologies, including cancer. TGFβ is an important regulatory tumor inhibitor in epithelial cells, inhibiting premature proliferation and inducing apoptosis. See FABREGAT, et. al., TGF-beta Signaling in Cancer Treatment, Curr. Pharm. Des. 20(17): pp. 2934-2947 (2014). That study showed that the development of therapeutic compounds that target or block the action of TGFβ may be useful in treating cancer. See HAQUE, et. al., Transforming growth factor-β: A Therapeutic Target for Cancer, Hum. Vaccin. Immunother., 13(8): pp. 1741-1750 (2017).

[0115] Based on the above, this disclosure describes a class of TGFβ inhibitors.

[0116] In one embodiment, the drug portion of this disclosure has the following chemical structure (indicated as TB4): [ka] It contains compounds that have the following properties.

[0117] In one embodiment, the drug portion of the present disclosure includes a protecting group intermediate that leads to the final TB4 prodrug shown in Figure 2.

[0118] In further embodiments, the drug portion of the present disclosure includes a protecting group intermediate that leads to the final TB4 prodrug shown in Figure 3.

[0119] In one embodiment, the drug portion of the present disclosure includes a protecting group intermediate that leads to the final TB4 prodrug (ALT-1) shown in Figure 20.

[0120] In further embodiments, the drug portion of the present disclosure includes a protecting group intermediate that leads to the final TB4 prodrug (ALT-1) shown in Figure 21.

[0121] In further embodiments, the drug portion of the present disclosure includes an intermediate chemical synthesis to arrive at the final TB4 prodrug (ALT-1) shown in Figure 22.

[0122] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure.

[0123] IV.) Lipids Generally speaking, and for the purposes of this disclosure, the term “lipid” is used in its broadest sense, but is not limited thereto, and includes several subcategories of lipids, including phospholipids / fatty acids. As recognized by those skilled in the art, phospholipids represent a certain class of lipids that are the main components of all cell membranes. Due to their amphiphilic properties, phospholipids can form lipid bilayers. 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 a simple organic molecule, such as choline, ethanolamine, or serine. These two components are usually linked together by a glycerol molecule. A representative list of the phospholipids / fatty acids of the present invention is shown in Table III.

[0124] As a brief background, at the most basic level, the properties of liposomes are determined by 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, and the properties of these bilayers can be tuned to modulate drug release and membrane stability. In a simplified bilayer model, acyl chain length dictates the bilayer thickness and phase transition temperature (Tm), acyl chain saturation controls the bilayer fluidity, and head group interactions influence intermolecular and intramolecular forces. Liposome behavior can be tuned by incorporating synthetic lipids, such as lipid prodrugs, fusionable lipids, and functionalizable lipids, into the bilayer. See KOHLI, et. al., J. Control Release, 0: pp. 274-287 (Sept. 28, 2014).

[0125] In one embodiment of the present disclosure, the TB prodrug comprises a monovalent lipid moiety.

[0126] In one embodiment, the TB prodrug includes a divalent lipid moiety.

[0127] In one embodiment, the lipid has the following chemical structure: [ka] It contains cholesterol.

[0128] In one embodiment, the lipid has the following chemical structure: [ka] Includes DPPG which has [specific properties].

[0129] In one embodiment, the lipid has the following chemical structure: [ka] Includes DMPG having [specific properties].

[0130] In one embodiment, the lipid has the following chemical structure: [ka] Includes Lyso PC.

[0131] In one embodiment, the lipid has the following chemical structure: [ka] Includes (Δ9-Cis)PG having

[0132] In one embodiment, the lipid has the following chemical structure: [ka] Includes Soy Lyso PC.

[0133] In one embodiment, the lipid has the following chemical structure: [ka] Includes PG which has

[0134] In one embodiment, the lipid has the following chemical structure: [ka] It contains C16 PEG2000 ceramide (Ceramde) which has [a specific characteristic].

[0135] In one embodiment, the lipid has the following chemical structure: [ka] It contains cholesteryl hemysuccinate ("CHEMS") which has the following properties.

[0136] For reference, a complete list of the chemical formulas and abbreviations of the lipids disclosed herein is provided in Table I.

[0137] In additional embodiments, the lipids include phospholipids / fatty acids disclosed herein and listed in Table III.

[0138] In further embodiments, the lipid includes stearic acid.

[0139] In addition, the TB prodrugs and / or liposomes(s) of this disclosure may comprise one or more helper lipids, also referred to herein as “helper lipid components.” The helper lipid components are preferably selected from the group comprising phospholipids and steroids. The phospholipids are preferably diesters and monoesters of phosphate. Preferred members of the phospholipids are phosphoglycerides and sphingolipids. The steroids, as used herein, are naturally occurring and synthetic compounds based on partially hydrogenated cyclopenta[a]phenanthrene. Preferably, the steroids contain 21 to 30 carbon atoms. A particularly preferred steroid is cholesterol.

[0140] While not bound by any theory, it should be noted that remarkable effects may be achieved due to a specific molar percentage of helper lipids (which may be either PEG-free or PEG-containing) contained in the lipid composition according to the present invention, and more particularly, when the content of any of these types of helper lipids falls within the concentration range specified herein.

[0141] In further embodiments of the present invention, the lipid composition, preferably existing 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 preferred embodiments, the lipid composition comprises any helper lipid or combination of helper lipids and any ALK5 inhibitor described herein (e.g., TB4). In further embodiments, the composition according to the present invention containing nucleic acids(s) forms a lipoplex. In preferred embodiments, the term lipoplex, as used herein, refers to a composition comprising neutral or anionic lipids, neutral helper lipids and the ALK5 inhibitor of the present invention. For references to the use of helper lipids in this 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. al., Biochimica et. Biophysica Acta, 1828, pp. 412-418 (2013).

[0142] In preferred embodiments, the helper lipids of the present invention include the helper lipids listed in Table II.

[0143] In one embodiment, the TB prodrug comprises the lipid of the present invention, where the lipid is CHEMS and the drug portion is TB4.

[0144] In one embodiment, the TB prodrug comprises the lipid of the present invention, where the lipid is CHEMS, the drug portion is TB4, and further comprises LU, where LU is a hydromethylcarbamate linker.

[0145] In one embodiment, the TB prodrug comprises the lipid of the present invention, where the lipid is CHEMS, the drug portion is TB4, and further comprises LU, where LU is a hydromethylcarbamate linker, and further comprises a helper lipid component, the helper lipid component comprising the helper lipids of Table II.

[0146] In one embodiment, the TB prodrug comprises the lipid of the present invention, where the lipid is CHEMS, the drug portion is TB4, and CHEMS is monovalent.

[0147] In one embodiment, the TB prodrug comprises the lipid of the present invention, where the lipid is stearic acid and the drug portion is TB4.

[0148] In one embodiment, the TB prodrug contains the lipid of the present invention, where the lipid is stearic acid, the drug portion is TB4, and the stearic acid is monovalent.

[0149] In one embodiment, the TB prodrug comprises the lipid of the present invention, where the lipid is stearic acid, the drug portion is TB4, and further comprises LU, where LU is a hydromethylcarbamate linker.

[0150] In one embodiment, the TB prodrug comprises the lipid of the present invention, where the lipid is stearic acid, the chemical composition is TB4, further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising a helper lipid component, the helper lipid component comprising the helper lipids of Table II.

[0151] In one embodiment, the TB prodrug comprises the lipid of the present invention, the lipid being stearic acid, the drug portion being TB4, and the TB prodrug having the following chemical structure: [ka] It has.

[0152] In one embodiment, the TB prodrug comprises the lipid of the present invention, the lipid being stearic acid, the drug portion being TB4, and the TB prodrug having the following chemical structure: [ka] It has.

[0153] In one embodiment, the TB prodrug comprises the lipid of the present invention, the lipid being stearic acid, the drug portion being TB4, and the TB prodrug having the following chemical structure: [ka] It has.

[0154] In one embodiment, the TB prodrug comprises the lipid of the present invention, the lipid being stearic acid, the drug portion being TB4, and the TB prodrug having the following chemical structure: [ka] It has.

[0155] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure.

[0156] V.) Linked Unit ("LU") In some embodiments, the subject matter of the present disclosure provides prodrugs comprising drug-lipid conjugates including biodegradable linkers, such as esters, thioesters, and other linkers known in the art.

[0157] Exemplary embodiments of ester chemistry are described herein. [ka]

[0158] In some embodiments, the prodrug is a drug-lipid conjugate, which is cleaved by an esterase.

[0159] In one embodiment, the prodrug of the present invention follows the scheme: [ka] Using a secondary amine, amide, or aniline, the LU is included. An example of synthesis is as follows: [ka] The cleavage of prodrug structures containing secondary amines, amides, or anilines is illustrated in the following exemplary synthesis: [ka] Under these conditions, obtained via esterase hydrolysis of secondary amines, amides, or aniline prodrugs. [In the formula, R1-NH-R2 can be any molecule containing a secondary amine, amide, or aniline.

[0160] In one embodiment, the secondary amide nitrogen of the TB4 drug portion is conjugated to CHEMS via a hydromethylcarbamate linker.

[0161] In one embodiment, the TB4 prodrug (ALT-1) has the following chemical structure: [ka] It conjugates to lipids via a pyrazole bond having [wherein B=-CH2-OC(O)-R, R=C 11 ~C 21 It is a saturated alkane.

[0162] In one embodiment, the TB4 prodrug (ALT-1) has the following chemical structure: [ka] It conjugates to lipids via an amide bond having [wherein B=-CH2-OC(O)-R, R=C 11 ~C 21 It is a saturated alkane.

[0163] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure.

[0164] VI.) Nanocarriers Generally speaking, and for the purposes of this disclosure, nanocarriers 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 materials. Because of their small size, nanocarriers can deliver drugs to sites in the body that would otherwise be inaccessible. 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").

[0165] In addition, nanocarriers are useful in drug delivery processes because they can deliver drugs to site-specific targets, ensuring that drugs are delivered to specific organs or cells rather than elsewhere. Site specificity offers significant therapeutic benefits by preventing the drug from being delivered to the wrong location. Furthermore, 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. Since chemotherapy drugs can be highly toxic to human cells, it is crucial that chemotherapy drugs are delivered to tumors without being released to other parts of the body.

[0166] Generally speaking, there are four ways in which nanocarriers can deliver drugs, and these methods include passive targeting, active targeting, pH specificity, and temperature specificity.

[0167] Passive targeting refers to the ability of nanocarriers to circulate within the tumor's vascular system, be captured, and accumulate in the tumor. This accumulation is caused by enhanced permeability and retention. The tumor's leaky vascular structure is a network of blood vessels formed within the tumor, and this network contains many small pores. These pores not only allow nanocarriers to enter but also contain many bends that can capture them. As more nanocarriers are captured, the drug accumulates at the tumor site. This accumulation allows for the direct delivery of large doses of the drug to the tumor site.

[0168] Active targeting involves incorporating targeting modules, such as ligands or antibodies, that are specific to certain types of cells throughout the body, onto the surface of a nanocarrier. Generally, nanocarriers have a high surface area-to-volume ratio, allowing for the incorporation of multiple ligands onto their surface.

[0169] In addition, certain nanocarriers release the drugs they contain only within a specific pH range. This 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 tissues have 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. The highly acidic environment causes drug release by degrading the nanocarrier's structure. Generally, these nanocarriers do not release drugs in neutral or basic environments, leaving normal somatic cells intact, while effectively targeting the acidic environment of tumors. This pH sensitivity can also be induced in micelle systems by adding copolymer chains to micelles that are determined to act independently of pH. 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 triggers a rapid release of the micelle polymers, rather than the gradual release seen with other drug treatments, allowing the majority of the drug to be released in a single dose.

[0170] In addition, some nanocarriers have been shown to deliver drugs more effectively at certain temperatures. Since tumor temperature is generally around 40°C, which is higher than the temperature of the rest of the body, this temperature gradient helps act as a safeguard for delivery to tumor-specific sites. See REZAEI, et. al., Polymer, 53(16): 3485-3497 (2012).

[0171] As disclosed herein, lipid-based nanocarriers, such as liposomes, are within the scope of the present invention. Lipid-based nanoparticles (LBNPs or LNPs), such as liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs) transport hydrophobic and hydrophilic molecules, exhibit very low toxicity or no toxicity at all, and can increase drug action duration by extending the drug half-life and using controlled release. Lipid nanoparticles may include 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 prepared into pH-sensitive formulations to facilitate drug release in acidic environments and can also associate with small molecules or antibodies or their receptors (e.g., folic acid (FoA)) that recognize tumor cells. Nanodrugs can also be used in combination with other therapeutic strategies to improve patient response. See GARCIA-PINEL, et. al., Nanomaterials 9(639) (2019).

[0172] In various embodiments, the silicasome drug carriers described herein include nanoparticles of porous silica (or other materials) coated with a lipid bilayer (e.g., silica bodies having a surface and defining multiple pores suitable for receiving molecules inside). The fact that the nanoparticles are called silica nanoparticles does not preclude the incorporation of non-silica materials into the silica nanoparticles. In some embodiments, the silica nanoparticles may be substantially spherical and have multiple pore openings on their surface providing access to the pores. However, in various embodiments, the silica nanoparticles may have shapes other than substantially spherical. Thus, for example, in certain embodiments, the silica nanoparticles may be substantially oval, rod-shaped, substantially regular polygonal, irregular polygonal, and so on.

[0173] Generally, silica nanoparticles contain silica bodies that define the outer surfaces and inner side walls between pore openings. Pores can extend across the entire silica body to another pore opening, or they can extend only partially across the silica body, having a bottom surface defined by the silica body.

[0174] In some embodiments, the silica material is mesoporous. In other embodiments, the silica material is microporous. As used herein, “mesoporous” means having pores with a diameter between about 2 nm and about 50 nm, while “microporous” means having pores with a diameter smaller than about 2 nm. Generally, the pores can be of any size, but in typical embodiments, they are large enough to contain one or more therapeutic compounds. In such embodiments, the pores allow small molecules, such as therapeutic compounds, such as anticancer compounds, to adhere to or bind to the inner surface of the pores and be released from the silica material when used for therapeutic purposes. In some embodiments, the pores are substantially cylindrical.

[0175] In certain embodiments, the nanoparticles contain multiple pores having pore diameters between approximately 1 nm and 10 nm or between approximately 2 nm and 8 nm. In certain embodiments, the nanoparticles contain multiple pores having pore diameters between approximately 1 nm and 6 nm or between approximately 2 nm and 5 nm. Other embodiments contain particles with pore diameters less than 2.5 nm.

[0176] In other embodiments, the pore size is between 1.5 and 2.5 nm. For example, silica nanoparticles with other pore sizes can be prepared by using various surfactants or swelling agents during the preparation of silica nanoparticles. In various embodiments, nanoparticles can include particles as large as approximately 1000 nm (e.g., average or median diameter (or other characteristic dimension)). However, in various embodiments, nanoparticles are typically less than 500 nm or about 300 nm, as particles larger than 300 nm may not be effective in 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 up to about 90 nm, or up to about 80 nm, or up to about 70 nm. In certain embodiments, nanoparticles range in size from about 60 nm to about 70 nm. Some embodiments include nanoparticles with an average maximum dimension between about 50 nm and about 1000 nm. Other embodiments include nanoparticles with an average maximum dimension between about 50 nm and about 500 nm. Still other embodiments include nanoparticles with an average maximum dimension between about 50 nm and about 200 nm.

[0177] 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 an average maximum dimension of 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, the size of a nanoparticle refers to the average or median size of the primary particle when 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).

[0178] Methods for preparing porous silica nanoparticles are well known to those skilled in the art. In certain embodiments, mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate (TEOS) with a template prepared from micellar rods. The result is an aggregate of nano-sized spheres or rods filled with regularly arranged pores. The template can then be removed by washing with a solvent adjusted to the appropriate pH (see, for example, TREWYN et al. (2007) Chem. Eng. J. 137(1): 23-29).

[0179] 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 additional polymer monomers as templates. In certain embodiments, 3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS.

[0180] In certain embodiments, mesoporous silica nanoparticles are cores synthesized by a modification of the sol / gel procedure described by MENG et. al. (2015) ACS Nemo, 9(4): 3540-3557.

[0181] The methods described herein have been demonstrated with respect to porous silica nanoparticles (e.g., mesoporous silica), but those skilled in the art will recognize that similar methods can be used with other porous nanoparticles. A number of 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 used.

[0182] 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.).

[0183] Similarly, in certain embodiments, mesoporous polymer particles can be utilized. The synthesis of highly ordered mesoporous polymers and carbon frameworks from organic-organic aggregates of triblock copolymers and soluble low molecular weight phenolic resin precursors (resols) by an evaporation-induced self-assembly strategy was reported by MENG, et. al. (2006) Chem. Mat. 6(18): 4447-4464.

[0184] The nanoparticles described herein are illustrative and non-limiting. Using the teachings provided herein, numerous other lipid bilayer-coated nanoparticles will be available to those skilled in the art.

[0185] In one embodiment, the present invention teaches a nanocarrier comprising a TB prodrug.

[0186] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, where the lipid comprises CHEMS.

[0187] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, where the lipid comprises stearic acid.

[0188] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, where the lipid comprises CHEMS and the liposomes further comprise a TB prodrug.

[0189] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, where the lipid comprises CHEMS and the liposomes further comprise TB4.

[0190] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise an ALK5 inhibitor.

[0191] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise TB4.

[0192] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise TB4 (designated as LNP-TB4).

[0193] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise TB4(ALT-1) (designated as LNP-TB4(ALT-1)).

[0194] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise TB4(ALT-2-pyrazole) (designated as LNP-TB4(ALT-2-pyrazole)).

[0195] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise TB4(ALT-3-amide) (designated as LNP-TB4(ALT-3-amide)).

[0196] In a further embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise TB4, and the liposomes are co-formulated with ID3 (designated as LNP-TB4-ID3).

[0197] In a preferred embodiment, the lipid particles comprise solid lipid nanoparticles (SLNP) comprising liposomes containing a TB4 prodrug.

[0198] In preferred embodiments, the lipid particles comprise solid lipid nanoparticles (SLNPs) containing liposomes that include the TB4 prodrug-ALT-1.

[0199] In a preferred embodiment, the lipid particles comprise solid lipid nanoparticles (SLNPs) containing liposomes comprising the TB4 prodrug-ALT-2-pyrazole.

[0200] In preferred embodiments, the lipid particles comprise solid lipid nanoparticles (SLNPs) containing liposomes comprising the TB4 prodrug-ALT-3-amide.

[0201] In one embodiment, the present invention teaches a nanocarrier comprising solid lipid nanoparticles ("SLNP"), wherein the solid lipid nanoparticles comprise stearic acid, and the solid lipid nanoparticles further comprise TB4 (indicated as SLNP-TB4).

[0202] In one embodiment, the present invention teaches a nanocarrier comprising solid lipid nanoparticles ("SLNP"), wherein the solid lipid nanoparticles comprise stearic acid, and further comprise TB4(ALT-1) (denoted as SLNP-TB4-ALT-1).

[0203] In further embodiments, the present invention teaches a nanocarrier comprising solid lipid nanoparticles ("SLNP"), wherein the solid lipid nanoparticles comprise stearic acid, further comprising TB4, and the SLNPs are co-formulated with ID3 (indicated as SLNP-TB4-ID3).

[0204] In further embodiments, the present invention teaches a nanocarrier comprising solid lipid nanoparticles ("SLNP"), wherein the solid lipid nanoparticles comprise stearic acid, further comprising TB4, and the SLNPs are co-formulated with IC1 (denoted as SLNP-TB4-IC1).

[0205] In a more preferred embodiment, the solid lipid nanoparticles of the present invention comprise a composition having the following ratio: [Table A] Here, lipid 1 comprises a TB4 prodrug, the lipid portion comprises stearic acid, the helper lipid is one of the helper lipids listed in Table II, the stabilizer is selected from the group consisting of polyvinyl alcohol (e.g., Molivol 488), poloxamer (e.g., Pluronic® F127), Tween® 80, PEG400, and Kolliphor RH40, and lipid 2 (lipid prodrug) comprises the lipid prodrugs of this 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 telratolimod.

[0206] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure.

[0207] In addition, the scope of this disclosure teaches three possible treatment modalities using the formulated prodrug of the present invention. See PCT Patent Application Publication WO2018 / 213631.

[0208] The first treatment modality involves combining a TB prodrug with another treatment (e.g., another formulated prodrug inhibiting ALK5 and other family members, a chemotherapeutic agent (e.g., ICD-inducing chemotherapy)) into a single liposome that enables systemic (or local) intracellular distribution and drug delivery to tumor sites. The dual delivery technique achieves synergistic enhancement of adaptive and innate immunity, significantly improving animal survival. In certain embodiments, the nanocarrier comprises vesicles (i.e., lipid bilayers encapsulating fluids).

[0209] The second treatment modality involves the local delivery of an agent that inhibits ALK5 and other family members to a tumor or the area surrounding a tumor in combination with a lipid (e.g., liposome) that includes an ALK5 signaling inhibitor.

[0210] The third treatment modality includes vaccination that utilizes dying cancer cells (e.g., KPC cells), where inhibition of ALK5 is induced ex vivo. Such vaccination can generate a systemic immune response that can interfere with tumor growth at a distant site and has been found to enable engraftment into non-immune animals. Those skilled in the art can recognize and implement the methods of the treatment modalities provided herein.

[0211] VII.) Liposomes In one aspect, the subject matter of the present disclosure is based on a technique for providing a TB prodrug of the present disclosure (see the section titled "Prodrugs") that is incorporated into a nanocarrier that includes a lipid coating layer to enhance delivery of the corresponding prodrug and is suitable for providing a combination therapy that includes the prodrug. Advantages of using the prodrugs of the present invention include facilitating controlled formulation into the LNPs (e.g., liposomes) of the present disclosure. Thereby, the prodrug is maintained in an inactive form in the systemic circulation, such that, for example, after phagocytosis by cells within a tumor, the liposome can release the active agent.

[0212] In certain embodiments, one or more TB prodrugs (e.g., any one or more of the TB prodrug inhibitors taught by Formula I, Formula II, Formula III, and / or the TB4 prodrug; see the section titled "Prodrugs") are formulated with a lipid moiety that can form a vesicle (e.g., liposome) structure in an aqueous solution or can form a component of a lipid bilayer that includes liposomes. The liposomes can be used and provided directly as a component in a combined formulation (e.g., in combination with another drug moiety or treatment modality disclosed herein).

[0213] In certain embodiments, the liposomes formulated with the TB prodrug contain lipids, PHGP, vitamin E, cholesterol, and / or fatty acids.

[0214] In one embodiment, the liposomes contain cholesterol.

[0215] In one embodiment, the liposome includes a DSPC.

[0216] In one embodiment, the liposomes include HSPCs.

[0217] In one embodiment, liposomes are DSPE-PEG 2000 Includes.

[0218] In one embodiment, the liposome contains DPPG.

[0219] In one embodiment, the liposome contains DMPG.

[0220] In one embodiment, the liposomes include Lyso PC.

[0221] In one embodiment, the liposome contains (Δ9-Cis)PG.

[0222] In one embodiment, the liposomes contain Soy Lyso PC.

[0223] In one embodiment, the liposome contains PG.

[0224] In one embodiment, the liposome contains PA-PEG3-mannose.

[0225] In one embodiment, the liposomes contain C16 PEG2000 ceramide.

[0226] In one embodiment, the liposome contains MPLA.

[0227] In one embodiment, the liposomes contain CHEMS.

[0228] In one embodiment, the liposome contains stearic acid.

[0229] In one embodiment, the liposomes contain the phospholipids listed in Table III.

[0230] In one embodiment, the liposome comprises TB4, further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0231] In one embodiment, the liposome comprises TB4, further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0232] In one embodiment, the liposome comprises TB4, further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0233] In one embodiment, the liposome comprises TB4, further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0234] In one embodiment, the liposomes of the present disclosure comprise a TB prodrug co-formulated with one or more additional immunomodulators, wherein the immunomodulators include, but are not limited to, immunogenic cell death-inducing chemotherapeutic agents, Toll receptor agonists, Sting agonists, IDO inhibitors, CTLA4 inhibitors, PD-1 inhibitors, and / or their prodrugs.

[0235] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with an ICD-inducing chemotherapeutic agent.

[0236] In a preferred embodiment, the liposome comprises a TB prodrug co-formulated with an ICD-inducing chemotherapeutic agent selected from the list of doxorubicin (DOX), mitoxantrone (MTO), oxaliplatin (OXA), cyclophosphamide (CP), bortezomib, carfilzomib, or paclitaxel.

[0237] In a preferred embodiment, the liposome contains a TB prodrug co-formulated with a Toll receptor TLR agonist / prodrug.

[0238] In a more preferred embodiment, the Toll receptor TLR agonist / prodrug is selected from the group consisting of TR3, TR4, TR5, and TR6.

[0239] In preferred embodiments, the liposomes contain a TB prodrug co-formulated with a Toll receptor (TLR) agonist / prodrug selected from the list of reximod (R848), gardikimod, 852A, DSR6434, tellaterimod, CU-T12-9, monophosphoryllipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD® or its prodrugs.

[0240] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a PD-1 inhibitor / prodrug.

[0241] In a preferred embodiment, the liposome comprises a TB prodrug co-formulated with a PD-1 inhibitor / prodrug selected from AUNP12, CA-170, or BMS-986189 or a list of its prodrugs.

[0242] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with an IDO-1 inhibitor / prodrug.

[0243] In a preferred embodiment, the liposome contains a TB prodrug co-formulated with an IDO-1 inhibitor / prodrug selected from the list of epacadostat, L-1-methyltryptophan (indoximod), D-1-methyltryptophan, linrhodostat mesylate (BMS986205), MK-7162, LY-3381916, KHK-2455, HTI-1090, DN-1406131, or BGB-5777.

[0244] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with doxorubicin (DOX).

[0245] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with mitoxantrone (MTO).

[0246] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with doxorubicin (DOX) and a PD-1 prodrug.

[0247] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with mitoxantrone (MTO) and a PD-1 prodrug.

[0248] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with doxorubicin (DOX) and an IDO-1 prodrug.

[0249] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with mitoxantrone (MTO) and IDO-1 prodrug.

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

[0251] In a preferred embodiment, the liposome comprises a TB prodrug co-formulated with mitoxantrone (MTO) and a TLR agonist / prodrug.

[0252] In preferred embodiments, the liposomes contain doxorubicin (DOX) and a TB prodrug co-formulated with a PD-1 prodrug and a TLR agonist / prodrug.

[0253] 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.

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

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

[0256] In a preferred embodiment, the liposomes contain a TB prodrug co-formulated with a CD1D agonist / prodrug.

[0257] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a TLR agonist / prodrug and a PD-1 prodrug.

[0258] In a preferred embodiment, the liposomes include a TB prodrug co-formulated with a TLR agonist / prodrug and an IDO-1 prodrug.

[0259] In a preferred embodiment, the liposomes contain a TB4 prodrug co-formulated with doxorubicin (DOX).

[0260] In a preferred embodiment, the liposomes contain a TB4 prodrug co-formulated with mitoxantrone (MTO).

[0261] In a preferred embodiment, the liposome comprises a TB4 prodrug co-formulated with doxorubicin (DOX) and / or an IDO prodrug and / or a TLR agonist / prodrug.

[0262] In a preferred embodiment, the liposome comprises a TB4 prodrug co-formulated with mitoxantrone (MTO) and / or an IDO prodrug and / or a TLR agonist / prodrug.

[0263] In a preferred embodiment, the liposome contains a TB4 prodrug (ALT-1) co-formulated with doxorubicin (DOX).

[0264] In a preferred embodiment, the liposome contains a TB4 prodrug (ALT-1) co-formulated with mitoxantrone (MTO).

[0265] In a preferred embodiment, the liposome comprises a TB4 prodrug (ALT-1) co-formulated with doxorubicin (DOX) and / or an IDO prodrug and / or a TLR agonist / prodrug.

[0266] In a preferred embodiment, the liposome comprises a TB4 prodrug (ALT-1) co-formulated with mitoxantrone (MTO) and / or an IDO prodrug and / or a TLR agonist / prodrug.

[0267] Those skilled in the art will recognize and understand that solubility is one of the most common problems they face in the drug development process. Chemical conjugations of drugs / anticancer agents via lipid molecules (i.e., lipid-based prodrugs) provide a platform for solving problems in formulating drugs into aqueous suspensions. The main advantage of delivering drugs using lipid conjugations (lipid-based prodrugs) lies in their ability to improve pharmacokinetics / half-life and targeted delivery.

[0268] Lipid-based prodrugs(s) can be integrated / formulated into liposomal formulations using techniques known in the art, by appropriately selecting lipid molecules, thereby offering many advantages over conventional drug delivery systems. (KOHLI, et. al., J. Control Release, 0:pp 274-287 (Sept. 28, 2014) and GARCIA-PINEL, et. al., Nanomaterials 9:638 (2019)). The advantages of combining lipid-prodrugs with liposomes are doubled because (i) liposomes containing lipid-prodrugs not only increase the solubility of the drug / prodrug itself, but also (ii) they have the ability to encapsulate multiple drugs (both hydrophilic and lipophilic) (see the section titled Nanocarriers).

[0269] For the purposes of this disclosure, the main advantages of liposomal formulations are as follows: i) The liposome formulation must be biocompatible / biodegradable and have no general toxicity. ii) The flexibility and manipulability of the size and surface charge may vary depending on the required purpose. For the purposes of this disclosure, liposome formulations may have a size range of 40 to 150 nm in diameter and a surface charge range of -40 to +40 mV. Furthermore iii) The liposomes of the present invention have one or more lipid-prodrugs as the lipid portion that is a component of the liposome(s). In addition, multiple drugs having different solubility profiles (hydrophilic or lipophilic) (e.g., acting by different mechanisms of action) can be formulated into these liposomes (either in the lipid bilayer or the hydrophilic core).

[0270] As will be recognized by those skilled in the art, all methods for producing liposomes include the following four basic steps: (i) The step of drying the lipids from the organic solvent, (ii) The step of dispersing lipids in an aqueous solution, (iii) the step of purifying the obtained liposomes, and (iv) The final product is analyzed. See AKBARZADEH, et. al., Nanoscale Research Letters, 8:102 (2013).

[0271] Another aspect of the present invention discloses liposome encapsulation technology (LET), a delivery technology used to permeate drugs. LET is a method for creating submicroscopic foamy structures called liposomes that encapsulate a number of materials. These “liposomes” form a barrier around their contents that is resistant to oral and gastric enzymes, alkaline solutions, digestive fluids, bile salts, as well as the intestinal flora and free radicals produced in the human body. Thus, the contents of the liposome are protected from oxidation and degradation. This protective phospholipid shield or barrier remains intact until the contents of the liposome are delivered to a precise target gland, organ, or system where they are utilized (see the section titled Nanocarriers).

[0272] In one embodiment, the liposome(s) of the Disclosure are synthesized using several different ratios of TB prodrugs, lipids, and / or lipid-prodrugs. As disclosed herein, the TB prodrug may include the helper lipids disclosed herein (see, for example, Table II).

[0273] In one embodiment, the liposome(s) of this disclosure are synthesized using several different ratios of TB prodrugs, lipids, and / or lipid-prodrugs. As disclosed herein, the TB prodrug may further comprise DSPE-PEG.

[0274] In a preferred embodiment, the liposome of the present invention comprises a composition having the following ratio: [Table B]

[0275] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratio: [Table C]

[0276] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratio: [Table D] Here, lipid 1 contains the TB4 prodrug, and the lipid portion contains CHEMS.

[0277] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratio: [Table E] Here, lipid 1 contains the TB4 prodrug, and the lipid portion contains stearic acid.

[0278] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure.

[0279] VIII.) Pharmaceutical Formulation As used herein, the term “drug” is synonymous with “pharmaceutical.” In certain embodiments, the liposomes of this disclosure are processed into encapsulated dosage forms and administered to a patient for the treatment of a disease.

[0280] Generally speaking, pharmaceutical formulation is the process of combining various chemical substances with a pure active pharmaceutical ingredient (API) to produce the final drug product. Formulation research involves developing drug preparations that are stable and tolerable for patients. For orally administered drugs, this typically involves incorporating the drug into tablets or capsules. It is important to fully understand that dosage forms contain various other substances besides the drug itself, and research must be conducted to ensure that the drug is compatible with these other substances.

[0281] Additives are inert substances used as carriers for the active ingredient of a drug product, in this case as liposomes containing the TB prodrug. In addition, additives can be used to assist in the process of manufacturing the drug product. The active ingredient is then dissolved or mixed with the additive. Additives are also sometimes used to increase the volume of formulations containing very potent active ingredients, thus enabling convenient and precise dosing. Once the active ingredient has been purified, it cannot remain in its purified form for extended periods. Often, the active ingredient denatures, separates from the solution, or adheres to the sides of the container.

[0282] Additives are added to stabilize the active ingredient, ensuring that the active ingredient remains active and stable for a sufficiently long period to ensure the product's shelf life is competitive with other products and safe for the end user. Examples of additives include, but are not limited to, anti-adhesives, binders, coatings, disintegrants, fillers, diluents, flavors, colorants, lubricants, and preservatives. The final formulation contains the active ingredient and additives, which are then encapsulated in a pharmaceutical dosage form.

[0283] Pre-formulation involves characterizing the physical, chemical, and mechanical properties of a drug in order to select which other components should be used in the preparation. Next, formulation studies consider factors such as stability, particle size, polymorphism, pH, and solubility, as all of these can affect bioavailability and therefore the activity of the drug. The drug must be combined with inert additives in a manner that ensures the amount of drug present is consistent in each dose unit (e.g., each vial). The doses should have a uniform appearance.

[0284] These studies are unlikely to be completed by the start of clinical trials. This means that simpler preparations will be developed early on for use in Phase I clinical trials. These typically consist of vials or hand-filled capsules containing small amounts of the drug and diluent. Evidence of long-term stability of these formulations is not necessary because they will only be used (tested) for a few days. However, long-term stability is crucial in supply chain management because the time from when the final formulation is packaged until it reaches patients can be months or even years. Something called drug load (i.e., the ratio of active drug to the total content of the dose) must be considered. A low drug load can cause homogeneity problems. A high drug load can lead to flow problems or require larger capsules if the bulk density of the compound is low. By the time Phase III clinical trials are reached, the drug formulation should be developed to be close to the final preparation used on the market.

[0285] Knowledge of stability is essential up to this stage, and conditions must be developed to ensure that the drug is stable in the preparation. If the drug is found to be unstable, the results of clinical trials will be invalid, as it would be impossible to know what the actual dose was. Stability studies are conducted to test whether temperature, humidity, oxidation, or photodegradation (ultraviolet or visible light) has any effect, and the preparation is analyzed to see if any degradation products have been formed. It is also important to check for any undesirable interactions between the preparation and the container. If a plastic container is used, tests are conducted to see if any of the components adsorb onto the plastic, and whether any plasticizers, lubricants, pigments, or stabilizers leach from the plastic into the preparation. The adhesive for the container label also needs to be tested to ensure that it does not leach from the plastic container into the preparation. The way the drug is formulated 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 an aqueous solution at a controlled rate. Factors such as particle size and crystal morphology can significantly affect dissolution. Rapid dissolution is not always ideal. For example, a slow dissolution rate can prolong the duration of action or avoid initial high plasma levels.

[0286] In some embodiments, nanocarriers (e.g., liposomes containing TB prodrugs) and / or liposomes co-formulated with an immunomodulatory agent, including a TB prodrug, are administered alone or in mixtures with a physiologically acceptable carrier (e.g., saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practices. For example, when used as an injectable, the nanocarrier can be formulated as a sterile suspension, dispersion, or emulsion using a pharmaceutically acceptable carrier. In certain embodiments, normal saline can be used as the pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, 5% glucose, etc., including glycoproteins to enhance stability, such as albumin, lipoproteins, and globulins. In compositions containing a carrier with saline or other salts, the carrier is preferably added after nanocarrier formation. Thus, after the nanocarrier is formed and loaded with the appropriate drug(s), the nanocarrier can be diluted with a pharmaceutically acceptable carrier, such as normal saline. Similarly, TB prodrug liposomes can be introduced into carriers that facilitate the suspension (e.g., emulsification, dilution, etc.) of nanomaterials.

[0287] The pharmaceutical composition may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions, suspensions, dispersions, emulsions, etc., may be packaged for use or filtered under sterile conditions. In certain embodiments, drug delivery nanocarriers (e.g., nanoparticles coated with LB) are lyophilized, and the lyophilized preparation is mixed with a sterile aqueous solution before administration. The composition may also contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0288] In addition, in certain embodiments, the pharmaceutical formulation may include lipid protectants that protect lipids from free radical and lipid peroxidation damage during storage. Suitable and intended herein are lipophilic free radical quenchers, such as alpha-tocopherol, and water-soluble iron-specific chelating agents, such as ferrioxamine. The concentration of nanocarriers (e.g., liposomes containing TB prodrugs) in the pharmaceutical formulation can vary widely, for example, from less than approximately 0.05%, typically from at least approximately 2-5% to as much as 10-50%, or up to 40%, or up to 30% by weight, and is selected mainly by fluid volume, viscosity, etc., according to a selected specific administration method. For example, the concentration may be increased to reduce the fluid load associated with the 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 depends on the specific drug used, the medical condition being treated, and the clinician's judgment, but is generally between approximately 0.01 mg and 50 mg per kilogram of body weight, preferably between approximately 0.1 mg and 5 mg per kg of body weight.

[0289] Those skilled in the art will recognize that the precise dosage varies depending on the specific TB prodrug and any co-formulated immunomodulator, as well as the desired medical effect, and patient factors such as age, sex, and overall condition. Those skilled in the art can take these factors into account and use them to easily establish effective therapeutic concentrations without excessive experimentation.

[0290] For the administration of the drugs to humans (or non-human mammals) in the curative, remission, retardative, or prophylactic treatment of the diseases described herein, the prescribing physician will ultimately determine the appropriate dosage of the drug 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 the drug delivered by the nanocarrier may be approximately the same as that used for the free drug. However, as stated above, the nanocarriers described herein can significantly reduce the toxicity of the drug(s) administered thereby and significantly increase the therapeutic range. Therefore, in some cases, a dosage exceeding that prescribed for the free drug(s) may be used.

[0291] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure.

[0292] IX.) Combination Therapy As is recognized and understood by those skilled in the art, the growth and survival of cancer cells may be influenced by multiple signaling pathways. Therefore, to treat such conditions, it is useful to combine various enzyme / protein / receptor inhibitors that exhibit different priorities to targets and modulate the activity of the targets. By targeting one or more signaling pathways (or one or more biological molecules involved in a given signaling pathway), the likelihood of drug resistance developing in a cell population and / or the toxicity of the treatment can be reduced.

[0293] Accordingly, liposomes containing the TB prodrugs of this 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 infections. Examples of diseases and indications that can be treated with combination therapy are described herein. Examples of cancers include, but are not limited to, solid tumors and humoral tumors, such as hematological malignancies. Examples of infections include viral infections, bacterial infections, fungal infections or parasitic infections.

[0294] For example, liposomes containing the TB prodrug of this 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), CSF IR, 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.

[0295] In further embodiments, liposomes comprising the TB prodrug 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 the compounds of this 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 Examples include enzymes (Tyro-3, Axl, and Mer), adenosine receptor antagonists (e.g., A2a / A2b receptor antagonists), HPK1 inhibitors, histone deacetylase inhibitors (HDACs), e.g., HDAC8 inhibitors, angiogenesis inhibitors, interleukin receptor inhibitors, bromo and extraterminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors, e.g., INCB54329 and INCB57643), poly-ADP-ribose polymerase (PARP) inhibitors, e.g., lucaparib, 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.

[0296] In addition, the liposomes comprising the TB prodrug of this disclosure can be used in combination with other methods of treating cancer, such as chemotherapy, radiotherapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, or surgery.

[0297] Examples of immunotherapies include cytokine therapy (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 virus therapy, and immunomodulatory small molecules including thalidomide or JAK1 / 2 inhibitors.

[0298] Liposomes containing TB prodrugs can be administered in combination with one or more anticancer drugs, such as chemotherapeutic agents. Examples of chemotherapeutic agents include avalerix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, olaparib, bortezomib, intravenous busulfan, oral busulfan, carsterone, capecitabine, carboplatin, carmustine, Cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin difutitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, Stramustine, 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, rebamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenylpropionatePhenpropionate), nelarabine, nofetumomab, olaparib, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobromane, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, ruxolitinib, rucaparib, sorafenib, streptozocin, s Examples include nitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tocitumomab, trastuzumab, tretinoin, uracil mustard, barrubicin, vinblastine, vincristine, vinorelbine, vorinostat, niraparib, beriparib, talazoparib, and zoledronate.

[0299] Other anticancer drugs (multiple options may be used) include antibody therapies such as trastuzumab (Herceptin), costimulatory molecules such as antibodies against CTLA-4 (e.g., ipilimumab), antibodies against 4-1BB (e.g., urelumab, utomirumab), antibodies against PD-1 and PD-L1 / L2, or antibodies against cytokines (IL-10, TGF-beta, etc.).

[0300] Examples of antibodies against PD-1 and / or PD-L1 / L2 that can be combined with the compounds of this 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.

[0301] In addition, liposomes comprising the TB prodrug of this disclosure can be used in combination with one or more immune checkpoint inhibitors for the treatment of diseases, such as cancer or infections. Exemplary immune checkpoint inhibitors include inhibitors against 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.

[0302] In some embodiments, the immune checkpoint molecule is a stimulant 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, liposomes containing the TB prodrug provided herein can be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGF beta inhibitors.

[0303] X.) Method for delivering TB prodrug-containing nanocarriers to ALK5-expressing cells As is well 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 a biologically effective amount of the TB prodrug of the present disclosure and / or the nanocarriers of the present disclosure containing the TB prodrug to a mammal having a tumor.

[0304] A typical embodiment is a method for delivering a therapeutic agent to cells expressing ALK5, comprising conjugating the drug portion of the Disclosure to a lipid of the Disclosure via a linking unit to form a TB prodrug, and exposing the cells to the TB prodrug.

[0305] In one embodiment, the TB prodrug comprises a drug moiety of formula I and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0306] In one embodiment, the TB prodrug comprises the drug moiety of formula I and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0307] In one embodiment, the TB prodrug comprises a drug portion of formula II and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0308] In one embodiment, the TB prodrug comprises the drug moiety of formula II and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0309] In one embodiment, the TB prodrug comprises a drug portion of formula III and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

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

[0311] In one embodiment, the TB prodrug comprises a TB4 prodrug, and the lipid portion comprises CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0312] In one embodiment, the TB prodrug comprises a TB4 prodrug, and the lipid portion comprises stearic acid conjugated via an LU containing a hydromethylcarbamate linker.

[0313] Another exemplary embodiment is a method for treating an individual suspected of having metastatic cancer, comprising the steps of parenterally administering to the individual a pharmaceutical composition containing a therapeutically effective amount of a TB prodrug, which is generated by conjugating a drug portion with a lipid of the Disclosure via a linking unit, thereby exposing cells to the TB prodrug.

[0314] In one embodiment, the TB prodrug comprises a drug moiety of formula I and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0315] In one embodiment, the TB prodrug comprises the drug moiety of formula I and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0316] In one embodiment, the TB prodrug comprises a drug portion of formula II and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0317] In one embodiment, the TB prodrug comprises the drug moiety of formula II and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0318] In one embodiment, the TB prodrug comprises a drug portion of formula III and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0319] In one embodiment, the TB prodrug comprises the drug moiety of formula III and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0320] In one embodiment, the TB prodrug comprises a TB4 prodrug, and the lipid portion comprises CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0321] In one embodiment, the TB prodrug comprises a TB4 prodrug, and the lipid portion comprises stearic acid conjugated via an LU containing a hydromethylcarbamate linker.

[0322] In one embodiment, the TB prodrug comprises a TB4 prodrug (ALT-1), and the lipid portion comprises stearic acid conjugated via an LU containing a hydromethylcarbamate linker.

[0323] In one embodiment, the TB prodrug comprises a TB4 prodrug (ALT-1), and the lipid portion comprises stearic acid conjugated via a LU containing a pyrazole linkage.

[0324] In one embodiment, the TB prodrug comprises a TB4 prodrug (ALT-1), and the lipid portion comprises stearic acid conjugated via an amide linkage-containing LU.

[0325] The TB prodrugs, liposomes, and co-formulated liposomes of this disclosure inhibit the activity / protein interactions of the TGFβ protein and are therefore useful for treating diseases and disorders associated with TGFβ activity as well as diseases and disorders associated with kinase inhibition. In further embodiments of this 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 enhancement of the response to vaccination.

[0326] In further embodiments, the Disclosure provides a method for inhibiting ALK5 T-cell function. The method involves 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 TB4 prodrug (ALT-1)), or any of the TB prodrugs, liposomes, SLNPs, and nanoencapsulated ALK5 inhibitor prodrugs listed in any of the claims and described herein, or pharmaceutically acceptable salts or stereoisomers thereof. The TB prodrugs, liposomes, SLNPs, and nanoencapsulated ALK5 inhibitor prodrugs of the Disclosure may be used alone, in combination with other agents or therapies, or as adjuvants or neoadjuvants to treat diseases or disorders, including cancer and other diseases. Any of the TB prodrugs, liposomes, and nanoencapsulated TB prodrugs of the Disclosure may be used, including any of the embodiments thereof, for the uses and methods described herein.

[0327] In addition, the TB prodrugs, liposomes, SLNPs, and nanoencapsulated TB prodrugs of this disclosure inhibit the function of ALK5 and / or T cells, thereby blocking the TGFβ pathway.

[0328] In further embodiments, the disclosure provides in vivo treatments of an individual or patient using TB prodrugs, liposomes, and nanoencapsulated TB prodrugs or salts or stereoisomers thereof to inhibit the growth of cancerous tumors.

[0329] TB prodrugs, liposomes, and nanoencapsulated TB prodrugs, or any of the formulas described herein (e.g., TB4 prodrug or TB4 prodrug (ALT-1)), or any of the claims listed herein, or any of the TB prodrugs, liposomes, SLNPs, and nanoencapsulated TB prodrugs, or their salts or stereoisomers, can be used to inhibit the growth of cancerous tumors.

[0330] Alternatively, the TB prodrugs, liposomes, SLNPs, and nanoencapsulated TB prodrugs of this disclosure, or any of the formulas described herein, or any of the compounds listed in any of the claims and described herein (e.g., TB4 prodrug or TB4 prodrug (ALT-1)), or their salts or stereoisomers, may be used in conjunction with other agents or standard cancer treatments as described herein.

[0331] In further embodiments, the Disclosure provides a method for inhibiting tumor cell growth in vitro. The method involves contacting tumor cells in vitro with any of the TB prodrugs, liposomes, and nanoencapsulated TB prodrugs of the Disclosure, or any of the formulas described herein (e.g., TB4 prodrug or TB4 prodrug (ALT-1)), or any of the TB prodrugs, liposomes, SLNPs, and nanoencapsulated TB prodrugs listed in any of the claims and described herein, or salts or stereoisomers thereof.

[0332] In further embodiments, the Disclosure provides a method for inhibiting the growth of tumor cells in a patient. The method involves contacting tumor cells with any of the TB prodrugs, liposomes, and nanoencapsulated TB prodrugs of the Disclosure, or any of the formulas described herein (e.g., TB4 prodrug or TB4 prodrug (ALT-1)), or any of the TB prodrugs, liposomes, SLNPs, and nanoencapsulated TB prodrugs listed in any of the claims and described herein, or their salts or stereoisomers.

[0333] XI.) Methods for treating cancer and other immunological disorders 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 of this disclosure (i.e., TB4 prodrug or TB4 prodrug (ALT-1)), a compound listed in any of the claims and described herein, or a salt thereof. Examples of cancer include cancers whose growth can be inhibited using the ALK5 inhibitors and TB prodrugs of this disclosure, as well as cancers that are typically responsive to immunotherapy.

[0334] In some embodiments, the present disclosure provides a method for enhancing, stimulating, and / or increasing a patient's immune response. The method involves administering to a patient a nanocarrier, a compound or composition listed in any of the claims and described herein, or a salt thereof, containing a therapeutically effective amount of a TB prodrug and / or its TB prodrug (i.e., TB4 prodrug or TB4 prodrug (ALT-1)).

[0335] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4 or a salt thereof to a patient.

[0336] In a further embodiment, the method includes administering a therapeutically effective amount of SLNP-TB4 or a salt thereof to a patient.

[0337] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4(ALT-1) or a salt thereof to a patient.

[0338] In further embodiments, the method includes administering a therapeutically effective amount of SLNP-TB4(ALT-1) or a salt thereof to a patient.

[0339] Non-limiting examples of cancers treatable with liposomes containing the TB prodrug of this disclosure, TB prodrugs, and co-formulated liposomes include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, and penile cancer. Examples of cancers include, but are not limited to, cancers, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia, solid tumors in childhood, lymphocytic lymphoma, bladder cancer, cancers of the kidney or urethra, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, and combinations of the said cancers. The compounds of this disclosure are also useful for treating metastatic cancers, particularly metastatic cancers expressing ALK5.

[0340] In some embodiments, cancers treatable with the liposomes or TB prodrugs of this disclosure include melanoma (e.g., metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), 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 carcinoma (e.g., bladder cancer), and high-frequency microsatellite instability (MSI). highThis includes cancer. In addition, this disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the liposomes or TB prodrugs or co-formulated liposomes of this disclosure.

[0341] In additional embodiments, cancers treatable with the formulations and / or co-formulated liposomes or TB prodrugs of the 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, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin lymphoma, or multiple myeloma) and combinations of the said cancers.

[0342] In further embodiments, cancers treatable with the formulations and / or co-formulated liposomes or TB prodrugs of the present disclosure include, but are not limited to, cholangiocarcinoma, triple-negative breast cancer, rhabdomyosarcoma, small cell lung cancer, leiomyosarcoma, hepatocellular carcinoma, Ewing's sarcoma, brain cancer, brain tumors, astrocytoma, neuroblastoma, neurofibroma, basal cell carcinoma, chondrosarcoma, epithelioid sarcoma, ocular cancer, fallopian tube cancer, gastrointestinal cancer, gastrointestinal stromal tumors, hairy cell leukemia, intestinal cancer, islet cell carcinoma, oral cancer, throat cancer, laryngeal cancer, lip cancer, mesothelioma, cervical cancer, nasal cavity cancer, eye cancer, intraocular melanoma, pelvic cancer, rectal cancer, renal cell carcinoma, salivary gland cancer, paranasal sinus cancer, spinal cord cancer, tongue cancer, tubular cancer, urethral cancer, and ureteral cancer.

[0343] In addition, in some embodiments, the formulations and / or co-formulated liposomes or TB prodrugs of the present disclosure can be used to treat sickle cell disease and sickle cell anemia.

[0344] Furthermore, in some embodiments, diseases and indications treatable with the formulations and / or co-formulated liposomes or TB prodrugs of this disclosure include, but are not limited to, hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary cancers, liver cancers, bone cancers, neurological cancers, gynecological cancers, and skin cancers.

[0345] Exemplary blood cancers include lymphomas and leukemias, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, non-Hodgkin lymphoma (including relapsed or refractory NHL and relapsed follicular lymphoma), Hodgkin 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).

[0346] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, hamartoma, and teratoma.

[0347] Examples of lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell, anaplastic small cell, anaplastic large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatoid hamartoma, and mesothelioma.

[0348] Similar types of gastrointestinal cancers include esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastric cancer (cancer, 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), colorectal cancer (adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma, leiomyoma), and colorectal cancer.

[0349] Examples of 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 (seminocarcinoma, teratoma, embryonic carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma).

[0350] Examples of liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0351] Examples of bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulosarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor.

[0352] Examples of neurological cancers include skull cancer (osteoma, hemangioma, granuloma, xanthomas, osteoosteitis), meningeal cancer (meningioma, meningiosarcoma, gliomas), brain cancer (astrocytoma, meduoblastoma, glioma, ependymoma, germ cell tumor (pineal glandoma), glioblastoma, glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), and spinal cord cancer (neurofibroma, meningioma, glioma, sarcoma), as well as neuroblastoma and Lhermitt-Dukuro disease.

[0353] Examples of gynecological cancers include uterine cancer (endometrial cancer), cervical cancer (cervical cancer, pre-tumor cervical dysplasia), ovarian cancer (ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified cancer), granulosa-theca cell tumor, Sertoli-Leydig cell tumor, undifferentiated germ cell tumor, malignant teratoma), vulvar cancer (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, staphylosarcoma (embryonic rhabdomyosarcoma)), and fallopian tube cancer.

[0354] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic mole, lipoma, angioma, dermatofibroma, and keloid. In some embodiments, diseases and indications treatable with the compounds of this disclosure include, but are not limited to, sickle cell disease (e.g., sickle cell anemia), triple-negative breast cancer (TNBC), myelodysplastic syndrome, testicular cancer, cholangiocarcinoma, esophageal cancer, and urothelial carcinoma.

[0355] In addition, the formulations and / or co-formulated liposomes of the present disclosure, or the blockade of TGFβ, ALK5, and / or kinase pathways using TB prodrugs, can also be used to treat infectious diseases, such as viral, bacterial, fungal, and parasitic infections.

[0356] This disclosure provides a method for treating infectious diseases, such as viral infections. The method comprises administering to a patient a therapeutically effective dose of any formulated and / or co-formulated liposome or TB prodrug or any of the formulas described herein (i.e., TB4 prodrug or TB4(ALT-1)), or a salt thereof, as listed in any of the claims and described herein.

[0357] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4 or a salt thereof to a patient.

[0358] In a further embodiment, the method includes administering a therapeutically effective amount of SLNP-TB4 or a salt thereof to a patient.

[0359] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4(ALT-1) or a salt thereof to a patient.

[0360] In further embodiments, the method includes administering a therapeutically effective amount of SLNP-TB4(ALT-1) or a salt thereof to a patient.

[0361] Examples of viruses that cause infectious diseases treatable by the methods of this disclosure include, but are not limited to, human immunodeficiency virus, human papillomavirus, influenza, hepatitis A, B, C, or D viruses, adenovirus, poxvirus, herpes simplex virus, human cytomegalovirus, severe acute respiratory syndrome virus, Ebola virus, and measles virus. In some embodiments, viruses causing infectious diseases 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), adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV viruses, dengue viruses, papillomaviruses, molluscum contagiosum viruses, polioviruses, rabies viruses, JC viruses, and arbovirus encephalitis viruses.

[0362] In addition, this disclosure provides a method for treating a bacterial infection. The method comprises administering to a patient a therapeutically effective dose of any of the formulated and / or co-formulated liposomes listed in any of the claims and described herein, or of any of the formulas described herein (i.e., TB4 prodrug or TB4(ALT-1)), or a salt thereof.

[0363] Examples of pathogens that cause infectious diseases treatable by the methods of this disclosure include, but are not limited to, Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus pneumoniae, Neisseria meningitidis and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria.

[0364] In addition, this disclosure provides a method for treating fungal infections. The method comprises administering to a patient a therapeutically effective dose of any of the formulated and / or co-formulated liposomes listed in any of the claims and described herein, or of any of the formulas described herein (i.e., TB4 prodrug or TB4(ALT-1)), or a salt thereof.

[0365] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4 or a salt thereof to a patient.

[0366] In a further embodiment, the method includes administering a therapeutically effective amount of SLNP-TB4 or a salt thereof to a patient.

[0367] Examples of pathogenic fungi that cause infections treatable by the methods of this disclosure include, but are not limited to, Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales (Mucor, absidia, rhizophus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0368] In addition, this disclosure provides a method for treating parasitic infections. The method comprises administering to a patient a therapeutically effective dose of any of the formulations and / or co-formulated liposomes listed in any of the claims and described herein, or of any of the formulas described herein (i.e., TB4 prodrug or TB4(ALT-1)), or a salt thereof.

[0369] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4 or a salt thereof to a patient.

[0370] In a further embodiment, the method includes administering a therapeutically effective amount of SLNP-TB4 or a salt thereof to a patient.

[0371] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4(ALT-1) or a salt thereof to a patient.

[0372] In further embodiments, the method includes administering a therapeutically effective amount of SLNP-TB4(ALT-1) or a salt thereof to a patient.

[0373] Examples of pathogenic parasites that cause infectious diseases treatable by the methods of this 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.

[0374] In a further set of embodiments within the scope of this disclosure, any formulation and / or co-formulated nanocarrier, liposome, SLNP, or TB prodrug, or any of the formulas described herein (i.e., TB4 prodrug or TB4(ALT-1)), is useful in preventing or reducing the risk of developing any of the diseases referred to herein, for example, in individuals who may be predisposed to a disease, condition, or disorder but have not yet experienced or shown any of the pathologies or overall symptoms of the disease.

[0375] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4 or a salt thereof to a patient.

[0376] In a further embodiment, the method includes administering a therapeutically effective amount of SLNP-TB4 or a salt thereof to a patient.

[0377] In one embodiment, the method includes administering a therapeutically effective amount of LNP-TB4(ALT-1) or a salt thereof to a patient.

[0378] In further embodiments, the method includes administering a therapeutically effective amount of SLNP-TB4(ALT-1) or a salt thereof to a patient.

[0379] XII.) Kits / Manufactured Products Kits for use in laboratory, prognostic, preventive, diagnostic, and therapeutic applications as described herein are within the scope of the present invention. Such a kit may include a carrier, package, or container partitioned to receive one or more containers, such as vials, tubes, etc., each container containing one of the separate elements used in the method, together with a label or insert containing instructions for use, for example, as described herein. For example, a container(s) may contain a formulated and / or co-formulated nanocarrier that is detectably labeled or can be detectably labeled and / or loaded with the TB prodrug of this disclosure. A kit may include a container containing a drug unit. A kit may contain all or part of the formulated and / or co-formulated nanocarrier, and / or the TB prodrug.

[0380] The kit of the present invention typically comprises the above-mentioned container and one or more other associated containers containing materials desirable from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, transport containers, packages, containers, vials, and / or tube labels and / or instructions for use listing the contents, as well as accompanying documentation containing instructions for use.

[0381] Labels may be present on or with the container to indicate that the composition is used for a specific therapeutic or non-therapeutic application, such as prognosis, prevention, diagnosis, or laboratory application, and may also indicate how to use it in vivo or in vitro, such as the methods of use described herein. Directions and other information may also be included on inserts or labels included with or on the kit. Labels may be on the container or associated with the container. Labels may be on the container if the letters, numbers, or other symbols forming the label are molded or etched onto the container itself. Labels may also be associated with the container, for example as an accompanying document, if they are also present in a container or transporter that holds the container. Labels may indicate that the composition is used for the diagnosis, treatment, prevention, or prognosis of a condition, such as cancer or other immunological disorders.

[0382] The terms "kit" and "manufactured product" can be used as synonyms.

[0383] In another embodiment of the present invention, articles(s) containing compositions, such as formulated and / or co-formulated nanocarriers, and / or TB prodrugs, are within the scope of the present disclosure. The articles typically comprise at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, such as glass, metal, or plastic. The containers can hold formulated and / or co-formulated nanocarriers loaded with TB prodrugs.

[0384] The container may, as an alternative, hold a composition effective for the treatment, diagnosis, prognosis, or prevention of a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous injection needle). The activator in the composition may be a TB prodrug and / or a formulation and / or co-formulated nanocarrier loaded with a TB prodrug disclosed herein.

[0385] The product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The product may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes, and / or accompanying documentation containing indications and / or instructions for use.

[0386] In one embodiment, the kit or product comprises LNP-TB4 and / or a therapeutically effective amount of LNP-TB4.

[0387] In one embodiment, the kit or product comprises SLNP-TB4 and / or a therapeutically effective amount of SLNP-TB4.

[0388] In one embodiment, the kit or product comprises LNP-TB4(ALT-1) and / or a therapeutically effective amount of LNP-TB4(ALT-1).

[0389] In one embodiment, the kit or product comprises SLNP-TB4(ALT-1) and / or a therapeutically effective amount of SLNP-TB4(ALT-1).

[0390] Exemplary Embodiments The embodiments provided include the following: 1) (i) Drug portion, (ii) lipid portion, and (iii) Linking unit ("LU") A TB prodrug composition comprising a drug portion containing a TGFβ antagonist, wherein the LU conjugates the drug portion with the lipid portion. 2) The TB prodrug according to claim 1, further comprising the chemical structure described in formula I. 3) The TB prodrug according to claim 1, further comprising the chemical structure described in formula II. 4) The TB prodrug according to claim 1, further comprising the chemical structure described in Formula III. 5) The TB prodrug according to claim 1, wherein the drug portion comprises the chemical structure described as TB4. 6) The TB prodrug according to claim 1, wherein LU is a hydromethylcarbamate linker. 7) The TB prodrug according to claim 1, wherein the lipid portion comprises the lipids listed in Table I. 8) The TB prodrug according to claim 1, wherein the lipid portion comprises the lipids listed in Table III. 9) The TB prodrug according to claim 1, wherein the lipid portion comprises CHEMS. 10) The TB prodrug according to claim 1, wherein the lipid portion comprises stearic acid. 11) The drug portion contains the chemical structure shown as TB4, the lipid portion contains stearic acid, and the compound has the following chemical structure: [ka] A TB prodrug according to claim 1, having the following characteristics. 12)(i) Drug portion containing TB4, (ii) Lipid portion containing CHEMS, and (iii) LU containing a hydromethylcarbamate linker A TB prodrug composition containing the above. 13)(i) Drug portion containing TB4, (ii) The lipid portion containing stearic acid, and (iii) LU containing a hydromethylcarbamate linker A TB prodrug composition containing the above. 14) The following chemical structure: [ka] A TB prodrug composition according to claim 13, having the following characteristics. 15) A nanocarrier containing a TB prodrug that releases an active ALK5 inhibitor after cleavage of the LU. 16) The nanocarrier according to claim 15, wherein LU is a hydromethylcarbamate linker. 17) The nanocarrier according to claim 15, further comprising a helper lipid, wherein the helper lipid is listed in Table II. 18) The nanocarrier according to claim 15, wherein the TB prodrug comprises TB4. 19) The nanocarrier according to claim 15, which is a liposome. 20) The liposome according to claim 19, wherein the TB prodrug comprises TB4 and is denoted as LNP-TB4. 21) A liposome according to claim 19, further co-formulated with one or more immunomodulators or their lipid-prodrugs, wherein the immunomodulators are 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 their prodrugs. 22) The liposome according to claim 19, 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. 23) The liposome according to claim 19, further comprising DOX. 24) The liposome according to claim 19, further comprising MTO. 25) The liposome according to claim 22, further comprising DOX. 26) The liposome according to claim 22, further comprising MTO. 27) A liposome according to claim 19, further co-formulated with a Toll receptor agonist or a lipid-prodrug, wherein the Toll receptor agonist is selected from the group consisting of reximod (R848), gardikimod, 852A, DSR6434, tellaterimod, CU-T12-9, monophosphoryllipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD®. 28) The liposome according to claim 19, further co-formulated with a PD-1 / PD-L1 antagonist or a lipid-prodrug, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of AUNP12, CA-170, or BMS-986189. 29) A kit comprising the liposomes described in any one of claims 15 to 28. 30) The nanocarrier according to claim 15, wherein the nanocarrier is a solid lipid nanoparticle (SLNP). 31) The SLNP according to claim 30, wherein the TB prodrug comprises TB4 and is denoted as SLNP-TB4. 32) The SLNP according to claim 30, wherein the SLNP is further co-formulated with one or more immunomodulators or their lipid prodrugs, the immunomodulators being 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 their prodrugs. 33) The SLNP according to claim 30, wherein the SLNP is further co-formulated with an ICD-inducing chemotherapeutic agent, the ICD-inducing chemotherapeutic agent being selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, or paclitaxel. 34) The SLNP according to claim 30, further comprising DOX. 35) The SLNP according to claim 30, further comprising MTO. 36) The SLNP according to claim 33, further comprising DOX. 37) The SLNP according to claim 33, further comprising MTO. 38) The SLNP according to claim 30, wherein the liposome is further co-formulated with a Toll receptor agonist or a lipid prodrug, the Toll receptor agonist being selected from the group consisting of rexiquimod (R848), gardikimod, 852A, DSR6434, tellaterimod, CU-T12-9, monophosphoryllipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD®. 39) The SLNP according to claim 30, wherein the liposome is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid-prodrug, the PD-1 / PD-L1 antagonist being selected from the group consisting of AUNP12, CA-170, or BMS-986189. 40) A kit comprising the SLNP described in any one of claims 30 to 39. 41) A method of treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of nanocarrier to a subject requiring such treatment, wherein the nanocarrier contains a TB prodrug. (ii) a pharmaceutically acceptable salt thereof, Methods that include... 42) The method according to claim 41, wherein the TB prodrug comprises a TB4 prodrug. 43) The method according to claim 41, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an ICD-inducing chemotherapeutic agent. 44) The method according to claim 41, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an immunomodulator. 45) The method according to claim 41, wherein the nanocarrier is a liposome. 46) The method according to claim 45, wherein the liposome is LNP-TB4. 47) The method according to claim 41, wherein the nanocarrier is a solid lipid nanoparticle. 48) The method according to claim 47, wherein the liposome is SLNP-TB4. 49) A method of treating a subject who has cancer or has been diagnosed with cancer, (iii) Administering an effective amount of nanocarrier to a subject requiring such treatment, wherein the nanocarrier contains a TB prodrug. (iv) a pharmaceutically acceptable salt thereof, Methods that include... 50) The method according to claim 49, wherein the TB prodrug comprises a TB4 prodrug. 51) The method according to claim 49, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an ICD-inducing chemotherapeutic agent. 52) The method according to claim 49, wherein the nanocarrier comprises a TB4 prodrug further co-formulated with an immunomodulator. 53) The method according to claim 49, wherein the nanocarrier is a solid lipid nanoparticle ("SLNP"). 54) The method according to claim 53, wherein SLNP is SLNP-TB4. 55) The method according to claim 49, wherein the nanocarrier is a liposome. 56) The method according to claim 55, wherein the liposome is LNP-TB4. 57) The following chemical structure: [ka] A TB4 prodrug containing [unclear]. 58) A liposome comprising the TB4 prodrug according to claim 57. 59) A liposome comprising the TB4 prodrug according to claim 57, further comprising a helper lipid. 60) The liposome according to claim 59, wherein the helper lipids are listed in Table II. 61) Solid lipid nanoparticles (SLNPs) comprising the TB4 prodrug described in claim 57. 62) The following chemical structure: [ka] A TB4 prodrug containing [unclear]. 63) A liposome comprising the TB4 prodrug according to claim 62. 64) A liposome comprising the TB4 prodrug according to claim 62, further comprising a helper lipid. 65) The liposome according to claim 64, wherein the helper lipids are listed in Table II. 66) The liposome according to claim 62, represented as LNP-TB4. 67) Solid lipid nanoparticles (SLNPs) comprising the TB4 prodrug described in claim 62. 68) The SLNP according to claim 67, indicated as SLNP-TB4. 69) The liposome according to claim 63, co-formulated with AR5. 70) The liposome according to claim 63, co-formulated with TR6. 71) The liposome according to claim 63, co-formulated with ID3. 72) The liposome according to claim 63, co-formulated with PD3. 73) The liposome according to claim 63, co-formulated with MTO. 74) The liposome according to claim 63, co-formulated with MTO and ID3. 75) The liposome according to claim 63, co-formulated with MTO and AR5. 76) The SLNP according to claim 68, co-formulated with MTO. 77) The SLNP according to claim 68, co-formulated with AR5. 78) The SLNP according to claim 68, co-formulated with ID3. 79) The SLNP according to claim 68, co-formulated with PD3. 80) The SLNP according to claim 68, co-formulated with MTO and ID3. 81) The SLNP according to claim 68, co-formulated with MTO and AR5. 82)(i) Drug portion, (ii) lipid portion, and (iii) Linking unit ("LU") A TB prodrug composition comprising a drug portion containing a TGFβ antagonist, wherein the LU conjugates the drug portion with the lipid portion. 83) The TB prodrug composition according to claim 82, wherein the drug portion comprises the chemical structure described as TB4. 84) The TB prodrug composition according to claim 82, wherein the lipid portion comprises stearic acid. 85) The following chemical structures: [ka] A TB prodrug composition according to claim 82, comprising a TB4 prodrug (ALT-1) having the following properties. 86) A nanocarrier containing a TB prodrug that releases an active ALK5 inhibitor after cleavage of the linking unit (LU). 87) The nanocarrier according to claim 86, further comprising a helper lipid, wherein the helper lipid is as listed in Table II. 88) The nanocarrier according to claim 86, wherein the TB prodrug comprises a TB4 prodrug (ALT-1). 89) The TB4 prodrug (ALT-1) has the following chemical structure: [ka] A nanocarrier according to claim 88, having the characteristics of the nanocarrier. 90) A method for treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of nanocarrier to a subject requiring such treatment, wherein the nanocarrier contains a TB prodrug. (ii) a pharmaceutically acceptable salt thereof, Methods that include... 91) The method according to claim 90, wherein the TB prodrug comprises a TB4 prodrug (ALT-1). 92) The TB4 prodrug (ALT-1) has the following chemical structure: [ka] The method according to claim 91, wherein the method is characterized by having the following: 93) The method according to claim 90, wherein the nanocarrier comprises a TB4 prodrug (ALT-1) further co-formulated with an ICD-inducing chemotherapeutic agent. 94) The method according to claim 93, wherein the ICD-inducing chemotherapy agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, or paclitaxel. 95) The method according to claim 90, wherein the nanocarrier comprises a TB4 prodrug (ALT-1) further co-formulated with an immunomodulator. 96) The method according to claim 95, wherein the nanocarrier is further co-formulated with an immunomodulator, the immunomodulator being selected from the group consisting of other TLR agonists and / or prodrugs, immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors, and / or prodrugs thereof. 97) The method according to claim 90, wherein the nanocarrier is further co-formulated with a Toll receptor agonist, the Toll receptor agonist being selected from the group consisting of reximod (R848), gardikimod, 852A, DSR6434, tellaterimod, CU-T12-9, monophosphoryllipid A (MPLA), monophosphorylhexaacyllipid A, 3-deacyl (synthetic), SMU127, Pam3CSK4, or 3-deacyl-phosphorylated hexa-acyl disaccharide. 98) The method according to claim 90, wherein the nanocarrier is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid-prodrug, the PD-1 / PD-L1 antagonist being selected from the group consisting of AUNP12, CA-170, or BMS-986189. 99) The method according to claim 90, wherein the nanocarrier comprises lipid nanoparticles (LNPs). 100) The method according to claim 90, wherein the nanocarrier comprises solid lipid nanoparticles (SLNPs). 101) The SLNP according to claim 100, wherein the SLNP is SLNP-TB4-IC1. 102) The method according to claim 90, wherein the subject is a human. 103) The method according to claim 90, wherein the cancer is breast cancer. 104) A kit comprising the nanocarrier described in claim 89. 105) A kit comprising the nanocarrier described in claim 92. [Examples]

[0391] Various aspects of the present invention will be further described and illustrated by the following examples, none of which are intended to limit the scope of the invention.

[0392] (Example 1) Chemical synthesis of TB4 prodrug containing stearic acid 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 obtain intermediate 3. Next, intermediate 3 is sequentially treated with DMF dimethyl acetal, then hydrazine hydrate, to obtain intermediate 4. Next, intermediate 4 is treated with trityl chloride to obtain intermediate 5. Next, intermediate 5 is treated with reagent (6) and palladium triphenylphosphine, followed by hydrolysis with sodium hydroxide to obtain intermediate 7. Next, intermediate 7 is treated with reagent (8) and EDCI / HOBt to obtain intermediate 9. Next, intermediate 9 is treated with lithium hexamethyl disilazide, followed by chloromethyl chloroformate (10), to obtain intermediate 11. Finally, intermediate 11 in DMF is sequentially treated with stearic acid, then silver carbonate, then sodium iodide at 80°C, and then HCl in methanol to obtain the final prodrug TB4(12) containing stearic acid (Figure 1). The synthesis described in this example yields the following chemical structure: [ka] A TB4 prodrug containing the following properties is obtained.

[0393] (Example 2) Chemical synthesis of protecting group intermediates leading to TB4 prodrugs The following protocol was used to synthesize the protecting group intermediate. Briefly, Boc2O (8.00 g, 36.7 mmol, 8.42 mL, 1.20 equivalents) and DMAP (746 mg, 6.11 mmol, 0.20 equivalents) were added to a solution of TB4 (13.0 g, 30.5 mmol, 1.00 equivalent) in DCM (1.50 L) at 15°C. After addition, the reaction mixture was stirred at 30°C for 12 hours. TLC (dichloromethane:methanol = 10:1) was used to separate TB4 (R f =0.2) is consumed, and one new major spot (R fIt was shown that a solution (=0.5) was formed. LC-MS confirmed the detection of the desired mass (RT=0.850 min). The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (SiO2, dichloromethane:methanol = 50:1~30:1, R f The compound was purified using a solution of 0.5 to obtain compound 2 (13.0 g, 24.7 mmol, yield 80.9%) as a white solid. The obtained compound is shown in Figure 2.

[0394] (Example 3) Chemical synthesis of protecting group intermediates leading to TB4 prodrugs In another embodiment, a further protecting group intermediate was synthesized by the following method. Briefly, TFA (16.6 g, 145 mmol, 10.8 mL, 20.0 equivalents) was added at 25°C to a solution of compound 3 (4.50 g, 7.28 mmol, 1.00 equivalent) in DCM (225 mL). After addition, the reaction mixture was stirred at 25°C for a further 4 hours. LC-MS confirmed that the reaction was complete and the desired mass (RT=0.874 min) was detected. The reaction mixture was adjusted to pH=7-8 with saturated NaHCO3 solution and extracted with DCM (150 mL x 2). The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered, and concentrated to obtain compound 4 (3.50 g, 6.76 mmol, yield 92.81%) as a yellow solid. The obtained compound is shown in Figure 3.

[0395] (Example 4) Chemical synthesis of TB4 prodrug containing stearic acid The chemical synthesis of TB4 prodrugs containing stearic acid was performed using the following protocol. First, Boc2O (8.00 g, 36.7 mmol, 8.42 mL, 1.20 equivalents) and DMAP (746 mg, 6.11 mmol, 0.20 equivalents) were added to a solution of TB4 (13.0 g, 30.5 mmol, 1.00 equivalent) in DCM (1.50 L) at 15°C. After addition, the reaction mixture was stirred at 30°C for 12 hours. TLC (dichloromethane:methanol = 10:1) was performed on TB4(R f=0.2) is consumed, and one major new spot (R f The reaction mixture was shown to have formed (=0.5). LC-MS showed that the desired mass (RT=0.850 min) was detected. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (SiO2, dichloromethane:methanol = 50:1~30:1, R). fThe compound was purified using (0.5) to obtain compound 2 (13.0 g, 24.7 mmol, yield 80.9%) as a white solid. Next, to a solution of compound 2 (9.00 g, 17.1 mmol, 1.00 equivalent) in DCM (1000 mL), LiHMDS (1 M, 37.6 mL, 2.20 equivalents) was added under N2 at -70°C for 1 hour. Compound 2a (9.36 g, 72.5 mmol, 6.45 mL, 4.24 equivalents) in DCM (50 mL) was added to the mixture at -70°C. The mixture was stirred at -70°C for 6 hours. Next, the mixture was stirred at 15°C for a further 6 hours. 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 a saturated NH4Cl solution (500 mL) and extracted with DCM (200 mL x 2). The combined organic phase was washed with brine (500 mL), dried over Na2SO4, concentrated, and confirmed by LC-MS. The crude product was purified by reversed-phase MPLC (ACN / H2O, TFA conditions) to remove ACN, and the aqueous phase was extracted with ethyl acetate (300 mL x 3). The combined organic layer was washed with brine (200 mL), dried over Na2SO4, concentrated under reduced pressure to obtain compound 3 (6.20 g, 10.0 mmol, yield 29.2%) as a yellow solid, and confirmed by LC-MS. Next, TFA (16.6 g, 145 mmol, 10.8 mL, 20.0 equivalents) was added at 25°C to a DCM (225 mL) solution of compound 3 (4.50 g, 7.28 mmol, 1.00 equivalent). After addition, the reaction mixture was stirred for a further 4 hours at 25°C. LC-MS indicated that the reaction was complete and the desired mass (RT=0.874 min) was detected. The reaction mixture was adjusted to pH=7-8 with saturated NaHCO3 solution and extracted with DCM (150 mL x 2). The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered, and concentrated to obtain compound 4 (3.50 g, 6.76 mmol, yield 92.81%) as a yellow solid, which was used directly in the next step without further purification.Finally, DIEA (2.62 g, 20.3 mmol, 3.53 mL, 3.00 equivalent) was added to a solution of compound 4 (3.50 g, 6.76 mmol, 1.00 equivalent) and stearic acid (2.88 g, 10.1 mmol, 3.41 mL, 1.50 equivalent) in ACN (175 mL). After the addition, the reaction mixture was stirred at 80°C for a further 36 hours. LC-MS showed that compound 4 had completed incompletely and the desired mass (RT=1.383 min) was detected. The mixture was concentrated under reduced pressure at 45°C to obtain the crude product, which was confirmed by LC-MS. The crude product was purified by reverse-phase MPLC (MeOH / H2O / TFA conditions) in combination with ET34822-17, and then concentrated to obtain 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 then by TLC (ethyl acetate:methanol = 10:1, R. f Detected by (=0.3). TB4 containing stearic acid (1.3g, 1.59 mmol, yield 21.2%, purity 93.9%) was obtained as a yellow, gum-like substance. 1 Confirmation was performed by 1H NMR, 1F NMR, LCMS, and HPLC. The obtained compound and synthesis are shown in Figure 7. The synthesis described in this example yields the following chemical structure: [ka] A TB4 prodrug containing the following properties is obtained.

[0396] (Example 5) Synthesis and Characterization of LNP-TB4 Liposomes In another experiment, liposomes containing the TB4 prodrug (indicated as LNP-TB4) were synthesized using the following method. 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). The TB4 prodrug (TB4 + stearic acid) stock solution was not soluble in ethanol, so it 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. The lipid mixture was heated to 55-60 degrees Celsius using a microfluidizer heating block attachment. Similarly, the aqueous phase containing 1 mM PBS buffer was preheated to 55–60 degrees Celsius and then passed through a microfluidic cartridge at a flow rate of 5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed from the DI water using a dialysis membrane (Sigma Aldrich) with a cutoff size of 12 kDa for at least 24 hours. During the 24 hours, the dialyzed water was changed at least 5 times to remove the solvent to the maximum extent. After solvent removal, LNP-TB4 was concentrated using an Amicon centrifugal filtration device (cutoff size 10 kDa, at 3000 g) as needed.

[0397] LNP-TB4 liposomes were characterized using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-TB4 liposomes (liposome concentration of 0.5–1 mg / ml) were placed in a transparent four-sided plastic cuvette and directly analyzed at 25°C. The results shown in Figure 8 indicate that the Z-mean size of the nanoparticles was approximately 87 nm and the PDI was approximately 0.265.

[0398] In addition, 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 of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-TB4 was approximately -15.1 mV (Figure 9).

[0399] (Example 6) Synthesis and Characterization of LNP-TB4-ID3 Liposomes In another experiment, liposomes containing the TB4 prodrug (indicated as 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 prepared separately in ethanol (20 mg / ml). Next, since the stock solutions of the TB4 prodrug (TB4 + stearic acid) and ID3 prodrug were insoluble in ethanol, they were prepared in acetonitrile (20 mg / ml). Then, 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 degrees Celsius using a microfluidizer heating block attachment. Similarly, the aqueous phase containing 1 mM PBS buffer was preheated to 50 degrees Celsius and then passed through a microfluidic cartridge at a flow rate of 4.5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed from the DI water using a dialysis membrane (Sigma Aldrich) with a cutoff size of 12 kDa for at least 24 hours. During the 24 hours, the dialyzed water was changed at least 5 times to maximize solvent removal. After solvent removal, LNP-TB4-ID3 was concentrated using an Amicon centrifugal filtration device (cutoff size 10 kDa, at 3000 g) as needed.

[0400] The LNP-TB4-ID3 liposomes were characterized using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-TB4-ID3 liposomes (liposome concentration of 0.5–1 mg / ml) were placed in a transparent four-sided plastic cuvette and directly analyzed at 25°C. The results shown in Figure 10 indicate that the Z-mean size of the nanoparticles was approximately 87 nm and the PDI was approximately 0.075.

[0401] In addition, 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 of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-TB4-ID3 was approximately -11.5 mV (Figure 11).

[0402] In addition, a summary table of additional co-formulated LNP-TB4 is provided as Table IV.

[0403] (Example 7) Synthesis and Characterization of SLNP-TB4 Solid Lipid Nanoparticles In another experiment, solid lipid nanoparticles (SLNPs) containing the TB4 prodrug (indicated as SLNP-TB4) were synthesized using the following method. Briefly, SLNPs containing the TB4 prodrug were prepared using several emulsifiers, including Moliwol 488 (polyvinyl alcohol), Pluronic F127, and Kolliphor RH40. In the first step, lipid stock solutions of POPC, CHOL, and DSPE-PEG were prepared in ethanol (20 mg / ml). Next, the TB4 prodrug stock solution was prepared in acetonitrile (20 mg / ml). The lipid mixture of POPC, CHOL, TB4, and DSPE-PEG was mixed together in a molar ratio of 51:29:15:5 and then diluted with ethanol to obtain a lipid concentration of 10 mg / ml. This lipid mixture was heated to 55 degrees Celsius using a microfluidizer heating block attachment. Similarly, the aqueous phase containing a 2% w / v Molivol 488 (or 2% w / v Pluronic F127 / Kolliphor RH40) solution was preheated to 55 degrees Celsius and then passed through a microfluidic cartridge at a flow rate of 5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed using a dialysis membrane (Sigma Aldrich) with a cutoff size of 12 kDa for at least 24 hours with the DI water. During the 24 hours, the dialyzed water was changed at least 5 times to remove the solvent to the maximum extent. After solvent removal, the SLNP was passed through a 0.2 micron filter membrane (cellulose acetate). The SLNP-TB4 was concentrated using an Amicon centrifugal filtration device (cutoff size 10 kDa, at 3000 g) as needed.

[0404] The characteristics of SLNP-TB4 were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-TB4 (SLNP concentration of 0.5–1 mg / ml) was placed in a transparent four-sided plastic cuvette and analyzed directly at 25°C. The results shown in Figure 12 indicate that the Z-mean size of the nanoparticles was approximately 90 nm and the PDI was approximately 0.074.

[0405] In addition, the zeta potential of SLNP-TB4 solid lipid nanoparticles in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results showed that the zeta potential of SLNP-TB4 was approximately -11.9 mV (Figure 13).

[0406] (Example 8) Synthesis and Characterization of SLNP-TB4-ID3 Solid Lipid Nanoparticles In another experiment, solid lipid nanoparticles (SLNPs) containing the TB4 prodrug co-formulated with ID3 (indicated as SLNP-TB4-ID3) were synthesized using the following method. Briefly, the SLNPs containing the TB4 prodrug were prepared using several emulsifiers, including Moliwol 488 (polyvinyl alcohol), Pluronic F127, and Kolliphor RH40. In the first step, lipid stock solutions of POPC, CHOL, and DSPE-PEG were prepared in ethanol (20 mg / ml). Next, TB4 and ID3 prodrug stock solutions were prepared in acetonitrile (20 mg / ml). The lipid mixture of POPC, CHOL, TB4, ID3, and DSPE-PEG was mixed together in a molar ratio of 52:29:7:7:5 and then diluted with ethanol to obtain a lipid concentration of 10 mg / ml. This lipid mixture was heated to 55 degrees Celsius using a microfluidizer heating block attachment. Similarly, the aqueous phase containing a 2% w / v Moliwol 488 (or 2% w / v Pluronic F127 / Kolliphor RH40) solution was preheated to 55 degrees Celsius and then passed through a microfluidic cartridge at a flow rate of 5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed using a dialysis membrane (Sigma Aldrich) with a cutoff size of 12 kDa for at least 24 hours with the DI water. During the 24 hours, the dialyzed water was changed at least 5 times 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 concentrated using an Amicon centrifugal filtration device (cutoff size 10 kDa, at 3000 g) as needed.

[0407] The characteristics of SLNP-TB4-ID3 were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-TB4-ID3 (SLNP concentration of 0.5–1 mg / ml) was placed in a transparent four-sided plastic cuvette and directly analyzed at 25°C. The results shown in Figure 14 indicate that the Z-mean size of the nanoparticles was approximately 104.3 nm and the PDI was approximately 0.119.

[0408] In addition, the zeta potential of SLNP-TB4-ID3 solid lipid nanoparticles in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results show that the zeta potential of SLNP-TB4-ID3 was approximately -10.3 mV (Figure 15).

[0409] In addition, a summary table of the additional co-formulated SLNP-TB4 is provided as Table V.

[0410] (Example 9) In vivo tumor inhibition of SLNP-TB4 using B16F10 cells. In this experiment, SLNP-TB4 was evaluated using the following protocol: 0.2 × 10⁶ mouse melanoma B16F10 cells. 6The individual molecules were subcutaneously inoculated into the right posterior ventral region of C57BL / 6 mice. Animals were treated twice weekly by IV injection with a vehicle control, 3 mg / kg of LNP-MTO (mitoxantrone dihydrochloride in liposomal form), 3 mg / kg of LNP-AR5 (AR5-stearic acid in liposomal form), a combination of 3 mg / kg of LNP-AR5 and LNP-TB4 (TB4-stearic acid in liposomal form), and a combination of 3 mg / kg of LNP-MTO and SLNP-TB4 (TB4-stearic acid in solid lipid nanoparticles). Tumor volume was measured three times using calipers in two dimensions, 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 tumor size data at day 15.

[0411] The results indicate that the combination of SLNP-TB4 with LNP-MTO yielded significant antitumor activity. The TGI was calculated to be 44.12% (all p<0.05). (Figure 16).

[0412] (Example 10) In vivo tumor inhibition of multiple LNP-TB4 combinations using B16F10 cells. In this experiment, LNP-TB4 was evaluated using the following protocol: 0.2 × 10⁶ mouse melanoma B16F10 cells. 6The individual doses were subcutaneously inoculated into the right posterior flank region of C57BL / 6 mice. Animals were compared to vehicle control, 3 mg / kg of LNP-MTO (liposomal mitoxantrone dihydrochloride), 3 mg / kg of a combination of LNP-TB4 (liposomal TB4-stearic acid) and LNP-TR6 (liposomal TR6-Chemes), a further combination of 3 mg / kg of LNP-TB4 and LNP-AR5 (liposomal AR5-stearic acid), a further combination of 3 mg / kg of LNP-TB4 and LNP-ID3 (liposomal ID3-stearic acid), and a further combination of 3 mg / kg of LNP-TB4 and LNP-PD3 (PD3-cholesterol). The tumors were treated twice weekly by intravenous injection with combinations of 3 mg / kg LNP-TB4, LNP-MTO, and LNP-ID3, and further combinations of 3 mg / kg LNP-TB4, LNP-MTO, and LNP-AR5. Tumor volume was measured three times using calipers in two dimensions, 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 at day 16.

[0413] The results indicate that treatment with LNP-MTO as a monotherapy at 3 mg / kg 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 also produced antitumor activity. The TGIs were calculated to be 32.86% (p<0.05) and 37.86% (p<0.05), respectively (Figure 17).

[0414] (Example 11) In vitro verification of the mechanism of action of LNP-TB4 and SLNP-TB4 In this experiment, the mechanisms of action of LNP-TB4 and SLNP-TB4 were evaluated in vitro using the following protocol. The following assays were performed to confirm that TB4 (LNP) in liposome form and TB4 (SLNP) in solid lipid nanoparticle form can have biological effects in vitro. Briefly, HEK-Blue® TGF-β cell and QUANTI-Blue® (InvivoGen, San Diego, CA) assays were used using standard methods. Stimulation of HEK-Blue® TGF-β cells with TGF-β induced activation of the TGF-β / Smad signaling pathway, leading to the formation of the Smad3 / Smad4 complex. This heterocomplex enters the nucleus, binds to the SBE site, and induces SEAP production. 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 incubation with each compound for 24 hours, the percentage of TGF-β inhibition (%) was assessed by measuring the level of optimal SEAP density (OD) using the QUANTI-Blue® assay and normalizing the data to a control group (cells treated with TGF-β alone).

[0415] The results showed that cell treatment with LNP-TB4 and SLNP-TB4 induced TGF-β inhibition (see Figure 18).

[0416] (Example 12) Chemical synthesis of TB4 prodrug (ALT-1) containing stearic acid The chemical synthesis of TB4 prodrug (ALT-1) containing stearic acid was performed using the following protocol. First, three reactions were carried out in parallel. To a solution of GW788388 (14.0 g, 32.9 mmol, 1.00 equivalent) in DCM (1.40 L), DMAP (401 mg, 3.29 mmol, 0.10 equivalent) was added, followed by the addition of Boc2O (12.9 g, 59.2 mmol, 13.6 mL, 1.80 equivalent) to the mixture at 25°C. After the addition, the reaction mixture was stirred at 25°C for 12 hours. LCMS (EW49768-8-P1A1) showed that 9.20% of GW788388 (RT=0.393 min) remained, and 86.2% of the desired mass (RT=0.473 min) was detected. The three reactions were combined and work-up-treated together. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The mixture was ground with (petroleum ether:ethyl acetate = 1:1, 800 mL) at 25°C for 12 hours. Next, it was filtered, and the filtered cake was concentrated to obtain the product. The resulting compound 2 (60.0 g, crude) was obtained as a brown solid and was confirmed by standard methods using LC-MS (see Figure 20).

[0417] Next, two reactions were carried out in parallel. Compound 3B (30.1 g, 182 mmol, 1.30 equivalents) was added dropwise to a mixture of compound 3A (40.0 g, 140 mmol, 47.3 mL, 1.00 equivalent), bisulfate; tetrabutylammonium (4.77 g, 14.0 mmol, 0.10 equivalent), and K2CO3 (77.7 g, 562 mmol, 4.00 equivalent) in DCM (400 mL) and H2O (400 mL), and the mixture was then stirred at 25°C for 12 hours. TLC (petroleum ether:ethyl acetate = 10:1) was obtained from compound 3A (R f =0.20) is completely consumed, and one new spot (R fThis showed that a compound (=0.50) was formed. The two reactions were combined and work-up was performed. The mixture was poured into H2O (800 mL) and extracted with DCM (800 mL x 3). The combined organic layer was washed with brine (300 mL x 2), dried over Na2SO4, and concentrated. The residue was analyzed by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0~1 / 1, TLC: petroleum ether / ethyl acetate = 10 / 1, R f It was purified by (=0.50). Compound 3 (55.0 g, 165 mmol, yield 58.7%) was obtained as a white solid. 1 This was confirmed using standard methods by 1H NMR (see Figure 21).

[0418] Finally, the two reactions were carried out in parallel. A solution of compound 2 (12.5 g, 21.5 mmol, 1.00 equivalent) in THF (557 mL) was cooled to -45°C on an acetone-dry ice bath. KHMDS (1 M, 32.3 mL, 1.50 equivalent) was added dropwise, and the mixture was stirred under N2 at -45°C for 30 minutes. Next, a solution of NaI (1.62 g, 10.8 mmol, 0.50 equivalent) and compound 3 (8.63 g, 25.9 mmol, 1.20 equivalent) in THF (184 mL) was added, the dry ice bath was removed, and the mixture was stirred at 25°C for 12 hours. LCMS (EW49768-19-P1A1) showed that 48.9% of compound 2 (RT=0.471 min) remained, and 28.8% of the desired mass (RT=0.723 min) was detected. The two reaction mixtures were combined and work-processed together. The reaction mixture was poured into water (800 mL) and extracted with ethyl acetate (600 mL x 3). The combined organic matter was washed with brine (300 mL x 2), then dried over Na₂SO₄, filtered, and concentrated to obtain the crude product. The crude product was purified by reverse-phase HPLC (TFA conditions), the fraction was adjusted to pH=11 with saturated Na₂CO₃ solution, and extracted with ethyl acetate (800 mL x 3). The combined organic matter was washed with brine (300 mL x 2), dried over Na₂SO₄, filtered, and concentrated to obtain the crude product. Target 1 (8.00 g, 10.0 mmol, yield 23.3%, purity 90.8%) was obtained as a white solid and confirmed by LC-MS using standard methods (see Figure 22).

[0419] The synthesis described in this example results in the following chemical structure: [ka] A TB4 prodrug (ALT-1) containing the above is obtained.

[0420] (Example 13) In vitro verification of the mechanism of action of LNP-TB4(ALT-1) and SLNP-TB4(ALT-1) In this experiment, to confirm that TB4 prodrugs (ALT-1) in LNP and SLNP forms (e.g., LNP-TB4(ALT-1) and SLNP-TB4(ALT-1)) have biological effects in vitro, HEK-Blue® TGF-β cells and QUANTI-Blue® (InvivoGen, San Diego, CA) assays were used under the following protocol. Specifically, stimulation of HEK-Blue® TGF-β cells with TGF-β induced activation of the TGF-β / Smad signaling pathway, leading to the formation of the Smad3 / Smad4 complex. This heterocomplex enters the nucleus, binds to the SBE site, and induces SEAP production. The amount of SEAP secreted into the supernatant can be easily assessed using QUANTI-Blue.

[0421] Following this reasoning, the following protocol was used. First, cells were incubated with various concentrations of TB4 (GW788388), LNP-TB4 (TB4 prodrug (ALT-1) in lipid nanoparticle form), and SLNP-TB4 (TB4 prodrug (ALT-1) in solid lipid nanoparticle form) in the presence of 10 ng / ml of TGF-β. After incubation with the compounds for 24 hours, the TGF-β inhibition percentage (%) was assessed by measuring the level of SEAP optimal density (OD) using the QUANTI-Blue® assay and normalizing the data to a control group (cells treated with TGF-β alone).

[0422] The results showed that treating cells with LNP-TB4(ALT-1) and SLNP-TB4(ALT-1) induced TGF-β inhibition (see Figure 23). In particular, this experiment was performed using the same sample as described in Figure 18. However, the sample was shown to be precisely TB4(ALT-1) (see Figures 19-22).

[0423] (Example 14) In vivo tumor inhibition of multiple combinations of SLNP-TB4 and SLNP-TB4-IC1 using EMT-6 cells. In this experiment, SLNP-TB4 and SLNP-TB4-IC1 were evaluated using the following protocol. For the purposes of this example, SLNP-TB4 and SLNP-TB4-IC1 are equivalent to SLNP-TB4(ALT-1) and SLNP-TB4-IC1(ALT-1). Mouse mammary cancer EMT6 cells (0.5 × 10⁻⁶) 6 The drug was subcutaneously inoculated into the right posterior ventral region of Balb / c mice. Animals were treated every other week by IV injection with a vehicle control, 2 mg / kg of SLNP-IC1 (doxorubicin-HCl prodrug in solid lipid nanoparticle form), 20 mg / kg of SLNP-TB, and 20-2 mg / kg of SLNP-TB4-IC1. Tumor volume was measured three times using calipers in two dimensions. 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)).

[0424] The results indicate that treatment with SLNP-TB4 has antitumor activity, and that this antitumor activity was enhanced when combined with IC1 (Figure 24).

[0425] (Example 15) In vivo tumor inhibition of SLNP-TB4-IC1 as a monotherapy using MC38 cells. In this experiment, SLNP-TB4-IC1 was evaluated using the following protocol. For the purposes of this example, SLNP-TB4-IC1 is equivalent to SLNP-TB4-IC1(ALT-1). Mouse mammary cancer MC38 cells (0.5 × 10⁻⁶) 6 The drug was subcutaneously inoculated into the right posterior flank region of C57 / BL6 mice. The animals were treated by IV injection every other week with vehicle controls of 20 mg / kg SLNP-TB4 and 20-2 mg / kg SLNP-TB4-IC1. Tumor volume was measured three times using calipers in two dimensions. 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)).

[0426] The results indicate that treatment using SLNP-TB4-IC1 has antitumor activity compared to the vehicle control group (Figure 25).

[0427] (Example 16) In vivo tumor inhibition of SLNP-TB4-IC1 as a monotherapy in multiple doses using EMT-6 cells. In this experiment, SLNP-TB4-IC1 was evaluated using the following protocol. For the purposes of this example, SLNP-TB4-IC1 is equivalent to SLNP-TB4-IC1(ALT-1). Mouse mammary cancer EMT6 cells (0.5 × 10⁻⁶) 6 The drug was subcutaneously inoculated into the right posterior ventral region of Balb / c mice. The animals were treated with vehicle control, SLNP-TB4-IC1 at 20-2 mg / kg or 40-4 mg / kg by IV injection every other week. Tumor volume was measured three times using calipers, in two dimensions. 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)).

[0428] The results show that SLNP-TB-IC1 has antitumor activity at both doses, but the antitumor activity was higher in the 40 / 4 mg / kg group (Figure 26).

[0429] (Example 17) Synthesis and Characterization of SLNP-TB4(ALT-1) Solid Lipid Nanoparticles In another experiment, solid lipid nanoparticles (SLNPs) containing the TB4(ALT-1) prodrug (indicated as SLNP-TB4(ALT-1)) were synthesized by the following method. In particular, those skilled in the art will understand that lipid nanoparticles of the TB4 prodrug (SLNP-TB4) and / or (SLNP-TB4(ALT-1)) can be synthesized by using a probe sonicator or by using magnetic stirring. In addition, SNLP-TB4 and / or SLNP-TB4(ALT-1) can also be synthesized with or without the use of emulsifiers such as Moliwol 488 (polyvinyl alcohol), Pluronic F127, or Kolliphor RH40.

[0430] In this experiment, lipid stock solutions of DSPC, CHOL, and DSPE-PEG were prepared separately in ethanol (20 mg / ml). A TB4(ALT-1) prodrug stock solution was prepared in ethanol (10 mg / ml). SNLP-TB4(ALT-1) can be synthesized with or without emulsifiers using DSPC, CHOL, TB4, and DSPE-PEG in various molar ratios.

[0431] In the second step, a lipid mixture of DSPC, CHOL, TB4, and DSPE-PEG in a molar ratio of 37:51:7:5 was obtained by mixing the aforementioned lipids and TB4(ALT-1) stock solution together. This lipid mixture was heated to 45-55 degrees Celsius. Next, this lipid mixture was added to DI water and stirred for approximately 4 hours using magnetic stirring. The solvent was removed using a tangential flow filtration (TFF) system (Pall Minimate, Pall Corporation) with Pall Minimate TFF capsules equipped with an omega 100K membrane.

[0432] The characteristics of SLNP-TB4(ALT-1) were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-TB4(ALT-1) (SLNP concentration of 1 mg / ml) was placed in a transparent four-sided plastic cuvette and analyzed directly at 25°C. The results shown in Figure 27 indicate that the Z-mean size of the nanoparticles was approximately 101 nm and the PDI was approximately 0.138.

[0433] In addition, the zeta potential of SLNP-TB4(ALT-1) solid lipid nanoparticles in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell available for use with the Zetasizer Nano series. Measurements were performed at 25°C. The results show that the zeta potential of SLNP-TB4(ALT-1) was approximately -21.1 mV (Figure 28).

[0434] (Example 18) Synthesis and Characterization of SLNP-IC1-TB4(ALT-1) Solid Lipid Nanoparticles In another experiment, solid lipid nanoparticles (SLNPs) containing TB4(ALT-1) co-formulated with the IC1 prodrug (indicated as SLNP-IC1-TB4(ALT-1)) were synthesized by the following method. In particular, those skilled in the art will understand that solid lipid co-formulated nanoparticles of the TB4 prodrug (SLNP-IC1-TB4) and / or (SLNP-IC1-TB4(ALT-1)) can be synthesized by using a probe sonicator or magnetic stirring. In addition, SNLP-IC1-TB4 and / or SLNP-IC1-TB4(ALT-1) can also be synthesized with or without the use of emulsifiers such as Moliwol 488 (polyvinyl alcohol), Pluronic F127, or Kolliphor RH40.

[0435] In this experiment, in the first step, lipid stock solutions of DSPC, CHOL, and DSPE-PEG were prepared separately in ethanol (20 mg / ml). Next, a TB4(ALT-1) prodrug stock solution was prepared in ethanol (10 mg / ml). Similarly, a stock solution of IC1 prodrug was prepared in ethanol (2.5 mg / ml). SNLP-IC1-TB4(ALT-1) can also be synthesized with or without emulsifiers using DSPC, CHOL, TB4, IC1, and DSPE-PEG in various molar ratios.

[0436] In the second step, the aforementioned lipids were mixed together with TB4(ALT-1) and IC1 stock solutions to obtain a lipid mixture of DSPC, CHOL, TB4, IC1, and DSPE-PEG in a molar ratio of 34:47:13:1:5. This lipid mixture was heated to 50-55 degrees Celsius. Next, this lipid mixture was added to DI water and stirred for approximately 4-5 hours using magnetic stirring. The solvent was removed using a tangential flow filtration (TFF) system (Pall Minimate, Pall Corporation) with Pall Minimate TFF capsules equipped with an omega 100K membrane.

[0437] The characteristics of SLNP-IC1-TB4(ALT-1) were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-IC1-TB4(ALT-1) (SLNP concentration of 1 mg / ml) was placed in a transparent four-sided plastic cuvette and analyzed directly at 25°C. The results shown in Figure 29 indicate that the Z-mean size of the nanoparticles was approximately 108 nm and the PDI was approximately 0.179.

[0438] In addition, the zeta potential of SLNP-IC1-TB4(ALT-1) solid lipid nanoparticles in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell available for use with the Zetasizer Nano series. Measurements were performed at 25°C. The results show that the zeta potential of SLNP-IC1-TB4(ALT-1) was approximately -14.65 mV (Figure 30).

[0439] (Example 19) Human clinical trials for the treatment of human cancers by the use of formulations and / or co-formulated liposomes containing TB prodrugs. The present invention utilizes formulated and / or co-formulated liposomes containing TB prodrugs, particularly those that accumulate in tumor cells and are used in the treatment of certain tumors as well as other immunological disorders and / or other diseases. For each of these indications, two clinical approaches are successfully explored.

[0440] I.) Adjuvant therapy: Adjuvant therapy involves treating patients with formulations and / or co-formulated liposomes containing TB prodrugs in combination with chemotherapeutic agents or pharmaceuticals or biopharmaceuticals or combinations thereof. Primary cancer targets are treated under standard protocols by the addition of formulations and / or co-formulated liposomes containing TB prodrugs. Protocol design addresses efficacy, as assessed by examples, including, but not limited to, reduction of tumor burden of primary or metastatic lesions, increased progression-free survival, overall survival, improved patient health, disease stabilization, and the ability to reduce the usual doses of standard chemotherapy and other biologic agents. These dose reductions enable additional and / or extended treatment by reducing dose-related toxicity of chemotherapeutic agents or biologic agents.

[0441] II.) Monotherapy: In connection with the use of formulations and / or co-formulated liposomes containing TB prodrugs in monotherapy of tumors, formulations and / or co-formulated liposomes containing TB prodrugs are administered to the patient without chemotherapeutic agents, pharmaceuticals, or bioagents. In one embodiment, monotherapy is clinically implemented in terminally ill cancer patients with extensive metastatic disease. The protocol design addresses efficacy, as evaluated by examples, including, but not limited to, reduction of tumor burden of primary or metastatic lesions, increased progression-free survival, overall survival, improvement of patient health, stabilization of disease, and the ability to reduce the usual doses of standard chemotherapy and other bioagents.

[0442] Dosage The dosage regimen may be adjusted to provide the optimal desired response. For example, a single formulation and / or co-formulated liposome containing the TB prodrug may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. “Dosage unit form,” as used herein, refers to a physically distinct unit suitable as a unit dose to the mammalian subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect along with the necessary pharmaceutical carrier. The specifications of the dosage unit form of the present invention are defined by and directly depend on (a) the inherent characteristics of the formulation and / or co-formulated liposome containing the TB prodrug, (b) the individual dynamics of the combination compound, if any, (c) the specific therapeutic or prophylactic effect to be achieved, and (d) the inherent limitations in the formulation technique of such compounds for treating the susceptibility of the individual.

[0443] Clinical Development Program (CDP) CDP is being developed in conjunction with treatments using formulated and / or co-formulated liposomes containing TB prodrugs, either as adjuvant therapy or monotherapy. The trial will first demonstrate safety, and then confirm the efficacy of repeated doses. The trial is open-label and will compare formulated and / or co-formulated liposomes containing TB prodrugs with standard chemotherapy and / or current treatment standards. As understood, one non-limiting criterion that may be available in connection with patient enrollment is the expression of TGFβ in tumors, as determined by standard detection methods known in the art.

[0444] Formulated and / or co-formulated liposomes, or any of the embodiments disclosed herein, are expected to have satisfactory pharmacological profiles and promising biopharmaceutical properties, such as toxicological profiles, metabolic and pharmacokinetic properties, solubility, and permeability. It will be understood that determining appropriate biopharmaceutical properties is within the knowledge of those skilled in the art, for example, determining the inhibition of a particular target or channel to determine cytotoxicity or potential toxicity in cells.

[0445] The scope of the present invention is not limited by the embodiments disclosed herein, which are intended as single examples of individual aspects of the invention, and any functionally equivalent ones fall within the scope of the invention. In addition to those described herein, various variations of the models, methods, and lifecycle methodologies of the invention will become apparent to those skilled in the art from the above description and teachings, and these too are intended to fall within the scope of the invention. Such variations or other embodiments can be implemented without departing from the true scope and spirit of the invention. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

Claims

1. (i) Drug portion, (ii) Lipid portion, and (iii) Linking unit ("LU") A TB prodrug composition comprising the drug portion comprising a TGFβ antagonist, wherein the LU conjugates the drug portion with the lipid portion.

2. The TB prodrug composition according to claim 1, wherein the drug portion comprises the chemical structure described as TB4.

3. The TB prodrug composition according to claim 1, wherein the lipid portion comprises stearic acid.

4. The following chemical structure: 【Chemistry 46】 A TB prodrug composition according to claim 1, comprising a TB4 prodrug (ALT-1) having the following properties.

5. A nanocarrier containing a TB prodrug, which releases an active ALK5 inhibitor after cleavage of its linking units (LUs).

6. The nanocarrier according to claim 5, further comprising a helper lipid, wherein the helper lipid is listed in Table II.

7. The nanocarrier according to claim 5, wherein the TB prodrug comprises a TB4 prodrug (ALT-1).

8. The TB4 prodrug (ALT-1) has the following chemical structure: 【Chemistry 47】 The nanocarrier according to claim 7, having the following characteristics.

9. A method for treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of nanocarrier to a subject requiring such treatment, wherein the nanocarrier contains a TB prodrug, (ii) The pharmaceutically acceptable salt thereof, Methods that include...

10. The method according to claim 9, wherein the TB prodrug comprises a TB4 prodrug (ALT-1).

11. The TB4 prodrug (ALT-1) has the following chemical structure: 【Chemistry 48】 The method according to claim 10, comprising:

12. The method according to claim 9, wherein the nanocarrier comprises a TB4 prodrug (ALT-1) further co-formulated with an ICD-inducing chemotherapeutic agent.

13. The method according to claim 12, wherein the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, or paclitaxel.

14. The method according to claim 9, wherein the nanocarrier comprises a TB4 prodrug (ALT-1) further co-formulated with an immunomodulator.

15. The method according to claim 14, wherein the nanocarrier is further co-formulated with an immunomodulator, the immunomodulator being selected from the group consisting of other TLR agonists and / or prodrugs, immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors, and / or prodrugs thereof.

16. The method according to claim 9, wherein the nanocarrier is further co-formulated with a toll receptor agonist, and the toll receptor agonist is selected from the group consisting of reximod (R848), gardikimod, 852A, DSR6434, tellaterimod, CU-T12-9, monophosphoryllipid A (MPLA), monophosphorylhexaacyllipid A, 3-deacyl (synthetic), SMU127, Pam3CSK4, or 3-deacylphosphorylated hexaacyl disaccharide.

17. The method according to claim 9, wherein the nanocarrier is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid-prodrug, and the PD-1 / PD-L1 antagonist is selected from the group consisting of AUNP12, CA-170, or BMS-986189.

18. The method according to claim 9, wherein the nanocarrier comprises lipid nanoparticles (LNPs).

19. The method according to claim 9, wherein the nanocarrier comprises solid lipid nanoparticles (SLNPs).

20. The SLNP according to claim 15, wherein the SLNP is SLNP-TB4-IC1.

21. The method according to claim 9, wherein the subject is a human being.

22. The method according to claim 9, wherein the cancer is breast cancer.

23. A kit comprising the nanocarrier described in claim 8.

24. A kit comprising the nanocarrier described in claim 11.

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