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

Abstract

Disclosed herein are formulated and / or co-formulated liposomes containing IDO prodrugs and methods for making the liposomes. The IDO prodrug compositions include a drug moiety, a lipid moiety, and a linking unit that inhibit IDO-1. The IDO prodrugs can be formulated and / or co-formulated into liposomes to provide a method for treating cancer, immune disorders, and other diseases by using a targeted drug delivery vehicle.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 974,086, filed on November 12, 2019, the contents of which are incorporated in full by reference herein.

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

[0003] Field of Invention The inventions described herein relate to prodrug compositions that inhibit the indoleamine-pyrrole 2,3-dioxygenase (IDO) enzyme after the release of an active inhibitor from the prodrug, and to nanoformulations containing such prodrugs. Specifically, the invention relates to prodrug compositions that are formulated within nanocarriers (e.g., liposomes) and used as vehicles for the treatment of cancer in humans. The invention further relates to the treatment of cancer and other immune disorders and diseases. [Background technology]

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

[0005] Several cancers are noted 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, at all ages, and in both men and women (GLOBOCAN 2018). These cancers, and virtually all other cancers, share the common fatal characteristic of metastasizing to sites distant from the primary tumor, and with very few exceptions, metastatic disease is fatal. Furthermore, even cancer patients who initially survive the primary cancer, according to general experience, experience a dramatic change in their lives. Many cancer patients experience intense anxiety due to the awareness of the possibility of recurrence or treatment failure. Many cancer patients also experience physical weakness after treatment. In addition, many cancer patients experience disease recurrence.

[0006] While cancer treatment has improved over the past few decades and survival rates have increased, the heterogeneity of cancers means that new treatment strategies utilizing multiple treatment modes are still needed. This is especially true for the treatment of solid tumors in anatomically important sites (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma), which may be limited to standard radiotherapy and / or chemotherapy. Nevertheless, the adverse effects of these therapies are chemotherapy resistance and radioresistance, which, in addition to severe side effects that reduce the patient's quality of life, promote local recurrence, distant metastasis, and another primary tumor.

[0007] Indoleamine-pyrrole 2,3-dioxygenase (IDO or INDO) is a heme-containing enzyme encoded by the IDO1 gene in humans. IDO is the first and rate-limiting enzyme in tryptophan catabolism via the kynurenine pathway, thus causing tryptophan depletion, which can slow the growth of microorganisms and T cells. Furthermore, IDO is an immune checkpoint molecule in the sense that it is an immunomodulatory enzyme produced by several selectively activated macrophages and other immunomodulatory cells (also used as an immune-destroying strategy by many tumors and chronic infectious viruses). IDO is known to suppress T cells and NK cells, generate and activate Treg and myeloid-derived suppressor cells, and promote the growth of new blood cells to supply tumors (angiogenesis). IDO selectively impairs T cell proliferation and survival by enabling tumor cells to escape the immune system through L-tryptophan depletion in the tumor microenvironment and the production of its catabolic product, kynurenine. A wide range of human cancers, including prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer, and lung cancer, overexpress human IDO (hIDO). IDO is involved in immunomodulation through its ability to restrict T cell function and enable mechanisms of immune tolerance. New evidence suggests that IDO is activated during tumorigenesis, helping malignant cells evade eradication by the immune system. (MUNN et al., Trends in Immunology, 37(3):pp.193-207 (March 2016) and PENDERGRAST et al., Cancer Immunol Immunother., 63(7):pp.721-735 (July 2014).

[0008] Furthermore, a prodrug is a drug or compound that, after administration, is metabolized (i.e., converted in the body) into a pharmacologically active drug. Instead of administering the drug directly, a corresponding prodrug is used to improve the absorption, distribution, metabolism, and / or elimination of the drug. Prodrugs are often designed to improve bioavailability, for example, when the drug itself is poorly absorbed from the gastrointestinal tract. Prodrugs can also be used to improve the degree to which a drug selectively interacts with cells or processes that are not its intended target. This reduces the harmful or unintended effects of a drug, which is particularly important in procedures such as chemotherapy, where severe unintended and undesirable side effects may occur. Thus, a prodrug can be considered a drug that contains a special non-toxic protecting group that is temporarily used to alter or eliminate undesirable properties of the parent molecule.

[0009] Finally, nanocarriers are nanomaterials used for transporting other substances, such as drugs. Many different types of nanocarriers exist. For example, some examples of nanocarriers include polymer conjugates, polymer nanoparticles, lipid-based carriers, and dendrimers. The different 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 major therapeutic advantage of nanocarriers. Nanocarriers are promising in drug delivery processes because they can deliver drugs to site-specific targets, delivering drugs to specific organs or cells and not to other organs or cells. Site specificity is a significant therapeutic advantage as it prevents drugs from being delivered to the wrong location. Furthermore, nanocarriers show specific potential for use in chemotherapy, as they may help reduce the harmful and more widespread toxicity of chemotherapy to rapidly growing healthy cells in 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.

[0010] From the above description, it is readily apparent to those skilled in the art that a new treatment paradigm is needed in the treatment of cancer and other immunological diseases. By using novel prodrugs in conjunction with the latest nanocarrier designs, new disease treatments can be achieved with the overall goals of more effective treatment, reduced side effects, and greater therapeutic utility in the treatment of cancer, particularly solid tumors. In light of the current shortcomings associated with cancer treatment, the object of this invention is to provide a novel and improved method for treating cancer, immunodeficiencies, and other diseases using prodrugs encapsulated within nanocarriers. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] MUNN et al., Trends in Immunology, 37(3):pp.193-207(March 2016) [Non-Patent Document 2] PENDERGRAST et al., Cancer Immunol Immunother.,63(7):pp.721-735(July 2014) [Overview of the project] [Means for solving the problem]

[0012] Summary of the Invention The present invention provides an IDO inhibitor prodrug ("IDO prodrug") composition comprising an IDO inhibitor agent, a lipid, and a biologically cleavable linker. In certain embodiments, a nanocarrier comprising the IDO prodrug is formulated for use as a delivery modality for treating human diseases such as cancer, including solid tumor cancer, and other immune disorders. In certain embodiments, the nanocarrier comprises a lipid bilayer (i.e., liposome) that can be incorporated into a drug delivery vehicle. In further preferred embodiments, the liposome comprises cholesterol hemisuccinate ("CHEMS"). In further preferred embodiments, the liposomes of the present invention comprise stearic acid.

[0013] In a further embodiment, the present invention provides a method for delivering an IDO inhibitor to a tumor, comprising: (i) synthesizing an IDO prodrug; (ii) formulating the IDO prodrug of the present invention into the nanocarrier of the present invention; and (iii) administering the nanocarrier to a patient.

[0014] In another embodiment, the present invention provides a method for delivering an IDO inhibitor to a tumor together with one or more additional immunomodulatory agents, comprising: (i) synthesizing an IDO prodrug; (ii) co-formulating the IDO prodrug of the present invention together with one or more additional immunomodulatory agents of the present invention into a nanocarrier; and (iii) administering the nanocarrier to a patient.

[0015] In another embodiment, the immunomodulatory agent comprises an immunogenic cell death-inducing chemotherapeutic agent, a PD-1 agonist, a toll receptor agonist, a STING agonist, a CTLA4 inhibitor, and / or prodrugs thereof.

[0016] In another embodiment, the present disclosure teaches a method for synthesizing an IDO prodrug.

[0017] In another embodiment, the present disclosure teaches a method for formulating an IDO prodrug within a nanocarrier, including but not limited to liposomes.

[0018] In another embodiment, the Disclosure teaches a method for treating cancer, immunodeficiency and other diseases in humans using the nanocarriers of the Disclosure. [Brief explanation of the drawing]

[0019] [Figure 1] General chemical synthesis of protected ID3 containing cholesterol hemisuccinate ("CHEMS").

[0020] [Figure 2] Chemical synthesis of ID3 prodrug intermediates.

[0021] [Figure 3] Chemical synthesis of ID3 prodrug intermediates.

[0022] [Figure 4] A synthesis scheme for IDO inhibitor prodrugs using carboxylic acid functional groups.

[0023] [Figure 5] A synthesis scheme for IDO inhibitor prodrugs using alcohol functional groups.

[0024] [Figure 6] A synthesis scheme for IDO inhibitor prodrugs using secondary amine, amide, or aniline functional groups.

[0025] [Figure 7] Chemical synthesis of ID3 prodrugs containing stearic acid.

[0026] [Figure 8] Chemical synthesis of ID3 prodrugs containing cholesterol hemisuccinate ("CHEMS").

[0027] [Figure 9] Determination of the properties of LNP-ID3 liposomes.

[0028] [Figure 10] Determination of the properties of LNP-ID3 liposomes (Zeta Potential).

[0029] [Figure 11] Determination of the properties of LNP-AR5-ID3 liposomes.

[0030] [Figure 12] Determination of the properties of LNP-AR5-ID3 liposomes (Zeta Potential).

[0031] [Figure 13] Determination of the properties of LNP-AR5-TR5-ID3 liposomes.

[0032] [Figure 14] Determination of the properties of LNP-AR5-TR5-ID3 liposomes (Zeta Potential).

[0033] [Figure 15] Determination of the properties of LNP-ID3-NK1 liposomes.

[0034] [Figure 16] Determination of the properties of LNP-ID3-NK1 liposomes (Zeta Potential).

[0035] [Figure 17] Determination of the properties of LNP-ID3-NK1-MTO liposomes.

[0036] [Figure 18] Determination of the properties of LNP-ID3-NK1-MTO liposomes (Zeta Potential).

[0037] [Figure 19]Tumor inhibition of LNP-ID3 in vivo using B16F10 cells in combination with other liposomes.

[0038] [Figure 20] Tumor inhibition of LNP-ID3 in combination with LNP-AR5 using B16F10 cells in vivo.

[0039] [Figure 21] In vivo tumor inhibition of LNP-ID3-NK1 using B16F10 cells.

[0040] [Figure 22] In vivo tumor inhibition of LNP-ID3 and LNP-ID3-NK1 using CT26 cells.

[0041] [Figure 23] In vivo tumor inhibition of LNP-ID3 using CT26 cells.

[0042] [Figure 24] In vivo tumor inhibition of LNP-ID3 using MC-38 cells. [Figure 25] Tumor inhibition of LNP-ID3 in combination with LNP-AR5 using H22 cells in vivo.

[0043] [Figure 26] Measurement of IDO-1 activity.

[0044] [Figure 27] Determination of the properties of SLNP-ID3-AR5-TR5.

[0045] [Figure 28] Determination of the properties of SLNP-ID3-AR5-TR5 (Zeta Potential). [Modes for carrying out the invention]

[0046] Detailed description of the invention Section Overview I.) Definition II.) Prodrugs III.) Chemical compounds IV.) Lipids V.) Connecting Unit ("LU") VI.) Nanocarriers VII.) Liposomes VIII.) Pharmaceutical Preparations IX.) Combination Therapy X.) Method for delivering liposomes containing prodrugs to cells XI.) Methods for treating cancer and other immune disorders XII.) Kits / Manufactured Products I.) Definition:

[0047] Unless otherwise defined, all technical terms, notations, and other scientific terms or terminology used herein are intended to have meanings that are generally understood by those skilled in the art, unless the context clearly indicates otherwise. In some cases, for clarity and / or for ease of reference, terms that have generally understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from those generally understood in the art.

[0048] Where a trade name is used herein, references to a trade name also refer to the product formulation, generic drugs, and active pharmaceutical ingredients of that product, unless otherwise indicated by the context.

[0049] Where used herein, the term “about” refers to a value or quantity of size (i.e., diameter), weight, concentration, or percentage, and is intended to encompass variations of ±20% or ±10%, ±5%, ±1%, and ±0.1% from a specified quantity, in one example, so as to be suitable for carrying out the disclosed method.

[0050] As used herein, the term "and / or," when used in the context of enumerating entities, means that the entities exist individually or in combination. Thus, for example, the phrase "A, B, C and / or D" includes A, B, C and D individually, but also any combination and partial combination of A, B, C and D.

[0051] In this specification, numerical ranges described by endpoints include 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).

[0052] As used herein, the phrase “essentially from” limits the scope of the claims to materials or processes that, in addition to the specified materials or processes, do not substantially affect the basic and novel features of the claimed subject matter.

[0053] 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 stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, nodule, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, as these patients have a substantially worse prognosis compared to patients with clinically localized (organ-limited) cancer.

[0054] 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, heptynyl, and allenyl groups, C1-C 20A linear (i.e., "straight-chain"), branched, or cyclic hydrocarbon chain (including both ends) can refer to a saturated or at least partially unsaturated, possibly unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chain. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl, or propyl, is bonded to a straight-chain alkyl group. "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 1~8 This refers to branched-chain alkyl groups.

[0055] The alkyl group may be optionally substituted with one or more alkyl substituents that may be identical or different ("substituted alkyl"). The term "alkyl group substituent" includes, but is not limited to, alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. In some embodiments, one or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms may be optionally inserted along the alkyl chain, and the nitrogen substituents may be hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl"), or aryl.

[0056] 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 functional group, including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0057] The term "aryl" is used herein to refer to an aromatic substituent that may be a single aromatic ring, or multiple aromatic rings that are condensed together, covalently bonded, or bonded to a common group such as, but not limited to, a methylene or ethylene moiety. The common linking group may also be a carbonyl, as in benzophenone, or oxygen, as in diphenyl ether, or nitrogen, as in diphenylamine. The term "aryl" specifically 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 a cyclic aromatic compound 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 that may be identical or different ("substituted aryl"), and 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.

[0058] As used herein, "heteroaryl" refers to an aryl group containing one or more non-carbon atoms (e.g., O, N, S, Se, etc.) in the ring structure's backbone. Nitrogen-containing heteroaryl moieties include, but are not limited to, pyridine, imidazole, benzimidazole, pyrazole, pyrazine, triazine, and pyrimidine.

[0059] The terms “anticancer agent,” “chemotherapeutic agent,” and “anticancer prodrug” refer to drugs (i.e., chemical compounds) or prodrugs that are known or thought to be capable of treating cancer (i.e., killing cancer cells, inhibiting the growth of cancer cells, or treating cancer-related symptoms). In some embodiments, the term “chemotherapeutic agent” as used herein refers to a non-PS molecule used to treat cancer and / or having cytotoxic activity. More traditional or conventional chemotherapeutic agents can be described by their 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 vinca alkaloids (e.g., vinblastine).

[0060] "Aralkyl" refers to an alkyl-aryl group in which the alkyl and / or aryl portions are substituted as needed.

[0061] "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. Alkylene groups can be linear, branched, or cyclic. Alkylene groups can also be unsaturated and / or substituted with one or more "alkyl group substituents" as needed. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be inserted along the alkylene group as needed (also referred to herein as "alkylaminoalkyl"), where the nitrogen substituent is the alkyl group described above. Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (-(CH2)3-); cyclohexylene (-C6H 10 -);-CH=CH-CH=CH-;-CH=CH-CH2-;-(CH2) q Each of -N(R)-(CH2), -, and q is an integer from 0 to about 20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, where R is hydrogen or a lower alkyl group; examples include methylenedioxyl (-O-CH2-O-) and ethylenedioxyl (-O-(CH2)2-O-). The alkylene group can have about 2 to about 3 carbon atoms, and can have a further 6 to 20 carbon atoms.

[0062] The term "arylene" refers to a divalent aromatic group, such as a divalent phenyl or naphthyl group. Arylene groups may be substituted with one or more aryl substituents as needed and / or may contain one or more heteroatoms.

[0063] The term "amino" refers to the group -N(R)2, where each R is independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, or substituted aralkyl. The terms "aminoalkyl" and "alkylamino" may refer to the group -N(R)2, where each R is H, alkyl, or substituted alkyl, and at least one R is alkyl or substituted alkyl. "Arylamine" and "aminoaryl" refer to the group -N(R)2, where each R is H, aryl, or substituted aryl, and at least one R is aryl or substituted aryl, such as aniline (i.e., -NHC6H5).

[0064] "Bioreactive nanomaterials" refer to engineered biomaterials that induce or catalyze biological responses. In certain embodiments, nanomaterials catalyze biological responses at various nano / bio interfaces by inducing a response through one or more properties selected from the group consisting of composition, size, shape, aspect ratio, solubility, electronic, redox, surface display, surface coating, hydrophobicity, hydrophilicity, atomically thin nanosheets, or functionalized surface groups. In certain embodiments, bioreactive nanomaterials have the ability to inhibit the IDO-1 biological response in cells (e.g., in tumor cells), as well as / or activate the innate immune system through the delivery of "danger signals" and adjuvant effects.

[0065] "Bulk" (also known as "API") refers to an API or pharmaceutical product that has not been filled into the final container for distribution. Final formulation bulk generally refers to a pharmaceutical product that has been formulated and is stored or held before filling. APIs may be stored or held as "bulk" or "concentrated bulk" before being formulated into pharmaceutical products.

[0066] The terms "carboxylate" and "carboxylic acid" refer to the group -C(=O)O, respectively. - It can also refer to -C(=O)OH. The term "carboxyl" can also refer to the -C(=O)OH group.

[0067] As used herein, the terms “conjugate” and “conjugated” may refer to the bonding (e.g., covalent bonding) of two or more components (e.g., chemical compounds, polymers, biomolecules, particles, etc.) to one another. In some embodiments, the conjugate may include monovalent portions derived from two different chemical compounds covalently bonded via a divalent linker portion (e.g., optionally substituted alkylene or arylene). In some embodiments, the linker may contain one or more biodegradable bonds such that one or more bonds in the linker may be cleaved when the prodrug is exposed to a particular physiological environment or enzyme (e.g., esterase).

[0068] The term “compound” refers to and includes not only chemical compounds (e.g., prodrugs) themselves, but also, unless the context makes it clear that the following should be excluded, whether explicitly stated or not: amorphous and crystalline forms of a compound, including polymorphic forms, which may be part of a mixture or in an isolated state; free acidic and free base forms of a compound, typically the forms shown in the structures provided herein; isomers of a compound, referring to optical isomers and tautomers, optical isomers including enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers including racemic and non-racemic mixtures in addition to isolated optical isomers; isomers may be in an isolated form or in a mixture with one or more other isomers; isotopes of a compound, including deuterium-containing compounds and tritium-containing compounds, and radioactive isotopes The compounds include elements, including radioactive isotopes effective for therapeutic and diagnostic purposes; polymeric forms of the compounds, including dimers, trimers, and other forms; salts of the compounds, preferably pharmaceutically acceptable salts, acid-added salts, and base-added salts, including salts having organic and inorganic counterions, including zwitterionic forms, where the compound is accompanied by two or more counterions, the two or more counterions may be identical or different; as well as solvates, semi-solvates, monosolvates, disolvates, etc., of the compounds, including organic solvents and inorganic solvates, where the inorganic solvate is a hydrate; where the compound is accompanied by two or more solvent molecules, the two or more molecules may be identical or different. In some examples, references to the compounds of the present invention herein include explicit references to one of the above forms or, for example, salts and / or solvates. However, these references are for emphasis only and should not be construed as excluding other forms specified above.

[0069] "Pharmaceuticals" generally refers to the final formulation containing an active drug component (i.e., a liposome containing an IDO inhibitor prodrug) accompanied by an inactive component. This term also includes final dosage forms that do not contain an active ingredient but are intended for use as a placebo.

[0070] The term "disulfide" can refer to the -SS- group.

[0071] The term "empty vesicle" refers to an unloaded lipid vesicle itself.

[0072] 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.

[0073] As used herein, the term "esterase" refers to a hydrolytic enzyme that breaks down esters into acids and alcohols.

[0074] "Excipients" refer to inert substances used as carriers for the active ingredient in drugs such as vaccines. Excipients are sometimes used to increase the volume of formulations containing highly potent active ingredients in order to enable convenient and precise dosages. Examples of excipients include, but are not limited to, antifouling agents, binders, coatings, disintegrants, fillers, diluents, flavoring agents, colorants, lubricants, and preservatives.

[0075] As used herein, the terms “halo,” “halide,” or “halogen” refer to fluoro, chloro, bromo, and iodo groups.

[0076] The terms "hydroxyl" and "hydroxy" refer to the -OH group.

[0077] As used herein, the terms “inhibit” or “inhibit” mean to reduce by a measurable amount or to completely suppress.

[0078] The terms “individual” and “patient” as used in the context of this disclosure may be used interchangeably.

[0079] As used herein, the term “ligand” generally refers to a species, such as a molecule or ion, that interacts with another species in some way, for example, by binding. See Martell, AE, and Hancock, RP, Metal Complexes in Aqueous Solutions, Plenum: New York (1996), which is incorporated herein in its entirety by reference.

[0080] 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 common lipids, phospholipids, cholesterol, PEG, and chemically functionalized lipids for ligand binding.

[0081] 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.

[0082] The terms “liposome,” “lipid vesicle,” or “vesicle” are used interchangeably to refer to an aqueous compartment surrounded by a lipid bilayer, as conventionally defined (see Stryer (1981) Biochemistry, 2d Edition, WH Freeman & Co., p. 213).

[0083] 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.

[0084] The terms "mercapto" or "thiol" refer to the -SH group.

[0085] The terms "metastatic cancer" and "metastatic disease" refer to cancer that has metastasized to regional lymph nodes or distant sites, and include stage D disease in the AUA system and stage T×N×M+ in the TNM system.

[0086] The terms “nanocarrier,” “nanoparticle,” and “nanoparticle drug carrier” are used interchangeably and refer to nanostructures having an aqueous, solid, or polymer internal core. In certain embodiments, a nanocarrier comprises a lipid bilayer that encloses (or surrounds or encloses) a porous particle core. In certain embodiments, the nanocarrier is a liposome, a lipid nanoparticle (“LNP”), or a solid-lipid nanoparticle (“SLNP”).

[0087] 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 less than about 20 nm). In some embodiments, the dimensions are approximately 20 nm to 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).

[0088] The term "nanopes" refers to "lipid vesicles" having a diameter (or an aggregate of vesicles having an average diameter) ranging from about 20 nm, or about 30 nm, or about 40 nm to about 500 nm, or up to about 400 nm, or up to about 300 nm, or up to about 200 nm, or up to about 150 nm, or up to about 100 nm, or up to about 80 nm. In certain embodiments, nanovesicles have a diameter ranging from about 40 nm to about 80 nm, or from about 50 nm to about 70 nm.

[0089] "Pharmacologically acceptable" means non-toxic, inactive, and / or composition that is physiologically compatible with humans or other mammals.

[0090] "Pharmaceutical preparation" refers to the process of manufacturing a final drug by combining different chemical substances with a pure active pharmaceutical ingredient (API).

[0091] 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 can be present or absent, and if present, it is selected from H, alkyl, aralkyl, or aryl.

[0092] The term "phosphate" refers to two -OP(=O)(OR') groups, where R' is either H or a negative charge.

[0093] The term “prodrug” means a drug or compound that is metabolized into a pharmacologically active drug after administration. For the purposes of this disclosure, the prodrug of the present invention comprises three components: (i) a drug portion; (ii) a lipid portion; and (iii) a linking unit ("LU").

[0094] The term "IDO prodrug" refers to the prodrug of the present invention, in which the drug portion comprises an IDO inhibitor.

[0095] The term "pyrolipid" refers to a conjugate of a lipid and a porphyrin, porphyrin derivative, or 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.

[0096] As used herein, the terms “specific,” “specifically binding,” and “specifically binding” refer to the selective binding of the nanocarriers of the present invention to target IDO-1.

[0097] The term “supported lipid bilayer” refers to a lipid bilayer surrounding a porous particle core. This definition as described in this disclosure is expressed because the lipid bilayer is located on the surface and is supported by the porous particle core. In certain embodiments, the lipid bilayer may have a thickness in the range of about 6 nm to about 7 nm, which includes a hydrophobic core with a thickness of 3 to 4 nm, a hydrated hydrophilic head base layer (each about 0.9 nm), and 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 bilayer that effectively encapsulates and seals the IDO inhibitor.

[0098] The term "thioalkyl" can refer to the group -SR, where R is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl. Similarly, the terms "thioaralkyl" and "thioaryl" refer to the -SR group, where R is aralkyl and aryl, respectively.

[0099] As used herein, “to treat” or “therapeutic” and grammatically related terms refer to any improvement of any outcome of a disease, such as an extension of survival, a reduction in morbidity, and / or a reduction in side effects that are byproducts of an alternative mode of treatment, and as readily understood in the art, complete eradication of the disease is desirable but not a requirement of a treatment act.

[0100] The term "therapeutic dose" refers to the amount of an active prodrug, nanoencapsulated prodrug, or pharmaceutically active substance that elicits a biological or medical response in a tissue, system, animal, individual, or human.

[0101] The term "unsupported lipid bilayer" refers to an uncoated lipid bilayer within a lipid vesicle or liposome. II.) Prodrugs

[0102] 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 "Lipid") via the LU (see the section titled "Conjugation Unit") of this disclosure. For the purposes of this disclosure, several strategies can be utilized for the formation of IDO prodrugs (see, for example, Figures 4, 5, and 6).

[0103] Therefore, in some embodiments, the prodrug is a drug-lipid moiety comprising the IDO inhibitor of the present disclosure.

[0104] In one embodiment, the prodrug comprises the following chemical structure represented by formula I: [ka] In the formula, in an exemplary embodiment of formula I: X1 = Cl, F, CN; X² = H, F; and A,B=H,CH3; Therefore, in one embodiment, the prodrug is a drug-lipid moiety comprising an IDO inhibitor of formula I.

[0105] In one embodiment, the prodrug is a drug-lipid moiety containing the IDO inhibitor shown in Figure 4.

[0106] In one embodiment, the prodrug is a drug-lipid moiety containing the IDO inhibitor shown in Figure 5.

[0107] In one embodiment, the prodrug is a drug-lipid moiety containing the IDO inhibitor shown in Figure 6.

[0108] In further embodiments, the IDO prodrug is a drug-lipid portion comprising the lipids of the present disclosure.

[0109] In a further embodiment, the IDO prodrug is a drug-lipid moiety in which the lipids are CHEMS.

[0110] In further embodiments, the IDO prodrug is a drug-lipid moiety comprising the LU of the present disclosure.

[0111] In a further embodiment, the IDO prodrug is a drug-lipid moiety in which LU is a hydromethylcarbamate linker.

[0112] In further embodiments, the prodrug is a drug-lipid moiety comprising the IDO inhibitor of the present invention, wherein the IDO inhibitor is a drug-lipid moiety comprising the chemical composition ID3.

[0113] In a further embodiment, the prodrug is a drug-lipid moiety comprising the IDO inhibitor of the present invention, wherein the IDO inhibitor comprises ID3 and further comprises CHEMS.

[0114] In a further embodiment, the prodrug is a drug-lipid moiety comprising the IDO inhibitor of the present invention, wherein the IDO inhibitor comprises ID3 and further comprises stearic acid.

[0115] In a further embodiment, the prodrug is a drug-lipid moiety comprising the IDO inhibitor of the present invention, wherein the IDO inhibitor comprises ID3, further comprises CHEMS, and LU is a hydromethylcarbamate linker.

[0116] In a further embodiment, the prodrug is a drug-lipid moiety comprising the IDO inhibitor of the present invention, wherein the IDO inhibitor comprises ID3, further comprises stearic acid, and LU is a hydromethylcarbamate linker.

[0117] In a further embodiment, the prodrug is a drug-lipid moiety comprising the IDO inhibitor of the present invention, wherein the IDO inhibitor comprises ID3 and further comprises stearic acid, and has the following structure: [ka] It is a drug-lipid portion that has the following characteristics:

[0118] In a further embodiment, the prodrug is a drug-lipid moiety comprising the IDO inhibitor of the present invention, wherein the IDO inhibitor comprises ID3 and further comprises a cholesterol hemisuccinate, and has the following structure: [ka] It is a drug-lipid portion that has the following characteristics:

[0119] In further embodiments of the present disclosure, the subject provides IDO inhibitor prodrugs comprising a lipid-conjugated therapeutic parent drug. 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, wherein the monovalent drug moiety and the monovalent lipid moiety are linked via the linker (e.g., covalently). The monovalent drug moiety and the monovalent lipid moiety may be a chemical compound and a monovalent derivative of a lipid, respectively. For example, the monovalent derivative may be a chemical compound or a deprotonated derivative of a lipid containing a hydroxyl, thiol, amino, or carboxylic acid group.

[0120] In further embodiments of the present disclosure, the subject provides an IDO inhibitor prodrug comprising a lipid-conjugated therapeutic parent drug. 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., covalently bonded). The divalent drug moiety and the divalent lipid moiety may be a chemical compound and a divalent derivative of a lipid, respectively. For example, the divalent derivative may be a chemical compound containing a hydroxyl, thiol, amino, or carboxylic acid group, or a deprotonated derivative of a lipid.

[0121] Those skilled in the art will understand and be able to modify and alter the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such modifications and alterations are intended within the scope of this disclosure. PRO.) Drug Section

[0122] Another aspect of the present invention provides a novel IDO prodrug compound having the following formula represented by ID3.

[0123] Those skilled in the art will understand that compounds are useful as IDO inhibitors (e.g., inhibiting IDO-1). As brief background, IDO-1 is involved in immunomodulation through its ability to limit T cell function and enable mechanisms of immune tolerance. See MUNN et al., Trends in Immunology, 34(3): pp. 137-143 (2012). New evidence suggests that IDO is activated during tumorigenesis, helping malignant cells evade eradication by the immune system. In mice, IDO has normal immune checkpoint function in immune tolerance during pregnancy and suppresses the maternal immune system. See YU et al., Cellular Physiology and Biochemistry, 49(1): pp. 134-143 (2018). The study showed that IDO overexpression was associated with earlier death in several tumors, including ovarian cancer, colorectal cancer, endometrial cancer, and esophageal cancer, but in kidney cancer and liver cancer, IDO overexpression appeared to be associated with better outcomes. Ibid.

[0124] Based on the above, this disclosure describes a certain class of IDO inhibitors.

[0125] In one embodiment, the drug portion of the present disclosure comprises a compound having the following chemical structure (represented by ID3): [ka]

[0126] Those skilled in the art will understand and be able to modify and alter the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such modifications and alterations are intended within the scope of this disclosure. IV.) Lipids

[0127] Generally speaking, and for the purposes of this disclosure, the term “lipid” is used in its broadest sense and includes several subcategories of lipids, including but not limited to phospholipids / fatty acids. As will be understood by those skilled in the art, phospholipids belong to a class of lipids that are major 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 phosphate groups that can be modified with simple organic molecules such as choline, ethanolamine, or serine. These two components are usually linked together by a glycerol molecule. A representative list of phospholipids / fatty acids of the present invention is shown in Table III.

[0128] As a brief background, at the most basic level, the properties of liposomes depend on the subtle physicochemical interactions between the various lipid species in their composition. Individual lipids can combine to form countless superstructures, including bilayers, and the properties of these bilayers can be tuned to regulate drug release and membrane stability. In a simplified bilayer model, acyl chain length determines the bilayer thickness and phase transition temperature (Tm), acyl chain saturation controls the bilayer fluidity, and head group interactions influence interlipid and intralipid molecular forces. Liposome behavior can be tuned by incorporating synthetic lipids, such as lipid prodrugs, membrane-fusionable lipids, and functionalizable lipids, into the bilayer. See KOHLI et al., J. Control Release, 0: pp. 274-287 (Sept. 28, 2014).

[0129] In one embodiment of the present disclosure, the IDO prodrug comprises a monovalent lipid moiety.

[0130] In one embodiment, the IDO prodrug contains a divalent lipid moiety.

[0131] In one embodiment, the lipid includes cholesterol having the following chemical structure. [ka]

[0132] In one embodiment, the lipid comprises DPPG having the following chemical structure. [ka]

[0133] In one embodiment, the lipid comprises DMPG having the following chemical structure. [ka]

[0134] In one embodiment, the lipid comprises lysoPC having the following chemical structure. [ka]

[0135] In one embodiment, the lipid comprises (Δ9-cis)PG having the following chemical structure. [ka]

[0136] In one embodiment, the lipid comprises soybean lysoPC having the following chemical structure. [ka]

[0137] In one embodiment, the lipid comprises a prostaglandin (PG) having the following chemical structure. [ka]

[0138] In one embodiment, the lipid comprises C16 PEG2000 ceramide having the following chemical structure. [ka]

[0139] In one embodiment, the lipid comprises cholesterol hemisuccinates ("CHEMS") having the following chemical structure. [ka]

[0140] In one embodiment, the lipid includes stearic acid having the following chemical structure. [ka]

[0141] For reference, Table I provides a complete list of the chemical formulas and abbreviations of the lipids disclosed herein.

[0142] In further embodiments, the lipids include phospholipids / fatty acids disclosed herein and shown in Table III.

[0143] Furthermore, the IDO prodrugs and / or liposomes of this disclosure may contain 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. Phospholipids are preferably diesters and monoesters of phosphate. Preferred members of the phospholipids are phosphoglycerides and sphingolipids. 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.

[0144] While we do not wish to be bound by any theory, it should be noted that depending on the specific molar percentage of the helper lipid contained in the lipid composition according to the present invention, this helper lipid may be either a PEG-free helper lipid or, in particular, a PEG-containing helper lipid, and more specifically, remarkable effects may be achieved if the content of any of these types of helper lipids falls within the concentration range specified herein.

[0145] In a further aspect 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 as described herein. In a preferred embodiment, the lipid composition comprises any helper lipid or combination of helper lipids, in addition to any IDO inhibitor as described herein. In a further embodiment, the composition of the present invention containing nucleic acids forms a lipoplex. In a preferred embodiment, the term lipoplex as used herein refers to a composition comprising the neutral or anionic lipid, neutral helper lipid and IDO inhibitor of the present invention. For reference regarding the use of helper lipids in this technical field, see, for example, U.S. Patent Application Publication No. 2011 / 0178164; OJEDA et al., Int. J. of Pharmaceutics (March 2016); DABKOWSKA et al., JRSoc. Interface 9, pp. 548-561 (2012); and MOCHIZUKI et al., Biochimica et. Biophysica Acta, 1828, pp. 412-418 (2013).

[0146] In a preferred embodiment, the helper lipid of the present invention includes the helper lipids listed in Table II.

[0147] In one embodiment, the IDO prodrug comprises the lipid of the present invention, wherein the lipid is CHEMS and the drug portion is ID3.

[0148] In one embodiment, the IDO prodrug comprises the lipid of the present invention, the lipid being CHEMS, the drug portion being ID3, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0149] In one embodiment, the IDO prodrug comprises the lipid of the present invention, wherein the lipid is CHEMS, the drug portion is ID3, 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.

[0150] In one embodiment, the IDO prodrug comprises the lipid of the present invention, the lipid being CHEMS, the drug portion being ID3, and the CHEMS being monovalent.

[0151] In one embodiment, the IDO prodrug contains the lipid of the present invention, wherein the lipid is stearic acid and the drug portion is ID3.

[0152] In one embodiment, the IDO prodrug contains the lipid of the present invention, the lipid being stearic acid, the drug portion being ID3, and the stearic acid being monovalent.

[0153] In one embodiment, the IDO prodrug comprises the lipid of the present invention, the lipid being stearic acid, the drug portion being ID3, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0154] In one embodiment, the IDO prodrug comprises the lipid of the present invention, wherein the lipid is stearic acid, the chemical composition is ID3, 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.

[0155] Those skilled in the art will understand and be able to modify and alter the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such modifications and alterations are intended within the scope of this disclosure. V.) Connecting Unit ("LU")

[0156] In some embodiments, the subject matter of this 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 comprises LU via a secondary amine, amide, or aniline using the following scheme. [ka] An example of synthesis is as follows: [ka] The cleavage of prodrug structures containing secondary amines, amides, or anilines is obtained by esterase hydrolysis of secondary amine, amide, or aniline prodrugs under the following exemplary synthesis. [ka] During the ceremony, R1 and R2 may be molecules that connect N via C.

[0160] In one embodiment, the secondary amide nitrogen of the ID3 drug moiety is conjugated to CHEMS via a hydromethylcarbamate linker.

[0161] In one embodiment, the secondary amide nitrogen of the ID3 drug moiety is conjugated to stearic acid via a hydromethylcarbamate linker.

[0162] Those skilled in the art will understand and be able to modify and alter the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such modifications and alterations are intended within the scope of this disclosure. VI.) Nanocarriers

[0163] 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. Due to their small size, nanocarriers can deliver drugs to parts of 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 and micelles.

[0164] Furthermore, nanocarriers are useful in drug delivery processes because they can deliver drugs to site-specific targets, delivering drugs to specific organs or cells and not to other organs or cells. Site specificity is a significant therapeutic advantage as it prevents drugs from being delivered to the wrong location. In addition, nanocarriers are promising for use in chemotherapy because they may help reduce the harmful and more widespread toxicity of chemotherapy to rapidly growing healthy cells in the body. Since chemotherapy drugs can be extremely toxic to human cells, it is important that chemotherapy drugs are delivered to tumors without being released to other parts of the body.

[0165] Generally speaking, there are four ways in which nanocarriers can deliver drugs: passive targeting, active targeting, pH specificity, and temperature specificity.

[0166] Passive targeting refers to the ability of nanocarriers to travel through the tumor's vascular system, be captured, and accumulate within the tumor. This accumulation is caused by enhanced permeability and retention effects. The tumor's leaky vascular system is a network of blood vessels formed within the tumor, containing many tiny pores. These pores allow nanocarriers to enter, but also contain many bends that allow the nanocarriers to be captured. As more nanocarriers are captured, the drug accumulates at the tumor site. This accumulation allows for the direct delivery of large quantities of the drug to the tumor site.

[0167] Active targeting involves incorporating targeting modules, such as ligands or antibodies, that are specific to a particular type of cell in the body, onto the surface of a nanocarrier. Generally, nanocarriers have a high surface area-to-volume ratio, which allows for the incorporation of multiple ligands onto their surface.

[0168] Furthermore, certain nanocarriers release the drug they contain only within a specific pH range. pH specificity also allows nanocarriers to directly deliver drugs to tumor sites. This is due to the fact that 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. A highly acidic environment degrades the nanocarrier's structure, leading to drug release. Generally, these nanocarriers do not release drugs in neutral or basic environments, effectively targeting the acidic environment of tumors while leaving normal somatic cells unaffected. This pH sensitivity can also be induced in micelle systems by adding copolymer chains to micelles, which have been shown to act in a pH-independent manner. See WU et al., Biomaterials, 34(4):1213-1222 (2012). These micelle-polymer complexes also help prevent cancer cells from developing multidrug resistance. A low pH environment triggers the rapid release of micelle polymers, causing the majority of the drug to be released at once, rather than gradually as with other drug treatments.

[0169] Furthermore, some nanocarriers have been shown to deliver drugs more effectively at specific 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 to act as a safety mechanism for tumor-specific site delivery. See REZAEI et al., Polymer, 53(16):3485-3497 (2012).

[0170] 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), can transport hydrophobic and hydrophilic molecules, exhibit extremely low toxicity or no toxicity, and increase the duration of drug action by extending the half-life and controlled release of drugs. Lipid nanoparticles may include chemical modifications (gangliosides or polyethylene glycol (PEG)) to evade detection by the immune system or to improve drug solubility. Furthermore, lipid nanoparticles can be prepared in pH-sensitive formulations to facilitate drug release in acidic environments and can also associate with small molecules or antibodies (such as folic acid (FoA)) that recognize tumor cells or their receptors. Nanodrugs can also be used in combination with other therapeutic strategies to improve patient response. See GARCIA-PINEL et al., Nanomaterials 9(639)(2019).

[0171] In various embodiments, the silicasome drug carriers described herein include porous silica (or other material) nanoparticles coated with a lipid bilayer (e.g., silica bodies having a surface that defines multiple pores suitable for receiving molecules). The fact that the nanoparticles are referred to as silica nanoparticles does not preclude the incorporation of non-silica materials within the silica nanoparticles. In some embodiments, the silica nanoparticles may be substantially spherical, having multiple pore openings through a surface that provides 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.

[0172] Generally, silica nanoparticles contain silica bodies that define the outer surfaces between pore openings and the side walls within the pores. Pores can extend through the silica bodies to another pore opening, or they can only extend partially through the silica bodies, such that the pore has a bottom surface defined by the silica bodies.

[0173] In some embodiments, the silica body is mesoporous. In other embodiments, the silica body is microporous. As used herein, “mesoporous” means having pores with a diameter of about 2 nm to about 50 nm, and “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 like anticancer compounds, to adhere to or bind to the inner surface of the pores and be released from the silica body when used for therapeutic purposes. In some embodiments, the pores are substantially cylindrical.

[0174] In certain embodiments, the nanoparticles contain pores having a diameter of approximately 1 nm to 10 nm or approximately 2 nm to 8 nm. In certain embodiments, the nanoparticles contain pores having a diameter of approximately 1 nm to 6 nm or approximately 2 nm to 5 nm. Other embodiments contain particles having a pore diameter of less than 2.5 nm.

[0175] In other embodiments, the pore size is 1.5 to 2.5 nm. Silica nanoparticles with other pore sizes can be prepared, for example, by using different surfactants or swelling agents during the preparation of the silica nanoparticles. In various embodiments, nanoparticles may include particles with a size of about 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 very effective when entering living cells or vascular fenestrations. In certain embodiments, nanoparticles are in the size range of about 40 nm, or about 50 nm, or 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 are in the size range of about 60 nm to about 70 nm. Some embodiments include nanoparticles with an average maximum dimension of about 50 nm to about 1000 nm. Other embodiments include nanoparticles with an average maximum dimension of about 50 nm to about 500 nm. Other embodiments include nanoparticles with an average maximum dimension of about 50 nm to about 200 nm.

[0176] In some embodiments, the average maximum dimensions are greater than about 20 nm, greater than about 30 nm, greater than 40 nm, or greater than about 50 nm. Other embodiments include nanoparticles having average maximum dimensions 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, nanoparticle size refers to the average or median size of primary particles 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).

[0177] Methods for producing porous silica nanoparticles are well known to those skilled in the art. In certain embodiments, mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate (TEOS) with a template made of micelle rods. The result is an aggregate of nano-sized spheres or rods filled with a regular arrangement of 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).

[0178] 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 together with additional polymer monomers as templates. In certain embodiments, 3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS.

[0179] In a particular embodiment, mesoporous silica nanoparticles are the core and are synthesized by a modification of the sol / gel procedure described by MENG et al., (2015)ACS Nemo, 9(4):3540-3557.

[0180] Although the methods described herein have been demonstrated with respect to porous silica nanoparticles (e.g., mesoporous silica), it will be recognized by those skilled in the art that similar methods can be used with other porous nanoparticles. Numerous 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 can be utilized.

[0181] 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.).

[0182] Similarly, mesoporous polymer particles can be utilized in certain embodiments. The synthesis of highly ordered mesoporous polymers and carbon frameworks from the organic-organic assembly of triblock copolymers with soluble low molecular weight phenolic resin precursors (resoles) by an evaporation-induced self-assembly strategy has been reported by MENG et al., (2006) Chem. Mat. 6(18):4447-4464.

[0183] The nanoparticles described herein are illustrative and non-limiting. Numerous other lipid bilayer-coated nanoparticles are available to those skilled in the art using the teachings provided herein.

[0184] In one embodiment, the present invention teaches a nanocarrier containing an IDO prodrug.

[0185] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises CHEMS.

[0186] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid.

[0187] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises CHEMS and the liposomes further comprise an IDO prodrug.

[0188] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises CHEMS and the liposomes further comprise ID3.

[0189] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise an IDO inhibitor.

[0190] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid and the liposomes further comprise ID3.

[0191] The scope of this disclosure teaches three possible treatment methods using the formulated prodrug of the present invention. See International Publication No. 2018 / 213631.

[0192] The first mode of treatment involves a combination of an IDO prodrug and another therapeutic agent (e.g., another formulated prodrug that inhibits IDO-1, a chemotherapeutic agent (e.g., ICD-inducing chemotherapy)) into a single liposome, enabling systemic (or local) in vivo distribution and drug delivery to the tumor site. The dual delivery approach achieved synergistic enhancement of adaptive and innate immunity, resulting in a significant improvement in animal survival. In certain embodiments, the nanocarrier comprises vesicles (i.e., lipid bilayers that encapsulate fluids).

[0193] The second mode of treatment involves local delivery of an IDO-1 inhibitor to the tumor or peritumoral region, combined with a lipid (e.g., liposomes) containing an IDO-1 inhibitor. Such local delivery of an IDO-1 inhibitor combined with an IDO prodrug has been demonstrated to induce cytotoxic tumor killing and tumor reduction at the local site. These adaptive immune responses involve enhancement of the innate immune system, reflected in CRT expression, as well as activation of DC populations particularly well-suited to generating cytotoxic T cell responses.

[0194] A third mode of treatment involves vaccination using dysentery cancer cells (e.g., KPC cells) in which IDO-1 inhibition is ex vivo induced. Such vaccination has been found to be able to inhibit tumor growth at distant sites and to generate a systemic immune response that enables adoptive transfer to non-immune animals. Those skilled in the art will understand and be able to implement the method modes of treatment provided herein. VII.) Liposomes

[0195] In one aspect, the subject matter of the present disclosure is based on an approach to provide prodrugs of the present disclosure (see the section titled "Prodrugs") that are suitable for incorporation into nanocarriers including a lipid coating layer in order to enhance the delivery of the corresponding prodrug and to provide combination therapies including the prodrug. The advantages of using the prodrugs of the present invention include the acceleration of controlled formulation into LNPs (e.g., liposomes) of the present disclosure. This allows the prodrug to be kept in an inactive form during systemic circulation, thereby enabling the liposome to release the active ingredient after phagocytosis by cells, for example, in tumors.

[0196] In certain embodiments, one or more IDO prodrugs (e.g., any one or more IDO prodrug inhibitors taught by formula I and / or ID3) (see the section titled "Prodrugs") are formulated with a lipid moiety that can form vesicular (e.g., liposome) structures in aqueous solution or form components of the lipid bilayer constituting liposomes. Liposomes can be used directly or provided as components in combined formulations (e.g., in combination with other drug moieties or therapeutic modes disclosed herein).

[0197] In certain embodiments, the liposomes formulated with the IDO prodrug contain lipids, PHGP, vitamin E, cholesterol, and / or fatty acids.

[0198] In one embodiment, the liposomes contain cholesterol.

[0199] In one embodiment, the liposome contains DPPG.

[0200] In one embodiment, the liposome contains DMPG.

[0201] In one embodiment, the liposome is lysoPC.

[0202] In one embodiment, the liposome is (Δ9-cis)PG.

[0203] In one embodiment, the liposome contains soy lysoPC.

[0204] In one embodiment, liposomes contain PG.

[0205] In one embodiment, the liposome contains PA-PEG3-mannose.

[0206] In one embodiment, the liposomes contain C16 PEG2000 ceramide.

[0207] In one embodiment, the liposome contains MPLA.

[0208] In one embodiment, the liposome contains CHEMS.

[0209] In one embodiment, liposomes contain stearic acid.

[0210] In one embodiment, the liposomes contain the phospholipids shown in Table III.

[0211] In one embodiment, the liposome comprises ID3, further comprising CHEMS, and further comprising LU, wherein LU is a hydromethylcarbamate linker.

[0212] In one embodiment, the liposome comprises ID3, further comprising stearic acid, and further comprising LU, wherein LU is a hydromethylcarbamate linker.

[0213] In one embodiment, the liposome comprises ID3, further comprising CHEMS, further comprising LU, wherein LU is a hydromethylcarbamate linker, and further comprising the helper lipids shown in Table II.

[0214] In one embodiment, the liposome comprises ID3, further comprising stearic acid, further comprising LU, wherein LU is a hydromethylcarbamate linker, and further comprising the helper lipids shown in Table II.

[0215] In one embodiment, the liposomes of the present disclosure comprise an IDO prodrug co-formulated with one or more additional immunomodulators, the immunomodulators including, but not limited to, immunogenic cell death-inducing chemotherapeutic agents, Toll receptor agonists, Sting agonists, CTLA4 inhibitors, PD-1 inhibitors, and / or prodrugs thereof.

[0216] In a preferred embodiment, the liposomes contain an IDO prodrug co-formulated with an ICD-inducing chemotherapeutic agent.

[0217] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with an ICD-inducing chemotherapeutic agent selected from the list of doxorubicin (DOX), mitoxantrone (MTO), oxaliplatin (OXA), cyclophosphamide (CP), bortezomib, carfildimib, or paclitaxel.

[0218] In a preferred embodiment, the liposome contains an IDO prodrug co-formulated with a Toll receptor TLR agonist / prodrug.

[0219] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with a Toll-like receptor (TLR) agonist / prodrug selected from the following list: resiquimod (R848), gardiquimod, 852A, DSR6434, telratolimod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4 or 3D-PHAD®.

[0220] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with a PD-1 inhibitor / prodrug.

[0221] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with a PD-1 inhibitor / prodrug selected from the list of AUNP12, CA-170 or BMS-986189 or prodrugs thereof.

[0222] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with doxorubicin (DOX).

[0223] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with mitoxantrone (MTO).

[0224] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with doxorubicin (DOX) and a PD-1 prodrug.

[0225] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with mitoxantrone (MTO) and a PD-1 prodrug.

[0226] In a preferred embodiment, the liposome comprises an IDO prodrug co-formulated with doxorubicin (DOX) and a TLR agonist / prodrug.

[0227] In a preferred embodiment, the liposomes contain an IDO prodrug co-formulated with mitoxantrone (MTO) and a TLR agonist / prodrug.

[0228] In a preferred embodiment, the liposomes contain doxorubicin (DOX) and an IDO prodrug co-formulated with a PD-1 prodrug and a TLR agonist / prodrug.

[0229] In a preferred embodiment, the liposomes contain mitoxantrone (MTO) and an IDO prodrug co-formulated with a PD-1 prodrug and a TLR agonist / prodrug.

[0230] In a preferred embodiment, the liposome contains an IDO prodrug co-formulated with a TLR agonist / prodrug.

[0231] In a preferred embodiment, the liposome contains an IDO prodrug co-formulated with a TLR agonist / prodrug.

[0232] In a preferred embodiment, the liposomes contain an IDO prodrug co-formulated with a TLR agonist / prodrug and a PD-1 prodrug.

[0233] In a preferred embodiment, the liposomes contain an IDO prodrug co-formulated with a TLR agonist / prodrug and a PD-1 prodrug.

[0234] In a preferred embodiment, the liposomes contain ID3 co-formulated with doxorubicin (DOX).

[0235] In a preferred embodiment, the liposomes contain ID3 co-formulated with mitoxantrone (MTO).

[0236] In a preferred embodiment, the liposome comprises ID3 co-formulated with doxorubicin (DOX) and / or an IDO prodrug and / or a TLR agonist / prodrug.

[0237] In a preferred embodiment, the liposome comprises ID3 co-formulated with mitoxantrone (MTO) and / or an IDO prodrug and / or a TLR agonist / prodrug.

[0238] In a preferred embodiment, the liposome comprises ID3 co-formulated with AR5.

[0239] In a preferred embodiment, the liposome comprises ID3 co-formulated with AR5 and TR5.

[0240] In a preferred embodiment, the liposome comprises ID3 co-formulated with NK1.

[0241] In a preferred embodiment, the liposome comprises ID3 co-formulated with NK1 and MTO.

[0242] In a preferred embodiment, the liposome comprises ID3 co-formulated with TR3.

[0243] In a preferred embodiment, the liposome comprises ID3 co-formulated with TR5.

[0244] In a preferred embodiment, the liposome comprises ID3 co-formulated with TB4.

[0245] In a preferred embodiment, the liposome comprises ID3 co-formulated with PD3.

[0246] In a preferred embodiment, the liposome comprises ID3 co-formulated with AR5 and TR3.

[0247] In a preferred embodiment, the liposomes include ID3 co-formulated with AR5 and TB4.

[0248] In a preferred embodiment, the liposomes contain ID3 co-formulated with AR5 and PD3.

[0249] In another preferred embodiment, the liposomes comprise solid-lipid nanoparticles (SLNPs) containing liposomes comprising an IDO prodrug.

[0250] Those skilled in the art recognize and understand that solubility is one of the most common problems they face in the drug development process. Chemical conjugation of drugs / anticancer agents via lipid molecules (i.e., lipid-based prodrugs) provides a basis for solving the problem of formulating drugs in aqueous suspensions. The main advantage of delivering drugs using lipid conjugations (lipid-based prodrugs) lies in its ability to improve pharmacokinetics / half-life and targeted delivery.

[0251] With the appropriate selection of lipid molecules, lipid-based prodrugs can be integrated / formulated into liposomal formulations using techniques known in the art, which offers many further advantages over conventional drug delivery systems. (KOHLI et al., J. Control Release, 0: pp274-287 (Sept. 28, 2014); and GARCIA-PINEL et al., Nanomaterials 9: 638 (2019). The advantages of combining lipid-prodrugs with liposomes consist of two elements: (i) liposomes containing lipid-prodrugs not only increase the solubility of the drug / prodrug itself, but also (ii) have the ability to encapsulate multiple drugs (both hydrophilic and lipophilic) (see the section titled Nanocarriers).

[0252] For the purposes of this disclosure, the main advantages of liposomal formulations are as follows: i) The liposomal formulation is biocompatible / biodegradable and free from general toxicity; ii) Flexibility and manipulation of size and surface charge according to 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; and iii) The liposomes of the present invention have one or more lipid-prodrugs as the constituent lipid portion of the liposome. Furthermore, multiple drugs (for example, those that function by different mechanisms of action) and drugs with different solubility profiles (hydrophilic or lipophilic) can be formulated into these liposomes (either in the lipid bilayer or the hydrophilic core).

[0253] As those skilled in the art will understand, all methods for producing liposomes involve four basic steps: (i) drying lipids from organic solvents; (ii) Dispersing lipids in an aqueous solution; (iii) Purify the obtained liposomes; and (iv) Analyze the final product. See AKBARZADEH et al., Nanoscale Research Letters, 8:102 (2013).

[0254] Another aspect of the present invention discloses liposome encapsulation technology (LET), a delivery technology used to deliver drugs. LET is a method of generating ultramicroscopic bubbles called liposomes that encapsulate a number of materials. These “liposomes” form a barrier around the liposome contents that is resistant to enzymes in the mouth and stomach, alkaline solutions, digestive fluids, bile salts, the gut microbiota produced in the human body, and free radicals. Thus, the contents of the liposome are protected from oxidation and degradation. This protective phospholipid shield or barrier remains undamaged until the contents of the liposome are delivered to the precise target gland, organ or system in which the contents are used (see the section titled Nanocarriers).

[0255] In one embodiment, the liposomes of the Disclosure are synthesized using several different ratios of IDO prodrugs, lipids, and / or lipid prodrugs. As disclosed herein, the IDO prodrugs may include helper lipids disclosed herein (see, for example, Table II).

[0256] In one embodiment, the liposomes of this disclosure are synthesized using several different ratios of IDO prodrugs, lipids, and / or lipid prodrugs. As disclosed herein, the IDO prodrug may further comprise DSPE-PEG.

[0257] In a preferred embodiment, the liposome of the present invention comprises a composition having the following ratio: [Table A]

[0258] In a further preferred embodiment, the liposome of the present invention comprises a composition having the following ratio: [Table B]

[0259] In a further preferred embodiment, the liposome of the present invention comprises a composition having the following ratio: [Table C] Here, lipid 1 includes ID3 and CHEMS.

[0260] In a further preferred embodiment, the liposome of the present invention comprises a composition having the following ratio: [Table D] Here, lipid 1 includes ID3 and stearic acid.

[0261] Those skilled in the art will understand and be able to modify and alter the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such modifications and alterations are intended within the scope of this disclosure. VIII.) Pharmaceutical Preparations

[0262] As used herein, the term “drug” is synonymous with “pharmaceutical.” In certain embodiments, the liposomes of this disclosure are prepared into encapsulated dosage forms and administered to a patient for the treatment of a disease.

[0263] Generally speaking, "pharmaceutical formulation" is the process of combining different chemical substances with a pure active pharmaceutical ingredient (API) to produce the final drug. Formulation research involves developing drug preparations that are stable and tolerable for patients. For orally administered drugs, this usually involves incorporating the drug into tablets or capsules. It is important to understand that dosage forms contain various other substances different from the drug itself, and research must be conducted to ensure that the drug is compatible with these other substances.

[0264] Excipients are inert substances used as carriers for liposomes containing the active ingredient of a pharmaceutical, in this case, an IDO prodrug. Furthermore, excipients can be used to assist in the manufacturing process of pharmaceuticals. The active ingredient is then dissolved or mixed with the excipient. Excipients are sometimes used to increase the volume of formulations containing extremely potent active ingredients to enable convenient and precise dosages. Once the active ingredient is purified, it cannot remain in its purified form for very long. Often, the active ingredient denatures, separates from the solution, or adheres to the sides of the container.

[0265] Excipients are added to stabilize the active ingredient, ensuring that the active ingredient remains stable for a sufficiently long period to maintain its activity and give the product a superior shelf life compared to other products, making it safe for end-users. Examples of excipients include, but are not limited to, antifouling agents, binders, coatings, disintegrants, fillers, diluents, flavoring agents, colorants, lubricants, and preservatives. The final formulation contains the active ingredient and excipients, which are then placed into a pharmaceutical dosage form.

[0266] Prior to formulation, the physical, chemical, and mechanical properties of the drug are determined in order to select what other components should be used in the preparation. Factors such as stability, particle size, polymorphism, pH, and solubility can all affect bioavailability and therefore drug activity; therefore, formulation studies then consider these factors. The drug must be combined with inert additives in a manner that ensures the amount of drug present is consistent in each dosing unit (e.g., each vial). The dosing amount should have a uniform appearance.

[0267] It is unlikely that these studies will be completed by the start of clinical trials. This means that simpler preparations will be developed first for use in Phase I clinical trials. These typically consist of vials, hand-filled capsules containing small amounts of drug and diluent. Since these formulations will be used (tested) within a few days, proof of long-term stability of these formulations is not required. However, long-term stability is important in supply chain management because the time the final formulation remains packaged before reaching patients can be several months or even years. The so-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 cause flow problems or require larger capsules if the compound has a low bulk density. By the time Phase III clinical trials are reached, the drug formulation should be developed to be close to the final formulation used in the market.

[0268] Stability knowledge is essential by this stage, and conditions must be developed to ensure that the drug is stable in the preparation. Since it is impossible to know the actual dose administered, if the drug is found to be unstable, the results obtained from clinical trials become invalid. Stability studies are conducted to test whether temperature, humidity, oxidation, or photodegradation (UV or visible light) have any effect, and the preparation is analyzed to see if degradation products have 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 to the plastic, and whether any plasticizers, lubricants, pigments, or stabilizers leach from the plastic into the preparation. Even the adhesive used for the container label needs to be tested to ensure that it does not leach through the plastic container into the preparation. The method by which 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 be soluble in aqueous solution at a controlled rate. Factors such as particle size and crystalline form can significantly affect dissolution. Rapid dissolution is not always ideal. For example, a slower dissolution rate may result in a longer duration of action or avoid initial high plasma levels.

[0269] In some embodiments, nanocarriers (e.g., liposomes containing an IDO prodrug) and / or liposomes containing an IDO prodrug co-formulated with an immunomodulator are administered alone or in mixtures with a physiologically acceptable carrier (such as saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. For example, when used as an injectable, the nanocarrier can be formulated as a sterile suspension, dispersion, or emulsion with a pharmaceutically acceptable carrier. In certain embodiments, ordinary 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., and glycoproteins to enhance the stability of albumin, lipoproteins, globulins, etc. In compositions containing saline or other salt-containing carriers, the carrier is preferably added after nanocarrier formation. Thus, after the nanocarrier has been formed and loaded with the appropriate drug, the nanocarrier can be diluted in a pharmaceutically acceptable carrier such as ordinary saline. Similarly, IDO prodrug liposomes can be introduced into carriers that facilitate the suspension of nanomaterials (e.g., emulsions, diluents, etc.).

[0270] 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., LB-coated nanoparticles) are lyophilized, and the lyophilized preparation is combined with a sterile aqueous solution prior to administration. The composition may also contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, isotonic adjusters, etc., including sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.

[0271] Furthermore, in certain embodiments, the pharmaceutical formulation may include lipid protectants to protect lipids from damage by free radicals and lipid peroxidation during storage. Lipophilic free radical scavengers such as α-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable and intended herein. The concentration of nanocarriers (e.g., liposomes containing IDO prodrugs) in the pharmaceutical formulation can vary widely, for example, from less than about 0.05% by weight, typically at least about 2-5% by weight to 10-50% by weight or ~40% by weight or ~30% by weight, and is selected mainly by fluid volume, viscosity, etc., according to the selected specific mode of administration. For example, the concentration may be increased to reduce the fluid load associated with the procedure. This may be particularly desirable in patients with atherosclerosis-associated 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 disease condition being treated, and the clinician's judgment, but is generally about 0.01 to 50 mg per kilogram of body weight, preferably about 0.1 to 5 mg per kg of body weight.

[0272] Those skilled in the art will understand that the precise dosage will vary depending on factors such as the specific IDO prodrug and any co-formulated immunomodulators and the desired medical effect, as well as patient factors such as age, sex, and general condition. Those skilled in the art can easily take these factors into consideration and use them to establish effective therapeutic concentrations without relying on excessive experimental manipulation.

[0273] For administration to humans (or non-human mammals) in the curative, remission, delayed, 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, and the appropriate dosage may vary depending on 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 can be approximately equal to the dosage used for the free drug. However, as described above, the nanocarriers described herein can significantly reduce the toxicity of the drug delivered by the nanocarrier and significantly increase the therapeutic range. Therefore, in some cases, dosages exceeding those prescribed for the free drug are utilized.

[0274] Those skilled in the art will understand and be able to modify and alter the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such modifications and alterations are intended within the scope of this disclosure. IX.) Combination Therapy

[0275] As those skilled in the art recognize and understand, the proliferation and survival of cancer cells can be influenced by multiple signaling pathways. Therefore, to address such symptoms, it is useful to combine different enzyme / protein / receptor inhibitors that exhibit different preferences at targets that modulate activity. Targeting more than one signaling pathway (or more than one biological molecule involved in a given signaling pathway) may reduce the potential for drug resistance to develop in a cell population and / or reduce the toxicity of the treatment.

[0276] Accordingly, liposomes containing the IDO prodrug of this disclosure can be used in combination with one or more other enzyme / protein / receptor inhibitors or one or more therapeutic agents to treat diseases such as cancer or infections. Examples of diseases and indications that can be treated with combination therapy are those described in this disclosure. Examples of cancer include, but are not limited to, solid tumors and liquid tumors such as hematological malignancies. Examples of infections include viral infections, bacterial infections, fungal infections or parasitic infections.

[0277] For example, liposomes containing the IDO prodrug of this disclosure are for the treatment of cancer, including the following kinases: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, PI3K(α, β, γ, δ), CSFIR, KIT, FLK-II, KDR / FLK-1, FLK-4 It can be combined with one or more inhibitors of 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.

[0278] In a further embodiment, liposomes comprising the IDO prodrug of this disclosure may be combined with one or more of the following inhibitors for the treatment of cancer or infection. Non-limiting examples of inhibitors that can be combined with the compounds of 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, PI3Kδ inhibitors (e.g., INCB50797 and INCB50465), PI3Kγ inhibitors, e.g., PI3Kγ selective inhibitors, Pim inhibitors (e.g., INCB53914), CSF1R inhibitors, TAM receptor tyrosine These include cytokinases (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, and adenosine receptor antagonists, or combinations thereof.

[0279] In one embodiment, the A2a receptor inhibitor is the following prodrug compound (denoted "AR5"): [ka] Includes.

[0280] In a further embodiment, the liposome containing the IDO prodrug further comprises α-galactosylceramide (α-GalCer) (denoted as "NK1").

[0281] Furthermore, liposomes comprising the IDO prodrug of this disclosure can be further used in combination with other methods of treating cancer, such as chemotherapy, radiotherapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, or surgery.

[0282] 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.

[0283] Liposomes containing IDO prodrugs can be administered in combination with one or more anticancer agents, such as chemotherapeutic agents. Examples of chemotherapeutic agents include avalerix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezomib, bortezomib, intravenous busulfan, oral busulfan, carsterone, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, and cytarabi. Dacarbazine, Dactinomycin, Dalteparin sodium, Dasatinib, Daunorubicin, Decitabine, Denileukin, Denileukin difutitox, Dexrazoxane, Docetaxel, Doxorubicin, Dromostanolone propionate, Eculizumab, Epirubicin, Erlotinib, Estramustine, Etoposide phosphate, Etoposide, Exemestane, Fentanyl citrate, Filgrastim, Phloxuridine, Fludarabine, Fluorouracil, Fulvestrant, Gefitinib, Gemcitabine, Gemtuzma Buozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon α2a, irinotecan, lapatinium nitosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisol, lomustine, mechloretamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, fenpropionate nandrolone, nelarabine Nofetumomab, olaparib, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobromane, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, ruxolitinib, rucaparib, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan,Examples include toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, barrubicin, vinblastine, vincristine, vinorelbine, vorinostat, niraparib, beriparib, talazoparib, and zoledronate.

[0284] Other anticancer drugs include antibody therapies such as trastuzumab (Herceptin), antibodies against costimulatory molecules such as CTLA-4 (e.g., ipilimumab) and 4-1BB (e.g., urelumab, utomirumab), antibodies against PD-1 and PD-L1 / L2, or antibodies against cytokines (IL-10, TGF-β, etc.).

[0285] 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 infections such as viral, bacterial, fungal, and parasitic infections include, but are not limited to, nivolumab, pembrolizumab, MPDL3280A, MEDI-4736, and SHR-1210.

[0286] Furthermore, liposomes containing the IDO 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 infectious diseases. Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, PD-1, PD-L1, and PD-L2.

[0287] 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 IDO prodrug provided herein can be used in combination with one or more activators selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGFRβ inhibitors. X.) Method for delivering liposomes containing an IDO prodrug to cells expressing IDO-1.

[0288] As is known in the art, a wide variety of compositions and methods for using prodrugs and / or liposomes to kill tumor cells are known in the art. In relation to cancer, a typical method involves administering a biologically effective amount of the IDO prodrug of the present disclosure and / or liposomes of the present disclosure containing the IDO prodrug to a mammal with a tumor.

[0289] A typical embodiment is a method for delivering a therapeutic agent to cells expressing IDO-1, comprising forming an IDO prodrug by conjugating the drug portion of the present disclosure with a lipid of the present disclosure via a linking unit, and exposing the cells to the IDO prodrug.

[0290] In one embodiment, the IDO prodrug comprises a drug moiety of formula I and CHEMS, conjugated via an LU containing a hydromethylcarbamate linker.

[0291] In one embodiment, the IDO prodrug comprises the drug moiety of formula I and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0292] In one embodiment, the IDO prodrug comprises ID3 and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0293] In one embodiment, the IDO prodrug comprises ID3 and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0294] 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 comprising a therapeutically effective amount of an IDO prodrug produced by conjugating a drug portion with a lipid of the present disclosure via a linking unit, and exposing cells to the IDO prodrug.

[0295] In one embodiment, the IDO prodrug comprises a drug moiety of formula I and CHEMS, conjugated via an LU containing a hydromethylcarbamate linker.

[0296] In one embodiment, the IDO prodrug comprises the drug moiety of formula I and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0297] In one embodiment, the IDO prodrug comprises ID3 and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0298] In one embodiment, the PD1 prodrug comprises ID3 and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0299] The IDO prodrugs, liposomes, and co-formulated liposomes of this disclosure inhibit the activity of IDO-1 protein / protein interactions and are therefore useful in the treatment of diseases and disorders related to IDO-1 activity, as well as diseases and disorders related to the kynurenine pathway, including interactions with other proteins such as IDO-2 and TDO. In further embodiments of this disclosure, IDO 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.

[0300] In further embodiments, the Disclosure provides a method for inhibiting IDO-1 T cell function. This method involves administering to an individual or patient an IDO prodrug, liposome, and / or any of the formulas described herein (e.g., ID3), or an IDO prodrug, liposome, or nano-encapsulated IDO inhibitor prodrug as described in any of the claims, or any pharmaceutically acceptable salt or stereoisomer thereof. The IDO prodrugs, liposomes, and nano-encapsulated IDO inhibitor prodrugs of the Disclosure may be used alone, in combination with other agents or therapies, or as immunostimulants or oncological immunostimulants for the treatment of diseases or disorders, including cancer and other diseases. Any of the IDO prodrugs, liposomes, and nano-encapsulated IDO prodrugs of the Disclosure, including any of their embodiments, may be used for the uses and methods described herein.

[0301] Furthermore, the IDO prodrugs, liposomes, and nano-encapsulated IDO inhibitor prodrugs of this disclosure inhibit the IDO-1 kynurenine pathway and / or T cell function, resulting in blockade of the IDO pathway. As is well known in the art, IDO is activated during tumorigenesis to help malignant cells evade eradication by the immune system. See MUNN et al., Trends in Immunology, 37(3)193-207 (2016).

[0302] In further embodiments, the disclosure provides in vivo treatment of individuals or patients using IDO prodrugs, liposomes, and nano-encapsulated IDO inhibitor prodrugs or salts or stereoisomers thereof to inhibit the growth of cancerous tumors.

[0303] IDO prodrugs, liposomes and nanoencapsulated IDO inhibitor prodrugs, or any of the formulas described herein (e.g., ID3), or any of the IDO prodrugs, liposomes and nanoencapsulated IDO inhibitor prodrugs, or salts or stereoisomers thereof described herein can be used to inhibit the growth of cancerous tumors.

[0304] Alternatively, the IDO prodrugs of this disclosure, liposomes and nanoencapsulated IDO prodrugs, or any of the formulas or claims described herein, and the compounds described herein (e.g., ID3), or salts or stereoisomers thereof, may be used in combination with other active agents or standard cancer treatments as described herein.

[0305] In a further embodiment, the Disclosure provides a method for inhibiting the proliferation of tumor cells in vitro. This method involves contacting tumor cells in vitro with the IDO prodrug, liposome and nano-encapsulated IDO inhibitor prodrug of the Disclosure, or any of the formulas described herein (e.g., ID3), or the IDO prodrug, liposome and nano-encapsulated IDO inhibitor prodrug, or salts or stereoisomers thereof as described in any of the claims and herein.

[0306] In further embodiments, the Disclosure provides a method for inhibiting the proliferation of tumor cells in a patient. This method involves contacting tumor cells with the IDO prodrugs, liposomes and nano-encapsulated IDO inhibitor prodrugs of the Disclosure, or any of the formulas described herein (e.g., ID3), or the IDO prodrugs, liposomes and nano-encapsulated IDO inhibitor prodrugs described herein, or salts or stereoisomers thereof, as described in any of the claims. XI.) Methods for treating cancer and other immune disorders

[0307] Another embodiment of the present disclosure is a method for treating cancer. This method involves administering to a patient a therapeutically effective amount of liposomes containing an IDO prodrug as described herein (i.e., ID3), a compound as described in any of the claims, or a salt thereof. Examples of cancers include cancers whose growth can be inhibited using the IDO inhibitors and IDO prodrugs of the present disclosure, as well as cancers that typically respond to immunotherapy.

[0308] In some embodiments, the present disclosure provides methods for enhancing, stimulating, and / or increasing the immune response in a patient. The methods include administering to a patient a therapeutically effective amount of liposomes containing an IDO prodrug and / or therein (i.e., ID3), a compound or composition as described in any of the claims and / or herein, or a salt thereof.

[0309] Non-limiting examples of cancers treatable with liposomes containing the IDO prodrug of this disclosure, IDO prodrugs, and co-formulated liposomes include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer. Examples of cancers that may be treated include, but are not limited to, cancers of the tract, cancers of the penis, acute myeloid leukemia, chronic myeloid leukemia, chronic or acute leukemia including acute lymphoblastic leukemia and chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, cancers of the bladder, cancers of the kidney or urethra, carcinomas of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, axial tumors of the spinal cord, gliomas of the brainstem, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of the said cancers. The compounds of this disclosure are also useful for treating metastatic cancers, particularly metastatic cancers that express IDO-1.

[0310] In some embodiments, cancers treatable with the liposomes or IDO 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, colorectal cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), squamous cell head and neck cancer, urothelial carcinoma (e.g., bladder cancer), and cancers with high microsatellite instability (MSI). high ) are included. Furthermore, this disclosure includes refractory or recurrent malignancies whose growth can be inhibited using the liposomes of this disclosure or IDO prodrugs or co-formulated liposomes.

[0311] In further embodiments, cancers treatable with the formulated and / or co-formulated liposomes or IDO prodrugs of the Disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colorectal cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancers, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., lymphoma, leukemias such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin lymphoma or multiple myeloma), and combinations of the said cancers.

[0312] In further embodiments, cancers treatable with the formulated and / or co-formulated liposomes or IDO prodrugs of this disclosure include, but are not limited to, cholangiocarcinoma, 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 tumor, hairy cell leukemia, intestinal cancer, islet cell carcinoma, oral cancer, mouth cancer, throat cancer, laryngeal cancer, lip cancer, mesothelioma, cervical cancer, nasal cavity cancer, eye cancer, ocular 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.

[0313] Furthermore, in some embodiments, the formulated and / or co-formulated liposomes or IDO prodrugs of this disclosure can be used to treat sickle cell disease and sickle cell anemia.

[0314] Furthermore, in some embodiments, diseases and indications treatable with the formulated and / or co-formulated liposomes or IDO 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.

[0315] 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).

[0316] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyosarcoma, rhabdomyosarcoma, fibroma, lipoma, hamartoma, and teratoma.

[0317] Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinomas (squamous, undifferentiated small cell, undifferentiated large cell, and adenocarcinomas), alveolar (bronchiolar) carcinomas, bronchial adenomas, chondrotoxic hamartomas, and mesotheliomas.

[0318] Exemplary gastrointestinal cancers include cancers of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, VIP-producing tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma, leiomyoma), and colorectal cancer.

[0319] Exemplary genitourinary cancers include cancers of the kidney (adenocarcinoma, Wilms' tumor [nephroblastoma]), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), and testes (seminocarcinoma, teratoma, embryonic carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma).

[0320] Typical liver cancers include hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0321] Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (osteochondroma exostosoma), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid, and giant cell tumors.

[0322] Exemplary neurological cancers include cancers of the skull (osteoma, hemangioma, granuloma, xanthomas, osteoosteitis), meninges (meningioma, meningiosarcoma, gliomas), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal glandoma), glioblastoma, glioblastoma multiforme, oligodendroglioma, Schwann cell tumor, retinoblastoma, congenital tumors), and spinal cord (neurofibroma, meningioma, glioma, sarcoma), as well as neuroblastoma and Lhermitt-Dukuro disease.

[0323] Exemplary gynecological cancers include cancers of the uterus (endometrial cancer), cervix (cervical cancer, preneoplastic cervical dysplasia), ovaries (ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-ennucleus cell tumor, Sertoli-Leydig cell tumor, undifferentiated germ cell tumor, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, staphylosarcoma (embryonic rhabdomyosarcoma)), and fallopian tubes (carcinoma).

[0324] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevus moles, lipomas, hemangiomas, dermatofibromas, and keloids. 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.

[0325] Furthermore, the formulations and / or co-formulated liposomes of this disclosure, or the blockade of the IDO-1 and / or kynurenine pathway by IDO prodrugs, can also be used to treat infections such as viral, bacterial, fungal, and parasitic infections.

[0326] This disclosure provides a method for treating infectious diseases, such as viral infections. The method comprises administering to a patient a therapeutically effective amount of a formulated and / or co-formulated liposome or IDO prodrug as described in any of the claims and / or herein, or any of the formulas herein (i.e., ID3), or a salt thereof.

[0327] 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.

[0328] Furthermore, this disclosure provides a method for treating a bacterial infection. This method comprises administering to a patient a therapeutically effective amount of a formulated and / or co-formulated liposome or IDO prodrug as described in any of the claims and / or herein, or any of the formulas herein (i.e., ID3), or a salt thereof.

[0329] Examples of pathogenic bacteria that cause infections treatable by the methods of this disclosure include, but are not limited to, Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Neisseria gonorrhoeae (conococci), Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria.

[0330] Furthermore, this disclosure provides a method for treating fungal infections. This method comprises administering to a patient a therapeutically effective amount of a formulated and / or co-formulated liposome or IDO prodrug as described in any of the claims and / or herein, or any of the formulas herein (i.e., ID3), or a salt thereof.

[0331] 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.), the genera Mucorales (Mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0332] Furthermore, this disclosure provides a method for treating parasitic infections. This method comprises administering to a patient a therapeutically effective amount of a formulated and / or co-formulated liposome or IDO prodrug as described in any of the claims and / or herein, or any of the formulas herein (i.e., ID3), or a salt thereof.

[0333] Examples of pathogenic parasites that cause infections 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.

[0334] In a further series of embodiments within the scope of this disclosure, formulated and / or co-formulated liposomes, or IDO prodrugs, or any of the formulas described herein (i.e., ID3) are 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, symptom, or disorder but have not yet experienced or shown the pathophysiology or overall symptoms of the disease. XII.) Kits / Manufactured Products

[0335] For use in the laboratory, prognostic, prophylactic, diagnostic, and therapeutic applications described herein, the kits are within the scope of the invention. Such kits may include a carrier, package, or container partitioned to receive one or more containers, such as vials or tubes, each container containing one of the distinct elements to be used in the method, along with a label or insert containing instructions for use, such as the uses described herein. For example, a container may contain formulated and / or co-formulated liposomes that are detectably labeled or may be detectably labeled and / or loaded with the IDO 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 liposomes and / or the IDO prodrug.

[0336] The kit of the present invention typically includes the above-mentioned container and one or more other containers accompanying the above-mentioned container, which contain equipment desirable from a commercial and user perspective, such as buffers, diluents, filters, needles, syringes, etc., labels for carriers, packages, containers, vials, and / or tubes that list the contents and / or instructions for use, and an accompanying document with instructions for use.

[0337] Labels may be present on or with the container to indicate that the composition is used for a specific therapeutic or non-therapeutic use, such as prognosis, prevention, diagnosis, or laboratory use, and may also indicate instructions for in vivo or in vitro use, such as those described herein. Instructions and other information may also be included with or on inserts or labels contained in the kit. Labels may be present on or associated with the container. If the letters, numbers, or other characters forming the label are molded or engraved onto the container itself, the label may be present on the container; if the label is present in a receptacle or carrier that also holds the container, for example as an accompanying document, the label may be associated with the container. Labels may indicate that the composition is used to diagnose, treat, prevent, or prognose conditions such as cancer or other immune disorders.

[0338] The terms "kit" and "manufactured product" can be used as synonyms.

[0339] In another embodiment of the present invention, a product containing a composition such as formulated and / or co-formulated liposomes and / or IDO prodrugs is within the scope of the present disclosure. The product typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be formed from a variety of materials such as glass, metal or plastic. The container can hold formulated and / or co-formulated liposomes loaded with IDO prodrugs.

[0340] Alternatively, the container may hold a composition effective for treating, diagnosing, prognosing, or preventing a symptom and may have a sterile access port (for example, the container may be a vial with a stopper that can be punctured by an intravenous solution bag or a subcutaneous needle). The active ingredients in the composition may be formulated and / or co-formulated liposomes loaded with the IDO prodrugs disclosed herein, as well as the IDO prodrugs themselves.

[0341] 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 equipment desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes, and / or accompanying documentation with instructions and / or explanations for use.

[0342] Exemplary Embodiments The embodiments provided include the following: 1) An IDO prodrug composition, wherein the IDO prodrug composition is (i) The drug portion and (ii) The lipid portion, (iii) Connecting unit ("LU") and Includes, An IDO prodrug composition in which the drug portion comprises an IDO antagonist, and the LU conjugates the drug portion with the lipid portion.

[0343] 2) The IDO prodrug according to claim 1, further comprising the chemical structure described in formula I.

[0344] 3) The IDO prodrug according to claim 1, wherein the drug portion comprises a chemical structure represented as ID3.

[0345] 4) The IDO prodrug according to claim 1, wherein the LU is a hydromethylcarbamate linker.

[0346] 5) The IDO prodrug according to claim 1, wherein the lipid portion comprises the lipids listed in Table I.

[0347] 6) The IDO prodrug according to claim 1, wherein the lipid portion comprises the lipids listed in Table III.

[0348] 7) The IDO prodrug according to claim 1, wherein the lipid portion comprises CHEMS.

[0349] 8) The IDO prodrug according to claim 1, wherein the lipid portion comprises stearic acid.

[0350] 9) An IDO prodrug composition, wherein the IDO prodrug composition is (i) A drug portion comprising a drug portion including ID3, (ii) The lipid portion, which includes a lipid portion containing CHEMS, (iii) LU comprising a hydromethylcarbamate linker An IDO prodrug composition containing the following:

[0351] 10) An IDO prodrug composition, wherein the IDO prodrug composition is (i) A drug portion comprising a drug portion including ID3, (ii) The lipid portion, which includes a lipid portion containing stearic acid, (iii) LU comprising a hydromethylcarbamate linker An IDO prodrug composition containing the following:

[0352] 11) A liposome comprising an IDO prodrug, wherein the liposome releases an active IDO inhibitor after cleavage of the LU.

[0353] 12) The liposome according to claim 11, wherein the LU is a hydromethylcarbamate linker.

[0354] 13) The liposome according to claim 11, further comprising a helper lipid, the helper lipid being shown in Table II.

[0355] 14) The liposome according to claim 11, wherein the IDO prodrug comprises ID3.

[0356] 15) The liposome according to claim 11, wherein the liposome is further co-formulated with an immunomodulator, the immunomodulator is selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, Toll receptor agonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors and / or prodrugs thereof.

[0357] 16) The liposome according to claim 11, wherein the liposome is further co-formulated with an ICD-inducing chemotherapeutic agent, and the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfildimib, or paclitaxel.

[0358] 17) The liposome according to claim 11, further comprising DOX.

[0359] 18) The liposome according to claim 11, further comprising MTO.

[0360] 19) The liposome according to claim 11, further comprising DOX.

[0361] 20) The liposome according to claim 11, further comprising MTO.

[0362] 21) A kit comprising the liposomes described in claim 11.

[0363] 22) A kit comprising the liposomes described in claim 15.

[0364] 23) A kit comprising the liposomes described in claim 16.

[0365] 24) A method for treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of liposomes to a subject requiring such treatment, wherein the liposomes contain an IDO prodrug, (ii) the pharmaceutically acceptable salt, Methods that include...

[0366] 25) The method according to claim 24, wherein the IDO prodrug comprises ID3.

[0367] 26) The method according to claim 24, wherein the liposome further comprises ID3 co-formulated with an ICD-inducing chemotherapeutic agent.

[0368] 27) The method according to claim 24, wherein the liposome further comprises ID3 co-formulated with an immunomodulator.

[0369] 28) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with AR5.

[0370] 29) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with AR5 and TR5.

[0371] 30) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with NK1.

[0372] 31) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with NK1 and MTO.

[0373] 32) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with TR3.

[0374] 33) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with TR5.

[0375] 34) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with TB4.

[0376] 35) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with PD3.

[0377] 36) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with AR5 and TR3.

[0378] 37) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with AR5 and TB4.

[0379] 38) The liposome according to claim 11, wherein the IDO prodrug comprises ID3 co-formulated with AR5 and PD3. [Examples]

[0380] Various aspects of the present invention are further described and illustrated by the following embodiments, none of which are intended to limit the scope of the invention.

[0381] Example 1: General chemical synthesis of a protected ID3 prodrug containing cholesterol hemisuccinate ("CHEMS") The chemical synthesis of protected ID3 prodrugs containing CHEMS is performed using the following protocol. First, quinoline (1) and boronate (2) (BARLIND, JG et al., J. Med. Chem., 55:10610-10629 (2012)) were coupled with cesium carbonate and a PEPPSI-IPr catalyst in dioxane / water at 100°C to obtain intermediate 3. Intermediate 3 was then hydrogenated on palladium carbon to obtain intermediate 4. Intermediate 4 was then hydrolyzed with lithium hydroxide in aqueous ethanol at 50°C to obtain intermediate 5. Next, intermediate 5 was coupled with a chiral auxiliary reagent (6) in THF using triethylamine and pivaloyl chloride, and the diastereomer was separated to obtain intermediate 7. Next, intermediate 7 was alkylated with methyl iodide and NaHMDS in THF at -50°C to obtain intermediate 8. Next, in an aqueous THF solution at 0°C, the chiral additive was cleaved from intermediate 8 using LiOH and hydrogen peroxide to obtain intermediate 9. Then, in ethyl acetate, intermediate 9 was coupled with aniline (10) using polyphosphonic anhydride and pyridine to obtain intermediate 11. Next, intermediate 11 was treated with reagent (12) in THF, and then treated with sodium iodide to obtain intermediate 13. Finally, intermediate 13 was treated with silver salt of CHEMS (14) under reflux in THF to obtain the final ID3 prodrug (Figure 1).

[0382] Example 2: Chemical synthesis of ID3 prodrug intermediate. The (if) chemical synthesis of the ID3 prodrug was carried out in the following manner. To a solution of the ID3 drug portion (6.00 g, 14.6 mmol, 1.00 equivalent) in THF (60 mL), LiHMDS (1 M, 29.2 mL, 2.00 equivalent) was added at -70°C, and the reaction mixture was stirred at -70°C for 0.5 hours. Then, a solution of compound 1 (3.24 g, 25.1 mmol, 2.23 mL, 1.72 equivalent) in THF (10 mL) was added to the mixture at -78°C, and the mixture was stirred for a further 1 hour at -78°C. TLC (petroleum ether:ethyl acetate = 2:1) was used to analyze the ID3 drug portion (R f =0.2) is consumed, and one major new spot (R fIt was shown that (= 0.6) was formed. The reaction mixture was poured into a saturated aqueous NH4Cl solution (150 mL) and extracted with ethyl acetate (100 mL × 2). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated to obtain crude compound 2 (7.60 g, unpurified) as a yellow oil. The obtained compound is shown in Figure 2.

[0383] Example 3: Alternative Chemical Synthesis of ID3 Prodrug Intermediate. In another experiment, an alternative chemical synthesis of the ID3 prodrug was carried out in the following manner. To a solution of the ID3 drug moiety (3.00 g, 7.30 mmol, 1.00 equiv) in THF (30.0 mL) was added LiHMDS (1 M, 14.6 mL, 2.00 equiv) at -70 °C. The reaction mixture was then stirred at -70 °C for 0.5 h. Then, a solution of compound 3a (6.57 g, 51.0 mmol, 4.53 mL, 6.98 equiv) in THF (15.0 mL) was added to the mixture at -70 °C and stirred for an additional 1 h. 1 1H NMR showed that the ID3 drug moiety was correct. LCMS showed that the drug moiety was completely consumed and the desired MS (RT = 0.877 min) was detected. The mixture was poured into a saturated solution of NH4Cl (200 mL) and extracted with ethyl acetate (200 mL × 3). The combined organics were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated to obtain a crude product. Compound 3 (4.00 g, unpurified) was obtained as a yellow oil. The obtained compound is shown in Figure 3.

[0384] Example 4: Chemical Synthesis of ID3 Prodrug Containing Stearic Acid In another experiment, an ID3 prodrug containing stearic acid was synthesized in the following manner: To a solution of stearic acid (3.39 g, 11.9 mmol, 4.01 mL, 0.80 equivalents) in DMF (400 mL), Ag2CO3 (6.16 g, 22.3 mmol, 1.01 mL, 1.50 equivalents) was added at 25°C. The reaction mixture was stirred at 25°C for 0.5 hours. Then, compound 2 (Figure 2) (7.50 g, 14.9 mmol, 1.00 equivalent) and NaI (3.35 g, 22.3 mmol, 1.50 equivalents) were added to the mixture. After the addition, the reaction mixture was stirred for a further 12 hours at 80°C. LCMS indicated that the reaction was complete, and the desired mass (RT = 1.309 min) was detected. The reaction mixture was cooled to 25°C, filtered through a celite pad, and washed with DCM (300 mL). The filtrate was concentrated at 50°C to obtain the crude product. The crude product was purified by reverse-MPLC (TFA conditions), and then concentrated under reduced pressure to obtain the crude product. Column chromatography (SiO2, petroleum ether:ethyl acetate = 5:1~2:1, R f The crude product is purified by (=0.5), and then TLC (petroleum ether:ethyl acetate = 2:1, R f When detected by (=0.5, PMA), (2.36g, 3.09 mmol, yield 20.7%, purity 98.3%) is obtained as a yellow solid. 1 1H NMR, 19 The compounds were identified by 1F NMR, LCMS, HPLC, and SFC. The obtained compounds are shown in Figure 7.

[0385] Example 5: Chemical synthesis of an ID3 prodrug containing cholesterol hemisuccinate ("CHEMS"). In another experiment, an ID3 prodrug containing cholesterol hemisuccinate ("CHEMS") was synthesized in the following manner. Briefly, a mixture of cholesterol hemisuccinate (5.80 g, 11.9 mmol, 1.50 equivalents) and Ag2CO3 (3.29 g, 11.9 mmol, 540 uL, 1.50 equivalents) in DMF (15.0 mL) was stirred at 25°C for 0.5 hours. Compound 3 (Figure 3) (4.00 g, 7.95 mmol, 1.00 equivalent) and NaI (1.43 g, 9.54 mmol, 1.20 equivalents) in DMF (15.0 mL) were added to the mixture at 25°C. The mixture was stirred at 80°C for 16 hours. LC-MS showed that compound 3 was completely consumed, and the desired MS (RT=1.286 min) was detected. The mixture was filtered and the filtrate was collected. The filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 10 to 1 / 4), and the major spot (petroleum ether / ethyl acetate = 2 / 1, R f A compound with a concentration of 0.4 was obtained. The unpurified compound (target 2) (3.00 g, 3.15 mmol, yield 39.6%) was obtained as a pale yellow solid. The obtained compound is shown in Figure 8.

[0386] Example 6: Synthesis and characterization of LNP-ID3 liposomes In another experiment, liposomes containing the ID3 prodrug (denoted as LNP-ID3) were synthesized as follows. Briefly, raw solutions of each lipid component of LNP-ID3 were prepared in ethanol at a concentration of 20 mg / ml. Therefore, ethanolic stock solutions of Hydro Soy PC (HSPC: L-α-phosphatidylcholine, hydrogenated (soybean)), cholesterol, and DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) were prepared at the above concentrations. A stock solution of the ID3 prodrug (20 mg / ml) was prepared in acetonitrile. Each lipid component (HSPC, CHOL, DSPE-PEG, and ID3 prodrug) was mixed in a molar ratio of 51:27:17:5 to synthesize LNP-ID3. The size of liposome LNP-ID3 depends on various parameters such as (i) flow rate, (ii) temperature, and (iii) concentration of the lipid mixture. The optimized ratio of the lipid mixture in a molar ratio of 51:27:17:5 was preheated at 50°C. Microfluidics cartridge (Precision) was used at a flow rate of 3:1 (aqueous phase:organic phase, lipid mixture). The aqueous phase containing 1 mM PBS buffer was preheated to 50°C before passing through (NanoSystems, Inc.). The solvent was removed for at least 24 hours using a dialysis membrane (Sigma Aldrich) that removes 12 kDa size particles from the DI water. To optimize solvent removal, the dialysate was changed at least 5 times during the 24-hour period. After solvent removal, LNP-ID3 was concentrated using an Amicon centrifugal filter (size 10 kDa, 3000 g).

[0387] The properties of LNP-ID3 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-ID3 liposomes (liposome concentration of 0.5–1 mg / ml) were placed in a four-sided transparent plastic cuvette and directly analyzed at 25°C. The results shown in Figure 9 indicate that the Zav size of the nanoparticles was approximately 80 nm and the PDI was approximately 0.203.

[0388] Furthermore, the zeta potential of LNP-ID3 liposomes in an aqueous dispersion was determined using the Malvern zeta seizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) were placed in a disposable capillary zeta potential cell available for use with the zeta seizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-ID3 was determined to be approximately -11.6 mV (Figure 10).

[0389] Example 7: Synthesis and characterization of LNP-AR5-ID3 liposomes In another experiment, liposomes containing the ID3 prodrug and the A2a receptor inhibitor prodrug (AR5) (denoted as LNP-AR5-ID3) were synthesized and their properties determined as follows. Briefly, the mixture was synthesized using a molar ratio of 52:27:8:8:5 for HSPC:CHOL:AR5:ID3:DSPE-PEG, with the AR5:ID3 molar ratio fixed at 1:1. The optimized ratio of the lipid mixture at the 52:27:8:8:5 molar ratio was preheated at 55-60°C. The final concentration of the lipid mixture was 2.5 mg / ml. The aqueous phase containing 1 mM PBS buffer was also preheated at 55-60°C before passing it through a microfluidics cartridge (Precision NanoSystems, Inc.) at a flow rate of 3:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed for a minimum of 24 hours using a dialysis membrane (Sigma Aldrich) that removes 12 kDa size particles relative to DI water. To optimize solvent removal, the dialysate was changed at least five times during a 24-hour period. After solvent removal, LNP-AR5-ID3 was concentrated using an Amicon centrifugal filter (cutoff size 10 kDa, 3000 g).

[0390] The properties of LNP-AR5-ID3 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-AR5-ID3 liposomes (liposome concentration of 0.5–1 mg / ml) were placed in a four-sided transparent plastic cuvette and analyzed directly at 25°C. The results shown in Figure 11 indicate that the Zav size of the nanoparticles was approximately 91 nm and the PDI was approximately 0.212.

[0391] Furthermore, the zeta potential of LNP-AR5-ID3 liposomes in an aqueous dispersion was determined using the Malvern zeta seizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) were placed in a disposable capillary zeta potential cell available for use with the zeta seizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-AR5-ID3 was approximately -14.1 mV (Figure 12).

[0392] Example 8: Synthesis and characterization of LNP-AR5-TR5-ID3 liposomes In another experiment, liposomes containing ID3 prodrug, A2a receptor inhibitor prodrug (AR5), and telluratrimod (TR5) (denoted as LNP-AR5-TR5-ID3) were synthesized and their properties determined as follows: Briefly, lipid stock solutions of HSPC, CHOL, DSPE-PEG, and AR5 prodrug were prepared. TR5 was prepared separately in ethanol (20 mg / ml). The ID3 prodrug stock solution was also prepared in acetonitrile (20 mg / ml). Using a molar ratio of 51.6:27:7:2.4:7:5, HSPC:CHOL:AR5:TR5:ID3:DSPE-PEG was synthesized. The optimized ratio of the lipid mixture at the molar ratio of 51.6:27:7:2.4:7:5 was preheated at 55-60°C. The final concentration of prodrug AR5 in the lipid mixture was 0.625 mg / ml. Before passing the 4.5:1 (aqueous phase:organic phase, lipid mixture) through a microfluidics cartridge (Precision NanoSystems, Inc.), the aqueous phase containing 1 mM PBS buffer was preheated to 55-60°C. The solvent was removed for a minimum of 24 hours using a dialysis membrane (Sigma Aldrich) that removes 12 kDa size particles from the DI water. To optimize solvent removal, the dialysate was changed at least 5 times during the 24-hour period. After solvent removal, LNP-AR5-TR5-ID3 was concentrated using an Amicon centrifugal filter (cutoff size 10 kDa, 3000 g).

[0393] The properties of LNP-AR5-TR5-ID3 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-AR5-TR5-ID3 liposomes (liposome concentration of 0.5–1 mg / ml) were placed in a four-sided transparent plastic cuvette and analyzed directly at 25°C. The results shown in Figure 13 indicate that the Zav size of the nanoparticles was approximately 96 nm and the PDI was approximately 0.117.

[0394] Furthermore, the zeta potential of LNP-AR5-TR5-ID3 liposomes in an aqueous dispersion was determined using the Malvern zeta seizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) were placed in a disposable capillary zeta potential cell available for use with the zeta seizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-AR5-TR5-ID3 was approximately -20.5 mV (Figure 14).

[0395] Example 9: Synthesis and characterization of LNP-ID3-NK1 liposomes In another experiment, liposomes containing the ID3 prodrug and α-galactosylceramide (α-GalCer) (NK1) (denoted as LNP-ID3-NK1) were synthesized and their properties determined as follows. Briefly, a stock solution of the ID3 prodrug (20 mg / ml) was prepared in acetonitrile, and a stock solution of NK1 (10 mg / ml) was prepared in DSMO. A lipid mixture of HSPC, CHOL, ID3, NK1, and DSPE-PEG was prepared in a molar ratio of 51:29:16:0.085:4, and all lipid stock solutions were mixed in appropriate amounts, then further diluted with ethanol to obtain a lipid concentration of 10 mg / ml. This lipid mixture was preheated at 50°C using a microfluidizer. Similarly, the aqueous phase containing 1 mM PBS buffer was preheated at 50°C before passing it through a microfluidics cartridge (Precision NanoSystems, Inc.) at a flow rate of 3:1 (aqueous phase:organic phase, lipid mixture). Therefore, the molar ratio of ID3:NK1 remained at 12:1. The solvent was removed for a minimum of 24 hours using a dialysis membrane (Sigma Aldrich) that removes 12 kDa size particles from the DI water. To optimize solvent removal, the dialysate was changed at least 5 times during the 24-hour period. After solvent removal, LNP-ID3-NK1 was concentrated using an Amicon centrifugal filter (cutoff size 10 kDa, 3000 g).

[0396] The properties of LNP-ID3-NK1 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-ID3-NK1 liposomes (liposome concentration of 0.5–1 mg / ml) were placed in a four-sided transparent plastic cuvette and analyzed directly at 25°C. The results shown in Figure 15 indicate that the Zav size of the nanoparticles was approximately 80 nm and the PDI was approximately 0.189.

[0397] Furthermore, the zeta potential of LNP-ID3-NK1 liposomes in an aqueous dispersion was determined using the Malvern zeta seizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) were placed in a disposable capillary zeta potential cell available for use with the zeta seizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-ID3-NK1 was approximately -13.1 mV (Figure 16).

[0398] Example 10: Synthesis and characterization of mitoxantrone-loaded LNP-ID3-NK1 liposomes In another experiment, liposomes containing mitoxantrone (MTO) were loaded with LNP-ID3-NK1 (denoted as LNP-ID3-NK1-MTO) and synthesized and their properties determined as follows. Briefly, a stock solution of MTO (0.6 mg / ml) was prepared in DI water. 10 ml of this stock solution was added to 20 ml of LNP-ID3-NK1 formulation containing 300 mM ammonium sulfate solution and incubated at 40°C for 2 hours. After the 2-hour incubation period, the entire liposome solution containing MTO was dialyzed (12 kDa dialysis membrane, 8-hour dialysis time) to remove free MTO (i.e., not incorporated into the liposomes). After dialysis, the MTO-loaded LNP-ID3-NK1 (LNP-ID3-NK1-MTO) was concentrated using an Amicon centrifugal filter (cutoff size 10 kDa, 2500 g).

[0399] The properties of LNP-ID3-NK1-MTO liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of LNP-ID3-NK1-MTO liposomes (liposome concentration of 0.5–1 mg / ml) were placed in a four-sided transparent plastic cuvette and analyzed directly at 25°C. The results shown in Figure 17 indicate that the Zav size of the nanoparticles was approximately 90 nm and the PDI was approximately 0.084.

[0400] Furthermore, the zeta potential of LNP-ID3-NK1-MTO liposomes in an aqueous dispersion was determined using the Malvern zeta seizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) were placed in a disposable capillary zeta potential cell available for use with the zeta seizer. Measurements were performed at 25°C. The results show that the zeta potential of LNP-ID3-NK1-MTO was determined to be approximately -11.6 mV (Figure 18).

[0401] Example 11: Determination of LNP-ID3-NK1-MTO liposome encapsulation and loading efficiency. In another experiment, the encapsulation and loading efficiency of LNP-ID3-NK1-MTO liposomes was determined by disintegrating the liposomes and measuring MTO using UV-vis spectroscopy as follows. Briefly, free MTO was separated by dialysis, and then the liposomes were disintegrated with dimethyl sulfoxide. The capture efficiency was determined using the following formula. Encapsulation efficiency (%) = [MTO] f / [MTO] t ×100 In the formula, [MTO] f This is the concentration of MTO encapsulated in LNP-ID3-NK1, and [MTO] tThis represents the total MTO concentration (total amount of MTO initially added: meaning loaded + free). The MTO concentration was then determined using a Nanodrop 2000C UV-vis spectrophotometer. The capture efficiency of MTO into LNP-ID3-NK1-MTO was found to be approximately 94%. Furthermore, the loading efficiency of MTO in LNP-ID3-NK1-MTO was determined using the following formula. Load efficiency (%) = [MTO] fwt / [LNP-ID3-NK1-MTO] fwt ×100 [MTO] fwt This is the total weight of the MTO sealed inside the MTO, [LNP-ID3-NK1-MTO] fwt This is the total weight of LNP-ID3-NK1 (LNP-ID3-NK1-MTO) containing MTO. The load efficiency of MTO in LNP-ID3-NK1-MTO was found to be approximately 4.1% w / w.

[0402] Example 12: Tumor inhibition of LNP-ID3 in combination with other liposomes using B16F10 cells in vivo The evaluation of LNP-ID3 in combination with various liposomes described herein was performed using the following protocol. In short, mouse melanoma carcinoma B16F10 cells (cells (0.2 × 10) 6LNP-MTO (liposomal mitoxantrone dihydrochloride) was subcutaneously inoculated into the right posterior flank region of C57BL / 6 mice. Animals were treated twice weekly by intravenous injection with the following: vehicle control, 3 mg / kg LNP-MTO (liposomal mitoxantrone dihydrochloride), 3 mg / kg liposomal LNP-ID3 and LNP-TB4 combination (TGF-β inhibitor - stearic acid), 3 mg / kg LNP-ID3 and LNP-TR6 combination (liposomal TLR1 / 2 agonist - CHEMS), and 3 mg / kg LNP-MTO, LNP-ID3, and LNP-TB4 combination. Tumor volume was measured three times in two dimensions using calipers, and the volume was calculated using the formula: V = (L × W × W) × 0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on tumor size data from day 16.

[0403] The results showed that LNP-MTO monotherapy demonstrated antitumor activity, with a TGI of 32.56% compared to the vehicle group (p<0.05). Furthermore, combination therapy with LNP-TB4 + LNP-MTO + LNP-ID3 showed significantly greater antitumor activity compared to the vehicle group, with a TGI of 32.86% (all p<0.05). (Figure 19).

[0404] Example 13: Tumor inhibition of LNP-ID3 in combination with LNP-AR5 using B16F10 cells in vivo In another experiment, LNP-ID3 was evaluated in combination with LNP-AR5 using the following protocol. In short, mouse melanoma cancer B16F10 cells (cells (0.2 × 10) 6LNP-AR5 was subcutaneously inoculated into the right posterior flank region of C57BL / 6 mice. Animals were treated twice weekly by intravenous injection with vehicle control, 3 mg / kg LNP-AR5, and a combination of 3 mg / kg LNP-AR5 and LNP-ID3. Tumor volume was measured three times in two dimensions using calipers, and the volume was calculated using the formula: V = (L × W × W) × 0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 15.

[0405] The results showed that the combination therapy of LNP-AR5 + LNP-ID3 had significantly mild antitumor activity compared to the vehicle group, with a TGI of 35.89% (all p<0.05). (Figure 20).

[0406] Example 14: In vivo tumor inhibition of LNP-ID3-NK1 using B16F10 cells. In another experiment, LNP-ID3-NK1 was evaluated using the following protocol. In short, mouse melanoma cancer B16F10 cells (0.2 × 10⁶ cells) 6 The drug was subcutaneously inoculated into the right posterior flank region of C57BL / 6 mice. Animals were treated twice weekly by intravenous injection with the following treatments: vehicle control, 2 mg / kg of LNP-MTO (mitoxantrone dihydrochloride in liposomal form), 3 / 0.25 mg / kg of LNP-ID3-NK1 (in liposomal form in a 1:12 ratio), a combination of 3 mg / kg of LNP-MTO and 3 / 0.25 mg / kg of LNP-ID3-NK1, and a combination of 3 / 0.25 mg / kg of LNP-ID3-NK1 and 5 mg / kg of PD3 (PD-1 receptor inhibitor cholesterol in liposomal form). Tumor volume was measured three times in two dimensions using calipers, and the volume was calculated using the formula: V = (L × W × W) × 0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on the tumor size data on day 18.

[0407] The results showed that treatment with LNP-ID3-NK1, LNP-MTO+LNP-ID3-NK1, and LNP-ID3-NK1+LNP-PD3 significantly inhibited tumor growth compared to the vehicle group, with TGI calculated to be 42.71% to 54.31%, respectively (p<0.05) (Figure 21).

[0408] Example 15: In vivo tumor inhibition of LNP-ID3 and LNP-ID3-NK1 using CT26 cells. In another experiment, LNP-ID3 and LNP-ID3-NK1 were evaluated using the following protocol. Briefly, mouse colorectal carcinoma CT26 cells (0.1 × 10⁶ cells) were subcutaneously inoculated into the right posterior flank region of Balb / C mice. The regimens included vehicle control, 3 mg / kg LNP-DOX (doxorubicin in liposomal form), 3 mg / kg LNP-ID3, 0.25 mg / kg LNP-NK1, a combination of 3 mg / kg LNP-DOX and LNP-ID3, 3 / 0.25 mg / kg LNP-ID3-NK1 (1:12 ratio), and 3 mg / kg LNP-DOX and 3 / 0.25 mg / kg LNP-ID3-NK1. Animals were treated twice a week by intravenous injection with the NK1 combination. Tumor volume was measured three times in two dimensions using calipers, and the volume was calculated using the formula: V = (L × W × W) × 0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). The study was terminated when the mean tumor size of the vehicle-treated group exceeded 2500 mm³ (27 days after tumor inoculation). Tumor growth inhibition (TGI) was calculated based on the tumor size data at 27 days.

[0409] The results showed that treatment with LNP-DOX, LNP-DOX+LNP-ID3, and LNP-DOX+LNP-ID3-NK1 significantly induced TGI compared to the vehicle group (p<0.01). TGI was recorded in 91%, 80%, and 89.2% of patients, respectively (Figure 22).

[0410] Example 16: In vivo tumor inhibition of LNP-ID3 using CT26 cells. In another experiment, LNP-ID3 was evaluated using the following protocol: mouse colon cancer CT26 cells (0.1 × 10⁶ cells). 6 The drug was subcutaneously inoculated into the right posterior ventral region of Balb / C mice. Animals were treated twice weekly by intravenous injection with the following treatments: vehicle control, 3 mg / kg LNP-DOX (liposomal doxorubicin), 3 mg / kg LNP-ID3, a combination of 3 mg / kg LNP-DOX and LNP-ID3, a combination of 3 mg / kg LNP-ID3 and 0.25 mg / kg LNP-NK1, a combination of 3 mg / kg LNP-ID3 and LNP-TR6, a combination of 3 mg / kg LNP-ID3 and 4 mg / kg LNP-TR5, and 3 mg / kg LNP-ID3 and 2 mg / kg LNP-TR8 (liposomal 3D-6-acyl-PHAD). Tumor volume was measured three times in two dimensions using calipers, and the volume was calculated using the formula: V = (L × W × W) × 0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L).

[0411] The results indicate that LNP-DOX alone or in combination with LNP-ID3 provided a therapeutic benefit by significantly extending the survival of CT26 tumor-bearing mice (Figure 23).

[0412] Example 17: In vivo tumor inhibition of LNP-ID3 using MC-38 cells. In another experiment, LNP-ID3 was further evaluated using the following protocol: mouse colorectal adenocarcinoma MC-38 cells (1 × 10⁶ cells) 6LNP-DOX was subcutaneously inoculated into the right posterior flank region of C57BL / 6 mice. Animals were treated twice weekly by intravenous injection with the following regimens: vehicle control, combination, 4 mg / kg LNP-DOX (liposomal doxorubicin), 10 mg / kg anti-PD1 antibody, 4 mg / kg LNP-ID3 and LNP-AR5 combination, 4 mg / kg LNP-DOX + LNP-ID3 + LNP-AR5 combination, and for the first dose, 3.5 mg / kg LNP-ID3, 3.5 mg / kg LNP-AR5 and 4 mg / kg LNP-TR5 combination, and for the remaining doses, 2 mg / kg. Animals were administered LNP-DOX only twice, then replaced with vehicle control. Tumor volume was measured three times in two dimensions using calipers, and the volume was calculated using the formula: V = (L × W × W) × 0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on the tumor size data on day 19.

[0413] The results showed that treatment with LNP-DOX, LNP-DOX+LNP-ID3+LNP-AR5, and LNP-ID3+LNP-AR5 demonstrated substantial TGI compared to the vehicle group (p<0.05). TGI was recorded in 74.39%, 90.40%, and 73.77%, respectively (Figure 24).

[0414] Example 18: Tumor inhibition of LNP-ID3 in vivo using H22 cells in combination with LNP-AR5. In another experiment, LNP-ID3 was evaluated in combination with LNP-AR5 using the following protocol. In short, mouse hepatocellular carcinoma H22 cells (2 × 10⁶ cells) 6The LNP-ID3+LNP-AR5 combination was subcutaneously inoculated into the right posterior flank region of Balb / C mice. Animals were treated twice weekly by intravenous injection with a vehicle control and a combination of 4 mg / kg of LNP-ID3 and LNP-AR5. Tumor volume was measured three times in two dimensions using calipers, and the volume was calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 14. The results show that LNP-ID3+LNP-AR5 induced TGI compared to the vehicle. TGI was recorded at 43.28% (Figure 25).

[0415] Example 19: Measurement of IDO-1 activity. IDO-1 activity was indirectly measured using kynurenine levels with the following protocol. Briefly, hIDO1-HEK293 recombinant cells were seeded overnight. The following day, the cells were left untreated or treated with 10 μM ID3, ID3-SA, or LNP-ID3-SA for 24 hours. IDO-1 activity was measured indirectly by kynurenine levels, which were determined by analyzing absorption at 480 nm using the IDO1 Cellular Activity QuickDetect® kit (PBSBioscience, San Diego, CA).

[0416] The results show that untreated cells exhibited high levels of IDO-1 activity, while treatment with ID3, ID3-SA, and LNP-ID3-SA inhibited IDO-1 activity. These results confirm ID3 activity in prodrug and liposome forms (Figure 26).

[0417] Example 20: Synthesis and property determination of SLNP-AR5-TR5-ID3 solid-lipid nanoparticles In another experiment, solid-lipid nanoparticles ("SLNPs") containing ID3-AR5-TR5 (denoted as SLNP-ID3-AR5-TR5) were prepared using Moliwol 488 (polyvinyl alcohol) as an emulsifier, following the protocol below. Briefly, lipid stock solutions of HSPC, CHOL, DSPE-PEG, and TR5 were prepared separately in ethanol (20 mg / ml). The ID3 prodrug stock solution was prepared in acetonitrile (20 mg / ml). The lipid mixture of HSPC, CHOL, AR5, TR5, ID3, and DSPE-PEG in a molar ratio of 51.6:27:7:2.4:7:5 was mixed together and then diluted with ethanol to obtain a lipid concentration of 5 mg / ml. This lipid mixture was heated at 50°C using a heating block attachment in a microfluidizer. Similarly, the aqueous phase containing 2% Moilwol 488 solution was preheated to 50°C before passing it through a microfluidics cartridge (Precision NanoSystems, Inc.) at a flow rate of 4:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed from the DI water for at least 24 hours using a dialysis membrane (Sigma Aldrich) with a cutoff size of approximately 12 kDa. To maximize solvent removal, the dialysate was changed at least 5 times during the 24-hour period. After solvent removal, the SLNPs were passed through a 0.2 micron filter membrane (cellulose acetate). SLNP-AR5-TR5-ID3 was concentrated using an Amicon centrifugal filter (cutoff size 10 kDa, 3000 g).

[0418] The properties of SLNP-AR5-ID3-TR5 solid-lipid nanoparticles were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-AR5-ID3-TR5 (concentration 1 mg / ml) was placed in a four-sided transparent plastic cuvette and analyzed directly at 25°C. The results shown in Figure 27 indicate that the nanoparticle Zav size was approximately 106 nm and the PDI was approximately 0.171.

[0419] Furthermore, the zeta potential of SLNP-AR5-ID3-TR5 solid-lipid nanoparticles in an aqueous dispersion was determined using the Malvern zeta seizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of the nano formulation (DI concentration in water approximately 3 mg / ml) was placed in a disposable capillary zeta potential cell available for use with the zeta seizer. Measurements were performed at 25°C. The results show that the zeta potential of SLNP-AR5-ID3-TR5 was determined to be approximately 9.87 mV (Figure 28).

[0420] Example 21: Human clinical trial for the treatment of human cancer using formulated and / or co-formulated liposomes containing an IDO prodrug. The present invention utilizes formulated and / or co-formulated liposomes containing an IDO prodrug, which specifically accumulates in tumor cells and is used in the treatment of certain tumors and other immunodisorders and / or other diseases. In relation to each of these indications, two clinical approaches are successfully carried out.

[0421] I.) Adjuvant therapy: In adjuvant therapy, patients are treated with formulated and / or co-formulated liposomes containing an IDO prodrug in combination with a chemotherapeutic agent or pharmaceutical or biopharmaceutical factor or a combination thereof. Primary cancer targets are treated under standard protocols by adding formulated and / or co-formulated liposomes containing an IDO prodrug. The protocol design addresses efficacy, which is assessed by cases including but not limited to reduction of tumor burden of primary or metastatic lesions, extension of progression-free survival, overall survival, improvement of the patient's health status, disease stabilization, and the ability to reduce the usual doses of standard chemotherapy and other biological agents. These dose reductions enable additional and / or long-term treatment by reducing the dose-related toxicity of the chemotherapeutic agent or biological agent.

[0422] II.) Monotherapy: With regard to the use of formulated and / or co-formulated liposomes containing IDO prodrugs in monotherapy for tumors, the formulated and / or co-formulated liposomes containing IDO prodrugs are administered to the patient without chemotherapeutic agents or pharmaceutical or biological factors. In one embodiment, monotherapy is clinically implemented in terminally ill cancer patients with widespread metastatic disease. The protocol design addresses efficacy, which is evaluated by examples including, but not limited to, reduction of tumor burden of primary or metastatic lesions, extension of progression-free survival, overall survival, improvement of the patient's health status, disease stabilization, and the ability to reduce the usual doses of standard chemotherapy and other biological agents. Dosage

[0423] The administration regimen may be adjusted to provide the optimal desired response. For example, a single formulated and / or co-formulated liposome containing the IDO 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. As used herein, “dosage unit form” refers to a physically separated unit suitable as a unit dose for the mammalian subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the dosage unit form of the present invention are determined by and directly depend on (a) the inherent characteristics of the formulated and / or co-formulated liposome containing the IDO prodrug, (b) the individual mechanisms of the co-combined compounds, if present, (c) the specific therapeutic or prophylactic effect to be achieved, and (d) the limitations inherent in the technique of formulating such compounds for the treatment of susceptibility in an individual. Clinical Development Program (CDP)

[0424] CDP will be developed in conjunction with treatments using formulated and / or co-formulated liposomes containing the IDO prodrug, either as adjuvant therapy or monotherapy. The trial will first demonstrate safety, and then confirm efficacy in repeated doses. The trial will be open-label and will compare standard chemotherapy and / or current standard treatment with the addition of formulated and / or co-formulated liposomes containing the IDO prodrug. As understood, one non-limiting criterion that may be available in connection with patient enrollment is the expression of IDO-1 in tumors, as determined by standard detection methods known in the art.

[0425] 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, such as cytotoxicity in cells or inhibition of a particular target or channel to determine potential toxicity, is within the scope of the knowledge of those skilled in the art.

[0426] The present invention should not be limited in scope by the embodiments disclosed herein, which are intended as single examples of individual aspects of the invention, and any functionally equivalent is within the scope of the invention. In addition to those described herein, various modifications to the models, methods, and lifecycle methods of the invention are intended to be obvious to those skilled in the art from the foregoing description and teachings, and are likewise within the scope of the invention. Such modifications or other embodiments can be implemented without departing from the true scope and spirit of the invention.

[0427] [Table 1] [Table 2] [Table 3] The present invention provides, for example, the following items: (Item 1) IDO prodrug composition, wherein the IDO prodrug composition is (i) The drug portion and (ii) The lipid portion, (iii) Connecting unit ("LU") and An IDO prodrug composition comprising, wherein the drug portion comprises an IDO antagonist, and the LU conjugates the drug portion with the lipid portion. (Item 2) The IDO prodrug described in item 1, comprising the chemical structure shown as ID3 for the drug portion. (Item 3) The IDO prodrug described in item 1, wherein the aforementioned lipid portion contains CHEMS. (Item 4) The IDO prodrug described in item 1, wherein the aforementioned lipid portion contains stearic acid. (Item 5) A liposome (LPN) containing an IDO prodrug, wherein the liposome releases an active IDO inhibitor after cleavage of the LU. (Item 6) The liposome described in item 5, wherein the IDO prodrug contains ID3. (Item 7) The liposome according to item 5, comprising the IDO prodrug ID3 and further comprising TR3. (Item 8) The liposome according to item 5, comprising the IDO prodrug ID3 and further comprising TR5. (Item 9) TR5 contains the liposome described in item 8, which contains telluratrimod. (Item 10) The liposome according to item 5, comprising the IDO prodrug ID3 and further comprising AR5. (Item 11) Liposomes as described in item 10, wherein AR5 contains an A2a receptor inhibitor. (Item 12) The liposome according to item 5, wherein the liposome is further co-formulated with an immunomodulator, the immunomodulator being selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, Toll receptor agonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors and / or prodrugs thereof. (Item 13) The liposome according to item 5, wherein the liposome is further co-formulated with an ICD-inducing chemotherapeutic agent, and the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfildimib, or paclitaxel. (Item 14) Liposomes, further containing DOX, as described in item 5. (Item 15) Liposomes as described in item 5, further containing MTO. (Item 16) Liposomes, further containing DOX, as described in item 13. (Item 17) Liposomes as described in item 13, further containing MTO. (Item 18) A method for treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of liposomes to a subject requiring such treatment, wherein the liposomes contain an IDO prodrug, (ii) the pharmaceutically acceptable salt, Methods that include... (Item 19) The method according to item 18, wherein the IDO prodrug comprises ID3. (Item 20) The method described in item 18, wherein the cancer is colorectal cancer.

Claims

1. A prodrug composition of an IDO inhibitor, wherein the prodrug composition of the IDO inhibitor is (i) the drug portion and (ii) Lipid portion, (iii) Connecting unit ("LU") and It comprises an IDO inhibitor prodrug, the IDO inhibitor prodrug having the following chemical structure: 【Chemistry 101】 A prodrug composition of an IDO inhibitor comprising the following:

2. The aforementioned lipid portion has the following chemical structure: 【Chemical Engineering 102】 The prodrug of the IDO inhibitor comprises a cholesterol hemisuccinate ("CHEMS") consisting of the following chemical structure: 【Chemical 102A】 A prodrug composition of an IDO inhibitor according to claim 1, comprising the above.

3. The lipid portion contains stearic acid, and the prodrug of the IDO inhibitor has the following chemical structure: 【Chemical 102B】 A prodrug composition of an IDO inhibitor according to claim 1, comprising the above.

4. A liposome (LPN) containing an IDO inhibitor prodrug, wherein the IDO inhibitor prodrug has the following chemical structure: 【Chemistry 103】 The prodrug of the IDO inhibitor consists of the following chemical structure: 【Chemical Engineering 103A】 A liposome (LPN) comprising a drug portion, wherein the liposome releases the drug portion after cleavage of the LU.

5. The liposome according to claim 4, wherein the liposome 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, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, and 3D-PHAD®.

6. The liposome according to claim 4, wherein the liposome is further co-formulated with telluratrimod (TR5).

7. The aforementioned liposome has the following chemical structure: 【Chemical 104】 The liposome according to claim 4, further co-formulated with an A2a receptor inhibitor (AR5) comprising the above.

8. The liposome according to claim 4, wherein the liposome is further co-formulated with an immunomodulator, the immunomodulator is selected from the group consisting of immunogenic cell death (ICD) inducing chemotherapeutic agents and toll receptor agonists, wherein the ICD inducing chemotherapeutic agent is selected from the group consisting of doxorubicin (DOX), mitoxantrone (MTO), oxaliplatin (OXA), cyclophosphamide (CP), bortezomib, carfilzomib, and paclitaxel, wherein the toll receptor agonist is selected from the group consisting of rexiquimod (R848), gardikimod, 852A, DSR6434, tellatrimod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, and 3D-PHAD®.

9. The liposome according to claim 4, wherein the liposome is further co-formulated with an ICD-inducing chemotherapeutic agent, and the ICD-inducing chemotherapeutic agent is selected from the group consisting of doxorubicin (DOX), mitoxantrone (MTO), oxaliplatin (OXA), cyclophosphamide (CP), bortezomib, carfilzomib, and paclitaxel.

10. The liposome according to claim 4, further comprising doxorubicin (DOX).

11. The liposome according to claim 4, further comprising mitoxantrone (MTO).

12. The liposome according to claim 9, further comprising doxorubicin (DOX).

13. The liposome according to claim 9, further comprising mitoxantrone (MTO).

14. A composition comprising liposomes for treating subjects who have cancer or have been diagnosed with cancer, wherein the liposomes comprise an IDO inhibitor prodrug or a pharmaceutically acceptable salt thereof, wherein the IDO inhibitor prodrug has the following chemical structure: 【Chemistry 105】 A composition consisting of the following.

15. The composition according to claim 14, wherein the cancer is colorectal cancer.

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