Formulated and / or co-formulated liposome compositions containing Toll-like receptor ("TLR") agonist prodrugs useful in cancer treatment, and methods thereof.

Abstract

The present invention provides a formulation and / or co-formulated liposome composition containing a Toll-like receptor ("TLR") agonist prodrug useful in the treatment of cancer, as well as a method for producing such a composition. [Solution] Formulated and / or co-formulated liposomes comprising a TLR prodrug and / or a TLR lipid moiety, as well as methods for producing such liposomes, are disclosed herein. The TLR prodrug composition comprises a drug moiety, a lipid moiety, and a linking unit, and inhibits Toll-like receptors (e.g., TLR1 / 2, TLR4, and / or TLR7). Formulated and / or co-formulated TLR prodrugs into liposomes and utilizing targeted drug delivery vehicles can provide methods for treating cancer, immunological disorders, and other diseases.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 974,746, filed December 20, 2019, the contents of which are incorporated in full by reference herein.

[0002] Statement of rights to the invention made under research funded by the federal government. Not applicable.

[0003] Field of Invention The present invention as described herein relates to prodrug compositions that inhibit Toll-like receptors ("TLRs") (plural) after an active inhibitor has been released from the prodrug, and to nanoformulations comprising such prodrugs. In particular, the present invention relates to prodrug compositions formulated within nanocarriers (e.g., liposomes) and used as vehicles for the treatment of cancer in humans. The present invention further relates to the treatment of cancer and other immunological disorders and diseases. [Background technology]

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

[0005] Several cancers stand out for their high mortality rates. In particular, lung cancer (18.4% of all cancer deaths), breast cancer (6.6% of all cancer deaths), colorectal cancer (9.2% of all cancer deaths), liver cancer (8.2% of all cancer deaths), and stomach cancer (8.2% of all cancer deaths) are the leading causes of cancer death worldwide for men and women of all ages (GLOBOCAN 2018). These cancers, and virtually all other cancers, share a common fatal characteristic: they metastasize to sites distal to the primary tumor, and are almost without exception fatal metastatic diseases. Furthermore, even cancer patients who have overcome primary cancer early on have shown a common experience of dramatically altered lives. Many cancer patients experience intense anxiety caused by the perception of potential recurrence or treatment failure. Many cancer patients also experience physical weakness after treatment. Additionally, many cancer patients experience disease recurrence.

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

[0007] Toll-like receptors ("TLRs") are a family of 10 identified pattern-recognition receptors that respond to pathogen-associated molecular patterns (PAMPs) and autologous damage-associated molecular patterns (DAMPs). TLRs then activate downstream pathways that trigger innate immune responses by generating inflammatory cytokines, type I interferons (IFNs), and other mediators.

[0008] TLR-class proteins are single transmembrane receptors typically expressed on sentinel cells, such as macrophages and dendritic cells, that recognize conserved molecules originating from microorganisms. Once these microorganisms have breached the body's barrier (e.g., skin or intestinal mucosa), they are recognized by TLRs, which then activate an immune cell response.

[0009] When activated, TLRs recruit adapter proteins (i.e., proteins that mediate other protein-protein interactions) into the cytosol of immune cells to propagate antigen-inducing signaling pathways. These recruited proteins then participate in the subsequent activation of other downstream proteins, including protein kinases (IKKi, IRAK1, IRAK4, and TBK1), which further amplify the signal, ultimately leading to upregulation or repression of genes that regulate inflammatory responses and other transcriptional events.

[0010] Several members of the TLR family deserve attention. TLR1 and TLR2 ("TLR1 / 2") are cell surface receptors that form heterodimers recognizing bacterial antigens, such as lipoproteins, as well as DAMPs, such as HMGB1, heat shock proteins, and proteoglycans. TLR1 / 2 are expressed in pre-dendritic cells, macrophages, and NK cells, where they mediate innate responses to PAMPS and DAMPS, upregulate inflammatory cytokines, and enhance antigen processing. TLR1 / 2 agonists, such as PAM3CSK4, have also been shown to suppress the immunosuppressive effects of Treg cells, thus enhancing adaptive immunity.

[0011] In addition, TLR4 is a cell surface receptor for various bacterial and viral components, among others, lipopolysaccharide (LPS). LPS is also known as an endotoxin and is a component of the cell wall of Gram-negative bacteria. LPS has been shown to be a natural adjuvant for specific immune responses, particularly antigen (Ag) specific antibody and T cell responses. The toxicity associated with LPS has hindered its use as an effective and safe vaccine adjuvant. However, monophosphorylated lipid A (monophosphorylated) is a metabolite of LPS. Lipid A (MPLA) has been found to retain many of the immunostimulatory functions of LPS, but is significantly less toxic than its parent. Therefore, MPLA functions well as a safe and effective vaccine adjuvant. Structural features of lipid A known to be responsible for the maintenance of adjuvant properties and loss of toxicity include the number of phosphate groups, as well as the number, type, and position of fatty acid residues. Synthetic MPLA and several functional analogs have been produced and characterized as immunostimulatory adjuvants. Many of these have been clinically used as adjuvants in vaccine cocktails, including illiciting anti-tumor immunity in conjunction with tumor antigens.

[0012] In addition, TLR4 is also a receptor for high-mobility box-1 (HMGB1), a protein secreted by tumor cells during immunogenic cell death, which enhances antitumor immunity by mobilizing dendritic cells and stimulating antigen processing by antigen-presenting cells (APCs) and secreting inflammatory cytokines. Therefore, co-delivery of TLR4 agonists and ICD-inducing chemotherapeutic agents to tumors enhances the antitumor immunity induced by ICD chemotherapeutic agents.

[0013] In addition, prodrugs are pharmaceuticals or compounds that are metabolized (i.e., converted in the body) into pharmacologically active drugs after administration. Instead of administering the drug directly, a corresponding prodrug is used as an alternative to improve how the drug is absorbed, distributed, metabolized, and / or eliminated. Prodrugs are often designed to improve bioavailability when the drug itself is poorly absorbed, for example, from the gastrointestinal tract. Prodrugs can also be used to improve how a drug selectively interacts with cells or processes that are not its intended target. This reduces the harmful or unintended effects of the drug, which can be particularly important in treatments such as chemotherapy, where severe unintended and undesirable side effects may occur. Therefore, prodrugs can be considered drugs containing special non-toxic protecting groups that are transiently used to alter or eliminate undesirable properties of the parent molecule.

[0014] Finally, nanocarriers are nanomaterials used as a means of transporting other substances, such as drugs. Many different types of nanocarriers exist. For example, some examples include polymer conjugates, polymer nanoparticles, lipid-based carriers, and dendrimers. The various types of nanomaterials used in nanocarriers enable the delivery of hydrophobic and hydrophilic drugs throughout the body. Since the human body is primarily water-based, the ability to effectively deliver hydrophobic drugs to humans is a significant therapeutic benefit of nanocarriers. Nanocarriers are promising in drug delivery processes because they deliver drugs to site-specific targets, ensuring that drugs are delivered to specific organs or cells rather than other organs or cells. Site specificity is a significant therapeutic benefit because it prevents the delivered drug from being delivered to the wrong location. In addition, nanocarriers are particularly promising for use in chemotherapy because they can help reduce the harmful, broader-scale toxicity of chemotherapy to rapidly growing healthy cells throughout the body. Since chemotherapy drugs can be extremely toxic to human cells, it is crucial that chemotherapy drugs are delivered to tumors without being released to other parts of the body.

[0015] From the above, it will be 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 modern nanocarrier designs, new disease treatments can achieve the overall goals of more effective treatment(s), reduced side effects, and higher therapeutic applicability in the treatment of cancer, particularly solid tumor cancers.

[0016] Given the current shortcomings associated with cancer treatment, one objective of the present invention is to provide a novel and improved method for treating cancer(s), immunological disorders, and other diseases using prodrugs encapsulated within nanocarriers.

[0017] In the present disclosure, the combined use of a TLR agonist and an ICD-inducing chemotherapeutic agent for illicitly eliciting an immune response directly in actual patient tumor cells (i.e., without the need to introduce tumor antigens or remove tumor cells for ex vivo treatment). These synergistic functional agents are packaged in a single nanocarrier vehicle that ensures co-delivery of the combination therapy and enhanced tumor selectivity. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEMS

[0018] SUMMARY OF THE INVENTION The present invention provides a TLR inhibitor prodrug (“TLR prodrug”) composition comprising a TLR inhibitor agent, a lipid, and a biologically cleavable linker. In certain embodiments, a nanocarrier comprising the TLR prodrug(s) is formulated for use as a delivery modality for treating human diseases such as cancer, including solid tumor cancers, and other immune disorders. In certain embodiments, the nanocarrier comprises a lipid bilayer that can be incorporated into a drug delivery vehicle (i.e., a liposome). In a further preferred embodiment, the liposome comprises cholesterol hemisuccinate (“CHEMS”). In a further preferred embodiment, the liposomes of the present invention comprise stearic acid.

[0019] In a further embodiment, the present invention includes a method of delivering a TLR inhibitor to a tumor, the method comprising: (i) synthesizing a TLR prodrug; (ii) formulating the TLR prodrug of the present invention into the nanocarrier of the present invention; and (iii) administering the nanocarrier to a patient.

[0020] In another embodiment, the invention is a method of delivering a TLR inhibitor to a tumor in combination with one or more additional immunomodulatory agents, comprising: (i) synthesizing a TLR prodrug; (ii) co-formulating the TLR prodrug of the invention with one or more additional immunomodulatory agents of the invention into a nanocarrier; and (iii) administering the nanocarrier to a patient.

[0021] In another embodiment, the immunomodulatory agent includes an agonist of another TLR, an immunogenic cell death (ICD)-inducing chemotherapeutic agent, a PD-1 / PD-L1 antagonist, an IDO antagonist, a STING agonist, a CTLA4 inhibitor, an iNKT cell agonist, and / or a prodrug thereof.

[0022] In another embodiment, the present disclosure teaches a method of synthesizing a TLR prodrug.

[0023] In another embodiment, the present disclosure teaches a method of formulating a TLR prodrug within a nanocarrier including, but not limited to, liposomes.

[0024] In another embodiment, the present disclosure teaches a method of treating human cancer, immunological disorders, and other diseases using the nanocarriers of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] [Figure 1] General chemical synthesis for TR5(B).

[0026] [Figure 2] General chemical synthesis for TR6.

[0027] [Figure 3] Synthesis scheme of TLR inhibitor prodrug using carboxylic acid functional group.

[0028] [Figure 4] Synthesis scheme of TLR inhibitor prodrug using alcohol functional group.

[0029] [Figure 5] A synthesis scheme for TLR inhibitor prodrugs using secondary amine, amide, or aniline functional groups.

[0030] [Figure 6] Chemical synthesis of TR6 prodrug intermediates.

[0031] [Figure 7] Chemical synthesis of TR6 prodrugs containing CHEMS.

[0032] [Figure 8] Chemical synthesis for TR3.

[0033] [Figure 9] Characterization of LNP-TR6 liposomes.

[0034] [Figure 10] Characterization of LNP-TR6 liposomes (zeta potential).

[0035] [Figure 11] Characterization of LNP-TR5 liposomes.

[0036] [Figure 12] Characterization of LNP-TR5 liposomes (zeta potential).

[0037] [Figure 13] Characterization of LNP-TR3 liposomes.

[0038] [Figure 14] Characterization of LNP-TR3 liposomes (zeta potential).

[0039] [Figure 15] Characterization of LNP-TR8 liposomes.

[0040] [Figure 16] Characterization of LNP-TR8 liposomes (zeta potential).

[0041] [Figure 17] Characterization of LNP-ID3-TR8 liposomes.

[0042] [Figure 18] Characterization of LNP-ID3-TR8 liposomes (zeta potential).

[0043] [Figure 19] Tumor inhibition of B16F10 cancer cells by a combination of LNP-TR5 and LNP-DOX.

[0044] [Figure 20] Tumor inhibition of the combination of LNP-TR6 and LNP-NK1, and the combination of LNP-TR6, LNP-TR8, and LNP-MTO in B16F10 cancer cells.

[0045] [Figure 21] Tumor inhibition of LNP-TR5 and LNP-AR5 combinations and LNP-TR6 and LNP-AR5 combinations in B16F10 cancer cells.

[0046] [Figure 22] Tumor inhibition of EMT6 cancer cells by a combination of LNP-TR5, LNP-AR5, and LNP-DOX.

[0047] [Figure 23] Tumor inhibition studies of LNP-TR5 and LNP-TR6 in multiple combinations in H22 cancer cells.

[0048] [Figure 24] Tumor inhibition studies of LNP-TR5 and LNP-TR6 in multiple combinations in colorectal cancer.

[0049] [Figure 25]In vitro verification of the mechanism of action of liposomal TR6 prodrugs.

[0050] [Figure 26] In vitro verification of the mechanism of action of TR8 in liposome form.

[0051] [Figure 27] In vitro verification of the mechanism of action of TR5 in liposome form.

[0052] [Figure 28] In vitro verification of the mechanism of action of liposome-form TR3. 28(A). Raw-Blue® incubated with TR5 and LNP-TR5. 28(B). Raw-Blue® incubated with TR3, TR3 + stearic acid, and LNP-TR3.

[0053] [Figure 29] In vitro verification of the mechanism of action of liposome-form TR3. 29(A). HEK-Blue TLR8 incubated with TR5 and LNP-TR5. 29(B). HEK-Blue TLR8 incubated with TR3, TR3 + stearic acid, and LNP-TR3 untreated groups. [Modes for carrying out the invention]

[0054] Detailed description of the invention Section Summary I.) Definition II.) Prodrugs PRO.) Drug Section IV.) Lipids V.) Linked Unit ("LU") VI.) Nanocarriers VII.) Liposomes VIII.) Pharmaceutical Formulation IX.) Combination Therapy X.) Method for delivering liposomes containing prodrugs to cells XI.) Methods for treating cancer(s) and other immunological disorders(s). XII.) Kits / Manufactured Products I.) Definition

[0055] Unless otherwise defined, all terms, notations, and other scientific or technical terms used herein are intended to have meanings that are generally understood by those skilled in the art, unless otherwise clearly indicated by the context. In some cases, terms that have generally understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from the generally understood meanings in the art.

[0056] Where a trademark is used herein, references to that trademark also refer to the product formulations, generic drugs, and the pharmaceutically active ingredients(s) of the trademarked product, unless otherwise specified by the context.

[0057] Where used herein, the term “about” means, when referring to a value or quantity of size (i.e., diameter), weight, concentration, or percentage, to include a variation of ±20% or ±10% in one example, ±5% in another example, ±1% in yet another example, and ±0.1% in yet another example from the specified quantity, because such variation is suitable for carrying out the disclosed method.

[0058] As used herein, the term "and / or" refers to entities that exist individually or in combination, when used in the context of a list of entities. Thus, for example, the phrase "A, B, C, and / or D" includes not only A, B, C, and D individually, but also any combination and partial combination of A, B, C, and D.

[0059] In this specification, the numerical range indicated by an endpoint includes all numbers and fractions contained within that range (for example, 1 to 5 includes, but is not limited to, 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5).

[0060] As used herein, the phrase “essentially from” limits the claims to the specified material or step, and any other essential and novel feature(s) of the claimed subject matter, in addition to the specified material or step(s).

[0061] The terms “advanced cancer,” “locally advanced cancer,” “progressive disease,” and “locally advanced disease” refer to cancer that has spread through the associated tissue capsule and include disease at stage C in the American Urological Association (AUA) system, disease at stages C1–C2 in the Whitmore-Jewett system, and disease at stages T3–T4 and N+ in the TNM (tumor, nodule, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, as these patients have substantially fewer favorable outcomes compared to patients with clinically localized (organ-limited) cancer.

[0062] As used herein, the term "alkyl" includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups, including C1-C1 groups at both ends. 20A linear (i.e., "straight-chain"), branched, or cyclic hydrocarbon chain can be defined as saturated or at least partially, and in some cases as unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains. "Branched" refers to an alkyl group in which a lower alkyl group, e.g., methyl, ethyl, or propyl, is bonded to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms (i.e., C1-C8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" specifically refers to a C1-C8 straight-chain alkyl group. In other embodiments, "alkyl" specifically refers to a C i~8 This refers to branched-chain alkyl groups.

[0063] The alkyl group may optionally be substituted with one or more alkyl substituents, which may be the same or different ("substituted alkyl"). The term "alkyl substituent" includes, but is not limited to, alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. In some embodiments, one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms may optionally be inserted along the alkyl chain, the nitrogen substituent being hydrogen, a lower alkyl (also referred to herein as "alkylaminoalkyl"), or an aryl.

[0064] Therefore, as used herein, the term “substituted alkyl” includes alkyl groups as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced by another atom or by a functional group including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate and mercapto.

[0065] The term “aryl” is used herein to refer to an aromatic substituent that may be a single aromatic ring, or may be fused together, covalently linked, or, but not limited to, multiple aromatic rings linked to a common group such as a methylene or ethylene moiety. The common linking group may also be a carbonyl group, as in benzophenone, or oxygen, as in diphenyl ether, or nitrogen, as in diphenylamine. The term “aryl” particularly encompasses heterocyclic aromatic compounds. Aromatic rings may include, among others, phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, and benzophenone. In certain embodiments, the term “aryl” means cyclic aromatic compounds containing about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5-membered and 6-membered aromatic rings and heteroaromatic rings. The aryl group may be optionally substituted with one or more aryl substituents, which may be the same or different ("substituted aryl"), where the "aryl substituents" include alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R'', where R' and R'' may independently be hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl. Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, and carbazole.

[0066] As used herein, "heteroaryl" refers to an aryl group whose ring structure contains one or more non-carbon atoms (e.g., O, N, S, Se, etc.). Nitrogen-containing heteroaryl moieties include, but are not limited to, pyridine, imidazole, benzimidazole, pyrazole, pyrazine, triazine, and pyrimidine.

[0067] The terms “anti-cancer drug,” “chemotherapeutic agent,” and “anti-cancer prodrug” refer to a drug (i.e., a chemical compound) or prodrug that is known to or is thought to be able to treat cancer (i.e., kill cancer cells, inhibit the growth of cancer cells, or treat symptoms associated with cancer). In some embodiments, the term “chemotherapeutic agent,” as used herein, refers to a non-PS molecule that is used to treat cancer and / or has cytotoxic activity. More traditional or conventional chemotherapeutic agents can be described by mechanism of action or by class of chemical compounds and may include, but are not limited to, alkylating agents (e.g., melphalan), anthracyclines (e.g., doxorubicin), cytoskeletal disruptors (e.g., paclitaxel), epothilons, histone deacetylase inhibitors (e.g., vorinostat), topoisomerase I or II inhibitors (e.g., irinotecan or etoposide), kinase inhibitors (e.g., bortezomib), nucleotide analogs or their precursors (e.g., methotrexate), peptide antibiotics (e.g., bleomycin), platinum-based drugs (e.g., cisplatin or oxaliplatin), retinoids (e.g., tretinoin), and vinka alkaloids (e.g., vinblastine).

[0068] "Aralkyl" refers to an alkyl-aryl group, and the alkyl and / or aryl parts are substituted as needed.

[0069] "Alkylene" refers to a linear or branched divalent aliphatic hydrocarbon group having 1 to about 20 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group may be linear, branched, or cyclic. The alkylene group may also be unsaturated as desired and / or substituted with one or more "alkyl substituents". Along the alkylene group, one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl") may be inserted as desired, the nitrogen substituent being alkyl as already described. Examples of alkylene groups include methylene (-CH2-), ethylene (-CH2-CH2-), propylene (-(CH2)3-), and cyclohexylene (-C6H 10 -), -CH=CH-CH=CH-, -CH=CH-CH2-, -(CH2) q Examples include -N(R)-(CH2)- (where each q is an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or a lower alkyl group), methylenedioxyl (-O-CH2-O-), and ethylenedioxyl (-O-(CH2)2-O-). The alkylene group can have about 2 to about 3 carbon atoms, and can also have 6 to 20 carbon atoms.

[0070] The term "arylene" refers to a divalent aromatic group, such as a divalent phenyl or naphthyl group. The arylene group may be substituted with one or more aryl substituents as needed and / or may contain one or more heteroatoms.

[0071] The term "amino" refers to a -N(R)² group, where each R is independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, or substituted aralkyl. The terms "aminoalkyl" and "alkylamino" may refer to a -N(R)² group, where each R is H, alkyl, or substituted alkyl, and at least one R is alkyl or substituted alkyl. "Arylamine" and "aminoaryl" refer to a -N(R)² group, where each R is H, aryl, or substituted aryl, and at least one R is aryl or substituted aryl, such as aniline (i.e., -NHC6H5).

[0072] "Bioreactive nanomaterials" refer to engineered biomaterials that induce or catalyze biological responses. In certain embodiments, nanomaterials catalyze biological responses at various nano / biointerfaces by inducing responses through one or more properties selected from the group consisting of composition, size, shape, aspect ratio, solubility, electrons, redox, surface presentation, surface coating, hydrophobicity, hydrophilicity, atomically thin nanosheet, or functionalized surface groups. In certain embodiments, bioreactive nanomaterials have the ability to inhibit the TLR-1 biological response in cells (e.g., tumor cells) and / or activate the innate immune system by transmitting "danger signals" and adjuvant effects.

[0073] "Bulk" (also known as drug substance) refers to drug substance or drug product that has not been filled into final containers for distribution. Final formulation bulk generally refers to drug products that have been formulated and are stored or held before filling. Drug substance may be stored or held as "bulk" or "concentrated bulk" before being formulated into drug products.

[0074] The terms "carboxylate" and "carboxylic acid" are -C(=O)O, respectively. -The term "carboxyl" can refer to the -C(=O)OH group.

[0075] The terms “conjugate” and “conjugated,” as used herein, can refer to two or more components (e.g., chemical compounds, polymers, biomolecules, particles, etc.) being linked to one another (e.g., covalently). In some embodiments, the conjugate may include monovalent portions derived from two different chemical compounds covalently linked via a divalent linker portion (e.g., alkylene or arylene, as may be substituted). In some embodiments, the linker may contain one or more biodegradable bonds, so that one or more of the linker bonds can be broken when the prodrug is exposed to a particular physiological environment or enzyme (e.g., esterase).

[0076] The term “compound” refers to and includes chemical compounds (e.g., prodrugs) themselves, as well as, unless the context makes it clear whether explicitly stated or not, the following: amorphous and crystalline forms of a compound, including polymorphic forms, where these forms may be part of a mixture or be isolated; free acid and free base forms of a compound, these are typically the forms shown in the structures provided herein; isomers of a compound, where optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers, as well as mixtures of optical isomers, including racemic and non-racemic mixtures, where isomers may be in isolated forms or in mixtures with one or more other isomers; the same Topologies, including deuterium-containing compounds and tritium-containing compounds, and compounds containing radioisotopes, including radioisotopes that are therapeutically and diagnostically effective; polymeric forms of compounds, including dimers, trimers, and the like; salts of compounds, preferably pharmaceutically acceptable salts, including acid-addition salts and base-addition salts, and salts having organic and inorganic counterions, and zwitterionic forms, where the compound associates with two or more counterions, the two or more counterions may be the same or different; and solvates of compounds, including hemisolvates, monosolvates, disolvates, and the like, and organic solvent hydrates and inorganic solvates, where the inorganic solvates include hydrates, where the compound associates with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some cases, references to the compounds of the present invention herein include an explicit reference to one or of the above forms, e.g., salts and / or solvates, but such references are for emphasis only and should not be construed as excluding any forms other than those specified above.

[0077] The term "drug product" generally refers to the final formulation containing an active drug component associated with an inactive component, although this association is not strictly necessary (i.e., a liposome containing a TLR inhibitor prodrug). The term also includes final dosage forms that do not contain an active ingredient but are intended to be used as a placebo.

[0078] The term "disulfide" can refer to the -SS- group.

[0079] The term "empty vesicle" refers to an unloaded lipid vesicle, in itself.

[0080] 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.

[0081] As used herein, the term "esterase" refers to a hydrolytic enzyme that breaks down esters into acids and alcohols.

[0082] "Excipients" refer to inert substances used as carriers for active ingredients in drugs, such as vaccines. Excipients are also sometimes used to increase the volume of formulations containing very potent active ingredients, enabling convenient and precise dosage. Examples of excipients include, but are not limited to, antiadherents, binders, coatings, disintegrants, fillers, diluents, flavorings, colorants, lubricants, and preservatives.

[0083] The terms "halo," "halide," or "halogen" as used herein refer to fluoro, chloro, bromo, and iodine groups.

[0084] The terms "hydroxyl" and "hydroxy" refer to the -OH group.

[0085] The terms “inhibit” or “inhibit” as used herein mean reducing or completely preventing a measurable amount.

[0086] The terms “individual” and “patient” may be used interchangeably when used in the context of this disclosure.

[0087] As used herein, the term “ligand” generally refers to a species, such as a molecule or ion, that interacts with another species in several ways, for example, by binding. See MARTELL, AE, and HANCOCK, RP, Metal Complexes in Aqueous Solutions, Plenum: New York (1996), which is incorporated herein by reference in its entirety.

[0088] As used herein, the term "lipid" refers to a class of naturally occurring (organic) compounds that are insoluble in polar solvents. In the context of this disclosure, lipids include conventional lipids, phospholipids, cholesterol, and lipids chemically functionalized for PEG-ligand binding.

[0089] 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.

[0090] The terms "liposome," "lipid vesicle," or "vesicle" are interchangeably used to refer to an aqueous compartment encapsulated by a lipid bilayer, as conventionally defined (see STRYER (1981) Biochemistry, 2d Edition, WH Freeman & Co., p. 213).

[0091] 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.

[0092] The terms "mercapto" or "thiol" refer to the -SH group.

[0093] The terms "metastatic cancer" and "metastatic disease" refer to diseases that have spread to local lymph nodes or distal sites and include stage D diseases in the AUA system and stage T×N×M+ in the TNM system.

[0094] The terms “nanocarrier,” “nanoparticle,” and “nanoparticle drug carrier” are used interchangeably and refer to nanostructures having an aqueous, solid, or polymeric inner core. In certain embodiments, the nanocarrier comprises a lipid bilayer enclosing (or surrounding or covering) a porous particle core. In certain embodiments, the nanocarrier is a liposome, lipid nanoparticle ("LNP"), or solid lipid nanoparticle ("SLNP").

[0095] The terms “nanoscale particles,” “nanomaterials,” “nanocarriers,” and “nanoparticles” refer to structures having at least one region with dimensions (e.g., length, width, diameter, etc.) less than about 1,000 nm. In some embodiments, the dimensions are smaller (e.g., less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, or even less than about 20 nm). In some embodiments, the dimensions are between approximately 20 nm and approximately 250 nm (for example, approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm).

[0096] The term “nanopes” refers to “lipid vesicles” (or groups of vesicles with an average diameter) having diameters ranging from about 20 nm, or about 30 nm, or about 40 nm, or about 50 nm, up to about 500 nm, or about 400 nm, or about 300 nm, or about 200 nm, or about 150 nm, or about 100 nm, or about 80 nm. In certain embodiments, nanovesicles have diameters ranging from about 40 nm to about 80 nm, or from about 50 nm to about 70 nm.

[0097] "Pharmacologically acceptable" refers to a composition that is non-toxic, inert, and / or physiologically compatible with humans or other mammals.

[0098] "Pharmaceutical formulation" refers to the process of combining various chemical substances with pure drug substances to produce the final drug product.

[0099] The term "phosphonate" refers to a -P(=O)(OR)2 group, where each R can independently be H, alkyl, aralkyl, aryl, or negatively charged (i.e., there is virtually no R group bonded to the oxygen atom, resulting in the presence of a lone pair of electrons on the oxygen atom). In other words, each R may or may not be present, and if present, it can be selected from H, alkyl, aralkyl, or aryl.

[0100] The term "phosphate" refers to -OP(=O)(OR')2 groups, where R' is H or a negative charge.

[0101] The term "prodrug" means a pharmaceutical or compound that is metabolized into a pharmacologically active drug after administration. For the purposes of this disclosure, the prodrug of the present invention comprises three components: (i) a drug moiety, (ii) a lipid moiety, and (iii) a linking unit ("LU").

[0102] The term "TLR prodrug" means the prodrug of the present invention, where the drug portion comprises a TLR inhibitor.

[0103] The term "pyrolipid" refers to a conjugate of a lipid with a porphyrin, a porphyrin derivative, or a porphyrin analog. In some embodiments, a pyrolipid may include a lipid conjugate in which a porphyrin or its derivative or analog is covalently bonded to a lipid side chain. See, for example, U.S. Patent Application Publication 2014 / 0127763.

[0104] As used herein, the terms “specific,” “specifically bind,” and “specifically bind to” refer to the selective binding of the nanocarrier of the present invention to the target TLR-1.

[0105] The term “supported lipid bilayer” refers to a lipid bilayer that encapsulates a porous particle core. This definition is given as such because the lipid bilayer is located on the surface and supported by the porous particle core, as described in this disclosure. In certain embodiments, the lipid bilayer may have a thickness in the range of about 6 nm to about 7 nm, including a hydrophobic core 3 to 4 nm thick, plus a layer of hydrated hydrophilic head groups (each about 0.9 nm thick), plus two partially hydrated regions each about 0.3 nm thick. In various embodiments, the lipid bilayer surrounding the liposome includes a continuous bilayer or substantially continuous bilayers, which effectively cover and seal the TLR inhibitor.

[0106] The term "thioalkyl" can refer to an -SR group, where R is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl. Similarly, the terms "thioaralkyl" and "thioaryl" refer to an -SR group, where R is aralkyl and aryl, respectively.

[0107] As used herein, “to treat” or “therapeutic” and grammatically related terms mean any improvement of any outcome of a disease, such as an extension of survival, a reduction in morbidity, and / or mitigation of side effects that are byproducts of an alternative mode of treatment, and, as readily recognized in the art, complete eradication of the disease is preferred, but this is not a requirement of a treating act.

[0108] The term "therapeutic dose" refers to the amount of an active prodrug, nanoencapsulated prodrug, or pharmaceutical product that elicits a biological or medical response in a tissue, system, animal, individual, or human.

[0109] The term "unsupported lipid bilayer" refers to an uncoated lipid bilayer in lipid vesicles or liposomes. II.) Prodrugs

[0110] As shown in this disclosure and for the purposes of the present invention, a suitable prodrug is formed by conjugating the drug portion of the present invention (see the section titled "Drug Portion") to the lipid portion of the present invention (see the section titled "Lipids") via the LU (see the section titled "Linking Units") of this disclosure. For the purposes of this disclosure, several strategies can be used to form the TLR prodrug (see, for example, Figures 3, 4, and 5).

[0111] Therefore, in some embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present disclosure.

[0112] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present disclosure, wherein the TLR inhibitor inhibits TLR1 / 2.

[0113] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present disclosure, wherein the TLR inhibitor inhibits TLR4.

[0114] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present disclosure, wherein the TLR inhibitor inhibits TLR8.

[0115] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present disclosure, wherein the TLR inhibitor inhibits TLR7 / 8.

[0116] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present disclosure, where the TLR inhibitor inhibits TLR1 / 2, and the prodrug comprises a prodrug of formula I.

[0117] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present disclosure, wherein the TLR inhibitor inhibits TLR7.

[0118] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present disclosure, wherein the TLR inhibitor inhibits TLR7, and the prodrug comprises a prodrug of formula II.

[0119] In one embodiment, the prodrug comprises the following chemical structure represented by formula I. [ka] [In the exemplary embodiment of formula I, X= [ka] And, Is Y=H or OH? or X=H, Y = CHEMS, or stearic acid, or CHEMS + linker, or stearic acid + linker.

[0120] In further embodiments, the prodrug comprises the following chemical structure represented by Formula II. [ka] [In the exemplary embodiment of formula II, X= [ka] And, Y = CH2, O, NH. A= [ka] It is.

[0121] Therefore, in one embodiment, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of formula I.

[0122] Therefore, in one embodiment, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of formula II.

[0123] In one embodiment, the prodrug is a drug-lipid moiety containing a TLR inhibitor as shown in Figure 1.

[0124] In one embodiment, the prodrug is a drug-lipid moiety containing a TLR inhibitor as shown in Figure 2.

[0125] In one embodiment, the prodrug is a drug-lipid moiety containing a TLR inhibitor as shown in Figure 7.

[0126] In one embodiment, the prodrug is a drug-lipid moiety containing a TLR inhibitor as shown in Figure 8.

[0127] In further embodiments, the TLR prodrug is a drug-lipid portion comprising the lipids of the present disclosure.

[0128] In a further embodiment, the TLR prodrug is a drug-lipid moiety, where the lipid is CHEMS.

[0129] In a further embodiment, the TLR prodrug is a drug-lipid moiety, where the lipid is stearic acid.

[0130] In further embodiments, the TLR prodrug is a drug-lipid moiety comprising the LU of the present disclosure.

[0131] In further embodiments, the TLR prodrug is a drug-lipid moiety, where LU is a hydromethylcarbamate linker.

[0132] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises the chemical composition TR6 and / or TR6(A).

[0133] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR6 and / or TR6(A), and further comprises CHEMS.

[0134] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR6 and / or TR6(A), and further comprises stearic acid.

[0135] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises the chemical compositions TR3, TR5, TR5(A) and / or TR5(B).

[0136] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR3, TR5, TR5(A) and / or TR5(B), and further comprises CHEMS.

[0137] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR3, TR5, TR5(A) and / or TR5(B), and further comprises stearic acid.

[0138] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR6, further comprising CHEMS, and LU is a hydromethylcarbamate linker.

[0139] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR6, further comprising stearic acid, and LU is a hydromethylcarbamate linker.

[0140] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR6 and further comprises stearic acid having the following structure. [ka]

[0141] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor, wherein the TLR inhibitor of the present invention inhibits TLR1 / 2, and the TLR inhibitor comprises TR6 and further comprises stearic acid having the following structure. [ka]

[0142] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR5, TR5(A) and / or TR5(B), further comprising CHEMS, and LU is a hydromethylcarbamate linker.

[0143] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR5, TR5(A) and / or TR5(B), further comprising stearic acid, and LU is a hydromethylcarbamate linker.

[0144] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR5(B) and further comprises stearic acid having the following structure. [ka]

[0145] In further embodiments, the prodrug is a drug-lipid moiety comprising a TLR inhibitor, wherein the TLR inhibitor of the present invention inhibits TLR7, and the TLR inhibitor comprises TR5(B) and further comprises stearic acid having the following structure. [ka]

[0146] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR6 and further comprises CHEMS having the following structure. [ka]

[0147] In one embodiment, the prodrug is a drug-lipid moiety containing a TLR inhibitor as shown in Figure 7.

[0148] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises the chemical composition TR3.

[0149] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR3 and further comprises CHEMS.

[0150] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR3 and further comprises stearic acid.

[0151] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR3, further comprising CHEMS, and LU is a hydromethylcarbamate linker.

[0152] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR3, further comprising stearic acid, and LU is a hydromethylcarbamate linker.

[0153] In further embodiments, the prodrug is a drug-lipid moiety comprising the TLR inhibitor of the present invention, wherein the TLR inhibitor comprises TR3 having the following structure. [ka]

[0154] In additional embodiments of the present disclosure, the subject provides a TLR inhibitor prodrug comprising a parent drug of a lipid-conjugated therapeutic agent. In some embodiments, the prodrug comprises (a) a monovalent drug moiety, (b) a monovalent lipid moiety, and (c) a divalent linker moiety comprising a linking unit that degrades in vivo, such as a disulfide bond, where the monovalent drug moiety and the monovalent lipid moiety are linked via a linker (e.g., covalently). The monovalent drug moiety and the monovalent lipid moiety may be monovalent derivatives of a chemical compound and a lipid, respectively. For example, the monovalent derivative may be a deprotonated derivative of a chemical compound or lipid containing a hydroxyl, thiol, amino, or carboxylic acid group.

[0155] In further embodiments of the present disclosure, the subject provides a TLR inhibitor prodrug comprising a parent drug of a lipid-conjugated therapeutic agent. In some embodiments, the prodrug comprises (a) a divalent drug moiety, (b) a divalent lipid moiety, and (c) a divalent linker moiety comprising a linkage that degrades in vivo, wherein the divalent drug moiety and the divalent lipid moiety are linked via a linker (e.g., by a covalent bond). The divalent drug moiety and the divalent lipid moiety may be divalent derivatives of a chemical compound and a lipid, respectively. For example, the divalent derivative may be a deprotonated derivative of a chemical compound or lipid containing a hydroxyl, thiol, amino, or carboxylic acid group.

[0156] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure. PRO.) Drug Section

[0157] Another aspect of the present invention provides novel TLR prodrug compounds (plural) having the following formulas: TR3, TR5, TR5(A), TR6, and TR6(A).

[0158] Those skilled in the art will fully understand that the compound is useful as a TLR inhibitor (e.g., inhibiting TLRs, e.g., TLR1 / 2, TLR4, TLR7, TLR8 and / or TLR7 / 8). As brief background, TLR-1 (CD281) recognizes pathogen-associated molecular patterns that are specific to Gram-positive bacteria. TLR-1 is found on the epithelial cell layer covering the small and large intestines and is an important player in the management of the gut microbiota and the detection of pathogens. TLR-1 is also found on the surface of macrophages and neutrophils. It has been demonstrated that TLR1, in cooperation with TLR2 (as a heterodimer), recognizes peptidoglycans and (triacyl)lipopeptides and interacts with TLR2. See FARHAT, et. al., J. Leukoc. Biol. 83(3): 692-701 (2007) and JIN, et. al., Cell. 130(6): 1071-1082 (2007).

[0159] TLR2 (CD282) is a protein encoded by the TLR2 gene in humans. TLR2 is a membrane protein expressed on the surface of certain cells that recognizes foreign substances and transmits appropriate signals to immune system cells. TLR2 is most abundantly expressed in peripheral blood leukocytes and, in response to NF-κB stimulation, mediates the host response to Gram-positive bacteria and yeast. (BARRELLO, et. al., Int. J. Immun.) See & Pharm. 24(3): 549-556 (2011). TLR2 is located on the plasma membrane and responds there to lipid-containing PAMPs, such as lipoteichoic acid, and lipopeptides containing di- and tri-acylated cysteine. TLR2 achieves this response by forming dimeric complexes with either TLR1 or TLR6 on the plasma membrane. See BOTOS, et. al., Structure 19(4): 447-459 (2011).

[0160] TLR4 (CD284) is another member of the TLR family. Its activation leads to the intracellular signaling pathway NF-κB and inflammatory cytokine production, which are involved in the activation of the innate immune system. TLR4 is best known for recognizing lipopolysaccharide (LPS), a component present in many Gram-negative bacteria (e.g., Neisseria species) and selected Gram-positive bacteria. Its ligands also include several viral proteins, polysaccharides, and various endogenous proteins, such as low-density lipoproteins, beta-defensins, and heat shock proteins. See BRUBAKER, et. al., Annual Rev. of Immun. 33:257-290 (2015). TLR4 signaling responds to signals by forming a complex using the extracellular leucine-rich repeat domain (LRR) and the intracellular toll / interleukin-1 receptor (TIR) ​​domain. LPS stimulation induces a series of interactions with several accessory proteins, thereby forming the TLR4 complex on the cell surface. LPS recognition is triggered by the binding of LPS to the LBP protein. Conformational changes in TLR4 induce the recruitment of intracellular adapter proteins containing the TIR domain, which is essential for activating downstream signaling pathways. (LU, et. al., Cytokine 42(2): 145-151 (2008)). TLR4 can activate the MAPK and NF-κB pathways, suggesting a potential direct role of cell-autonomous TLR4 signaling in regulating oncogenesis, particularly through increased tumor cell proliferation, inhibition of apoptosis, and metastasis.

[0161] TLR7 is another member of the TLR family. TLR7 recognizes endosomal single-stranded RNA, a common feature of viral genomes that are internalized by macrophages and dendritic cells. TLR7 recognizes single-stranded RNA of viruses, such as HIV and HCV. See HEIL, et. al., Science 303(5663): 1526-1529 (2004). TLR7 can recognize GU-rich single-stranded RNA. Ibid. However, the presence of GU-rich sequences in single-stranded RNA is insufficient to stimulate TLR7. TLR7 has been shown to play a crucial role in the pathogenesis of autoimmune disorders, such as systemic lupus erythematosus (SLE), and in the regulation of antiviral immunity. In addition, TLR7 agonists have been studied for cancer immunotherapy due to their ability to induce robust production of anti-cancer cytokines, such as interleukin-12.

[0162] Based on the above, this disclosure describes a class of TLR inhibitors.

[0163] In one embodiment, the TLR inhibitor of that class inhibits TLR1 / 2.

[0164] In one embodiment, the TLR inhibitor of that class inhibits TLR7.

[0165] In one embodiment, the drug portion of the present disclosure comprises a compound having the following chemical structure (indicated as TR6). [ka]

[0166] In alternative embodiments, the drug portion of the present disclosure comprises a compound having the following chemical structure (indicated as TR6(A)). [ka]

[0167] In one embodiment, the drug portion of the present disclosure comprises a compound having the following chemical structure (indicated as TR5). [ka]

[0168] In alternative embodiments, the drug portion of the present disclosure comprises a compound having the following chemical structure (indicated as TR5(A)). [ka]

[0169] In one embodiment, the drug portion of the present disclosure comprises a compound having the following chemical structure (multiple) (indicated as TR3). [ka] [In the formula, Y = CH3, n-Pr, OEt, or NHEt, R = saturated C12-C24 alkyl group.

[0170] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure. IV.) Lipids

[0171] Generally speaking, and for the purposes of this disclosure, the term “lipid” is used in its broadest sense, but is not limited thereto, and includes several subcategories of lipids, including phospholipids / fatty acids. As recognized by those skilled in the art, phospholipids represent a certain class of lipids that are the main components of all cell membranes. Due to their amphiphilic properties, phospholipids can form lipid bilayers. The structure of a phospholipid molecule generally consists of two hydrophobic fatty acid “tails” and a hydrophilic “head” consisting of a phosphate group that can be modified with a simple organic molecule, such as choline, ethanolamine, or serine. These two components are usually linked together by a glycerol molecule. A representative list of the phospholipids / fatty acids of the present invention is shown in Table III.

[0172] As a brief background, at the most basic level, the properties of liposomes are determined by subtle physicochemical interactions among the various lipid species in their composition. Individual lipids can be combined to form a wide variety of superstructures, including bilayers, and the properties of these bilayers can be tuned to modulate drug release and membrane stability. In a simplified bilayer model, acyl chain length determines the bilayer thickness and phase transition temperature (Tm), acyl chain saturation controls the bilayer fluidity, and head group interactions influence intermolecular and intramolecular forces. Liposome behavior can be tuned by incorporating synthetic lipids, such as lipid prodrugs, fusionable lipids, and functionalizable lipids, into the bilayer. See KOHLI, et. al., J. Control Release, 0: pp. 274-287 (Sept. 28, 2014).

[0173] In one embodiment of the present disclosure, the TLR prodrug comprises a monovalent lipid moiety.

[0174] In one embodiment, the TLR prodrug includes a divalent lipid moiety.

[0175] In one embodiment, the lipid includes cholesterol having the following chemical structure. [ka]

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

[0177] In one embodiment, the lipid comprises DMPG having the following chemical structure. [ka]

[0178] In one embodiment, the lipid comprises Lyso PC having the following chemical structure. [ka]

[0179] In one embodiment, the lipid includes (Δ9-Cis)PG.

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

[0181] In one embodiment, the lipid includes a prostaglandin (PG) having the following chemical structure. [ka]

[0182] In one embodiment, the lipid comprises C16 PEG2000 ceramide (Ceramide) having the following chemical structure. [ka]

[0183] In one embodiment, the lipid comprises hemysuccinate cholesterol ("CHEMS") having the following chemical structure. [ka]

[0184] In one embodiment, the lipids include a class of lipids having the following chemical structure represented by formula III. [ka] [In the exemplary embodiment of formula III, X= [ka] And, Y= [ka] And, Z= [ka] It is.

[0185] In one embodiment, the lipid portion of the present disclosure comprises a compound having the following chemical structure (indicated as TR8). [ka] See GIGG J., et. al., Carb. Res. 141(1):pp. 91-97 (1985).

[0186] In one embodiment, the lipids include a class of lipids having the following chemical structure represented by formula IV. [ka] [In the exemplary embodiment of formula IV, X= [ka] It is.

[0187] In one embodiment, the lipid portion of the present disclosure includes a compound having the following chemical structure (indicated as TR11). [ka] See KAUR, et. al., RSC Advances 8, pp. 9587-9686 (2018).

[0188] In further embodiments, the lipid portion of the disclosure comprises a class of invariant natural killer T (iNKT) cells.

[0189] In further embodiments, the lipid portion of the disclosure comprises alpha-galactosylceramide (α-GalCer).

[0190] For reference, a further list of the chemical formulas and abbreviations(s) of the lipids disclosed herein is provided in Table I.

[0191] In additional embodiments, the lipids include phospholipids / fatty acids disclosed herein and listed in Table III.

[0192] In further embodiments, the lipid includes stearic acid.

[0193] In addition, the TLR prodrugs and / or liposomes(s) of this disclosure may comprise one or more helper lipids, also referred to herein as “helper lipid components.” The helper lipid components are preferably selected from the group comprising phospholipids and steroids. The phospholipids are preferably diesters and monoesters of phosphate. Preferred members of the phospholipids are phosphoglycerides and sphingolipids. The steroids, as used herein, are naturally occurring and synthetic compounds based on partially hydrogenated cyclopenta[a]phenanthrene. Preferably, the steroids contain 21 to 30 carbon atoms. A particularly preferred steroid is cholesterol.

[0194] Without adhering to any particular theory, it should be noted that remarkable effects may be achieved due to a specific molar percentage of helper lipids (which may be either PEG-free or PEG-containing) contained in the lipid composition according to the present invention, and more particularly, when the content of any of these types of helper lipids falls within the concentration range specified herein.

[0195] In further embodiments of the present invention, the lipid composition, preferably existing as a lipoplex or liposome, preferably exhibits a neutral or overall anionic charge. The anionic lipid is preferably any neutral or anionic lipid described herein. In preferred embodiments, the lipid composition comprises any helper lipid or combination of helper lipids and any TLR inhibitor described herein. In further embodiments, a composition according to the present invention containing nucleic acid(s) forms a lipoplex. In preferred embodiments, the term lipoplex, as used herein, refers to a composition comprising neutral or anionic lipids, neutral helper lipids and the TLR inhibitor of the present invention. For references to the use of helper lipids in this art, see, for example, U.S. Patent Application Publication No. 2011 / 0178164, OJEDA, et. al., Int. J. of Pharmaceutics (March 2016), DABKOWSKA, et. al., JR Soc. Interface 9, pp. 548-561 (2012), and MOCHIZUKI, et. al., Biochimica et. Biophysica Acta, 1828, pp. 412-418 (2013).

[0196] In preferred embodiments, the helper lipids of the present invention include the helper lipids listed in Table II.

[0197] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is CHEMS and the drug portion is TR6.

[0198] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is CHEMS, the drug portion is TR6, and further comprises LU, where LU is a hydromethylcarbamate linker.

[0199] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is CHEMS, the drug moiety is TR6, further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising a helper lipid component, and the helper lipid component comprises the helper lipids of Table II.

[0200] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is CHEMS, the drug moiety is TR6, and CHEMS is monovalent.

[0201] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid and the drug moiety is TR6.

[0202] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the drug moiety is TR6, and stearic acid is monovalent.

[0203] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the drug moiety is TR6, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0204] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the chemical composition is TR6, further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising a helper lipid component, and the helper lipid component comprises the helper lipids of Table II.

[0205] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is CHEMS and the drug moiety is TR5(B).

[0206] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is CHEMS, the drug moiety is TR5(B), and further comprising LU, where LU is a hydromethylcarbamate linker.

[0207] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is CHEMS, the drug moiety is TR5(B), further comprising LU, and LU is a hydroxymethylcarbamate linker, further comprising a helper lipid component, and the helper lipid component comprises the helper lipid of Table II.

[0208] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is CHEMS, the drug moiety is TR5(B), and CHEMS is monovalent.

[0209] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid and the drug moiety is TR5(B).

[0210] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the drug moiety is TR5(B), and stearic acid is monovalent.

[0211] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the drug moiety is TR5(B), and further comprising LU, and LU is a hydroxymethylcarbamate linker.

[0212] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the chemical composition is TR5(B), and further comprising LU, and LU is a hydroxymethylcarbamate linker, further comprising a helper lipid component, and the helper lipid component comprises the helper lipid of Table II.

[0213] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is CHEMS and the drug moiety is TR3.

[0214] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is CHEMS, the drug portion is TR3, and further comprises LU, where LU is a hydromethylcarbamate linker.

[0215] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is CHEMS, the drug portion is TR3, and further comprises LU, where LU is a hydromethylcarbamate linker, and further comprises helper lipid components, the helper lipid components comprising the helper lipids of Table II.

[0216] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is CHEMS, the drug portion is TR3, and CHEMS is monovalent.

[0217] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is stearic acid and the drug portion is TR3.

[0218] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is stearic acid, the drug portion is TR3, and the stearic acid is monovalent.

[0219] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is stearic acid, the drug portion is TR3, and further comprises LU, where LU is a hydromethylcarbamate linker.

[0220] In one embodiment, the TLR prodrug comprises the lipid of the present invention, where the lipid is stearic acid, the chemical composition is TR3, further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising a helper lipid component, the helper lipid component comprising the helper lipids of Table II.

[0221] One skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without changing the functions and purposes of the invention disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure. V.) Linking Unit (“LU”)

[0222] In some embodiments, the subject matter of the present disclosure provides prodrugs that include biodegradable linkages, such as esters, thioesters, and other linkers known in the art, in drug-lipid conjugates.

[0223] Exemplary embodiments of ester chemistry are described herein. [Chemical formula]

[0224] In some embodiments, the prodrug is a drug-lipid conjugate, where the drug-lipid conjugate is cleaved by an esterase.

[0225] In one embodiment, the prodrug of the present invention includes a LU via a secondary amine, amide, or aniline using the following scheme. [Chemical formula] Exemplary syntheses are as follows. [Chemical formula] Cleavage of prodrug structures containing a secondary amine, amide, or aniline is obtained via esterase hydrolysis of the secondary amine, amide, or aniline prodrug under the following exemplary syntheses: [Chemical formula] [wherein, R1 and R2 can be molecules that are bonded to N via C.

[0226] In one embodiment, the secondary amide nitrogen of the TR6 drug moiety is conjugated to CHEMS via a hydromethylcarbamate linker.

[0227] In one embodiment, the secondary amide nitrogen of the TR6(A) drug moiety is conjugated to CHEMS via a hydromethylcarbamate linker.

[0228] In one embodiment, the secondary amide nitrogen of the TR5 drug moiety is conjugated to CHEMS via a hydromethylcarbamate linker.

[0229] In one embodiment, the secondary amide nitrogen of the TR5(A) drug moiety is conjugated to CHEMS via a hydromethylcarbamate linker.

[0230] In one embodiment, the secondary amide nitrogen of the TR5(B) drug moiety is conjugated to CHEMS via a hydromethylcarbamate linker.

[0231] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure. VI.) Nanocarriers

[0232] Generally speaking, and for the purposes of this disclosure, nanocarriers are within the scope of the present invention. Nanocarriers are nanomaterials used as transport modules for other substances, such as drugs. Commonly used nanocarriers include micelles, polymers, carbon-based materials, liposomes, and other materials. Because of their small size, nanocarriers can deliver drugs to sites in the body that would otherwise be inaccessible. Nanocarriers may include polymer conjugates, polymer nanoparticles, lipid-based carriers, dendrimers, carbon nanotubes, and gold nanoparticles. Lipid-based carriers include both liposomes and micelles.

[0233] In addition, nanocarriers are useful in drug delivery processes because they can deliver drugs to site-specific targets, ensuring that drugs are delivered to specific organs or cells rather than elsewhere. Site specificity offers significant therapeutic benefits by preventing the drug from being delivered to the wrong location. Furthermore, nanocarriers are promising for use in chemotherapy because they can help reduce the harmful, broader-scale toxicity of chemotherapy to rapidly growing healthy cells throughout the body. Since chemotherapy drugs can be highly toxic to human cells, it is crucial that chemotherapy drugs are delivered to tumors without being released to other parts of the body.

[0234] Generally speaking, there are four ways in which nanocarriers can deliver drugs, and these methods include passive targeting, active targeting, pH specificity, and temperature specificity.

[0235] Passive targeting refers to the ability of nanocarriers to circulate within the tumor's vascular system, be captured, and accumulate in the tumor. This accumulation is caused by enhanced permeability and retention. The tumor's leaky vascular structure is a network of blood vessels formed within the tumor, and this network contains many small pores. These pores not only allow nanocarriers to enter but also contain many bends that can capture them. As more nanocarriers are captured, the drug accumulates at the tumor site. This accumulation allows for the direct delivery of large doses of the drug to the tumor site.

[0236] Active targeting involves incorporating targeting modules, such as ligands or antibodies, that are specific to certain types of cells throughout the body, onto the surface of a nanocarrier. Generally, nanocarriers have a high surface area-to-volume ratio, allowing for the incorporation of multiple ligands onto their surface.

[0237] In addition, certain nanocarriers release the drugs they contain only within a specific pH range. This pH specificity also allows nanocarriers to deliver drugs directly to tumor sites. This is because tumors are generally more acidic than normal human cells, with a pH of approximately 6.8. Normal tissues have a pH of approximately 7.4. Therefore, nanocarriers that release drugs only within a specific pH range can be used to release drugs only within the acidic tumor environment. The highly acidic environment causes drug release by degrading the nanocarrier's structure. Generally, these nanocarriers do not release drugs in neutral or basic environments, leaving normal somatic cells intact, while effectively targeting the acidic environment of tumors. This pH sensitivity can also be induced in micelle systems by adding copolymer chains to micelles that are determined to act independently of pH. See WU, et. al., Biomaterials, 34(4): 1213-1222 (2012). These micelle-polymer complexes also help prevent cancer cells from developing multidrug resistance. The low pH environment triggers a rapid release of the micelle polymers, rather than the gradual release seen with other drug treatments, allowing the majority of the drug to be released in a single dose.

[0238] In addition, some nanocarriers have been shown to deliver drugs more effectively at certain temperatures. Since tumor temperature is generally around 40°C, which is higher than the temperature of the rest of the body, this temperature gradient helps act as a safeguard for delivery to tumor-specific sites. See REZAEI, et. al., Polymer, 53(16): 3485-3497 (2012).

[0239] As disclosed herein, lipid-based nanocarriers, such as liposomes, are within the scope of the present invention. Lipid-based nanoparticles (LBNPs or LNPs), such as liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs) transport hydrophobic and hydrophilic molecules, exhibit very low toxicity or no toxicity at all, and can increase drug action duration by extending the drug half-life and using controlled release. Lipid nanoparticles may include chemical modifications to avoid detection by the immune system (gangliosides or polyethylene glycol (PEG)) or to improve drug solubility. In addition, lipid nanoparticles can be prepared into pH-sensitive formulations to facilitate drug release in acidic environments and can also associate with small molecules or antibodies or their receptors (e.g., folic acid (FoA)) that recognize tumor cells. Nanodrugs can also be used in combination with other therapeutic strategies to improve patient response. See GARCIA-PINEL, et. al., Nanomaterials 9(639) (2019).

[0240] In various embodiments, the silicasome drug carriers described herein include nanoparticles of porous silica (or other materials) coated with a lipid bilayer (e.g., silica bodies having a surface and defining multiple pores suitable for receiving molecules inside). The fact that the nanoparticles are called silica nanoparticles does not preclude the incorporation of non-silica materials into the silica nanoparticles. In some embodiments, the silica nanoparticles may be substantially spherical and have multiple pore openings on their surface providing access to the pores. However, in various embodiments, the silica nanoparticles may have shapes other than substantially spherical. Thus, for example, in certain embodiments, the silica nanoparticles may be substantially oval, rod-shaped, substantially regular polygonal, irregular polygonal, and so on.

[0241] Generally, silica nanoparticles contain silica bodies that define the outer surfaces and inner side walls between pore openings. Pores can extend across the entire silica body to another pore opening, or they can extend only partially across the silica body, having a bottom surface defined by the silica body.

[0242] In some embodiments, the silica material is mesoporous. In other embodiments, the silica material is microporous. As used herein, “mesoporous” means having pores with a diameter between about 2 nm and about 50 nm, while “microporous” means having pores with a diameter smaller than about 2 nm. Generally, the pores can be of any size, but in typical embodiments, they are large enough to contain one or more therapeutic compounds. In such embodiments, the pores allow small molecules, such as therapeutic compounds, such as anticancer compounds, to adhere to or bind to the inner surface of the pores and be released from the silica material when used for therapeutic purposes. In some embodiments, the pores are substantially cylindrical.

[0243] In certain embodiments, the nanoparticles contain multiple pores having pore diameters between approximately 1 nm and 10 nm or between approximately 2 nm and 8 nm. In certain embodiments, the nanoparticles contain multiple pores having pore diameters between approximately 1 nm and 6 nm or between approximately 2 nm and 5 nm. Other embodiments contain particles with pore diameters less than 2.5 nm.

[0244] In other embodiments, the pore size is between 1.5 and 2.5 nm. For example, silica nanoparticles with other pore sizes can be prepared by using various surfactants or swelling agents during the preparation of silica nanoparticles. In various embodiments, nanoparticles can include particles as large as approximately 1000 nm (e.g., average or median diameter (or other characteristic dimension)). However, in various embodiments, nanoparticles are typically less than 500 nm or about 300 nm, as particles larger than 300 nm may not be effective in entering living cells or vascular fenestrations. In certain embodiments, nanoparticles range in size from about 40 nm, or from about 50 nm, or from about 60 nm to about 100 nm, or up to about 90 nm, or up to about 80 nm, or up to about 70 nm. In certain embodiments, nanoparticles range in size from about 60 nm to about 70 nm. Some embodiments include nanoparticles with an average maximum dimension between about 50 nm and about 1000 nm. Other embodiments include nanoparticles with an average maximum dimension between about 50 nm and about 500 nm. Still other embodiments include nanoparticles with an average maximum dimension between about 50 nm and about 200 nm.

[0245] In some embodiments, the average maximum dimension is greater than about 20 nm, greater than about 30 nm, greater than about 40 nm, or greater than about 50 nm. Other embodiments include nanoparticles having an average maximum dimension of less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 75 nm. As used herein, the size of a nanoparticle refers to the average or median size of the primary particle when measured by transmission electron microscopy (TEM) or similar visualization techniques known in the art. Further examples of mesoporous silica nanoparticles include, but are not limited to, MCM-41, MCM-48, and SBA-15. See KATIYARE, et. al., J. Chromotog. 1122(1-2): 13-20 (2006).

[0246] Methods for preparing porous silica nanoparticles are well known to those skilled in the art. In certain embodiments, mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate (TEOS) with a template prepared from micellar rods. The result is an aggregate of nano-sized spheres or rods filled with regularly arranged pores. The template can then be removed by washing with a solvent adjusted to the appropriate pH (see, for example, TREWYN et al. (2007) Chem. Eng. J. 137(1): 23-29).

[0247] In certain embodiments, mesoporous particles can also be synthesized using a simple sol-gel method (see, for example, NANDIYANTO, et al. (2009) Microporous and Mesoporous Mat. 120(3): 447-453). In certain embodiments, tetraethyl orthosilicate can also be used with additional polymer monomers as templates. In certain embodiments, 3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS.

[0248] In certain embodiments, mesoporous silica nanoparticles are cores synthesized by a modification of the sol / gel procedure described by MENG et. al. (2015) ACS Nemo, 9(4): 3540-3557.

[0249] The methods described herein have been demonstrated with respect to porous silica nanoparticles (e.g., mesoporous silica), but those skilled in the art will recognize that similar methods can be used with other porous nanoparticles. A number of other mesoporous materials that can be used in drug delivery nanoparticles are known to those skilled in the art. For example, in certain embodiments, mesoporous carbon nanoparticles could be used.

[0250] 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.).

[0251] Similarly, in certain embodiments, mesoporous polymer particles can be utilized. The synthesis of highly ordered mesoporous polymers and carbon frameworks from organic-organic assemblies of triblock copolymers and soluble low molecular weight phenolic resin precursors (resoles) by an evaporation-induced self-assembly strategy was reported by MENG, et. al. (2006) Chem. Mat. 6(18): 4447-4464.

[0252] The nanoparticles described herein are illustrative and non-limiting. Using the teachings provided herein, numerous other lipid bilayer-coated nanoparticles become available to those skilled in the art.

[0253] In one embodiment, the present invention teaches a nanocarrier containing a TLR prodrug.

[0254] In one embodiment, the present invention teaches a nanocarrier comprising a TLR prodrug, wherein the TLR prodrug comprises TR3.

[0255] In one embodiment, the present invention teaches a nanocarrier comprising a TLR prodrug, wherein the TLR prodrug comprises TR6.

[0256] In one embodiment, the present invention teaches a nanocarrier comprising a TLR prodrug, wherein the TLR prodrug comprises TR5.

[0257] In one embodiment, the present invention teaches a nanocarrier containing liposomes, wherein the lipids include CHEMS.

[0258] In one embodiment, the present invention teaches a nanocarrier containing liposomes, wherein the lipid contains stearic acid.

[0259] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises CHEMS, and the liposomes further comprise a TLR prodrug.

[0260] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises CHEMS, and the liposomes further comprise TR6.

[0261] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises CHEMS, and the liposomes further comprise TR3.

[0262] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises CHEMS, and the liposomes further comprise TR5.

[0263] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises CHEMS, and the liposomes further comprise TR5(B).

[0264] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid, and the liposomes further comprise a TLR inhibitor.

[0265] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid, and the liposomes further comprise TR3.

[0266] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid, and the liposomes further comprise TR6.

[0267] In one embodiment, the present invention teaches a nanocarrier comprising liposomes, wherein the lipid comprises stearic acid, and the liposomes further comprise TR5(B).

[0268] In one embodiment, the present invention teaches a nanocarrier containing a TLR lipid moiety.

[0269] In one embodiment, the present invention teaches a nanocarrier comprising a TLR lipid moiety, wherein the lipid moiety further comprises TR8.

[0270] In one embodiment, the present invention teaches a nanocarrier comprising a TLR lipid moiety, wherein the lipid moiety further comprises TR11.

[0271] In one embodiment, the present invention teaches a nanocarrier comprising a TLR lipid moiety, wherein the lipid moiety further comprises alpha-galactosylceramide (α-GalCer).

[0272] The scope of this disclosure teaches three non-limiting possible modes of treatment using the formulated prodrug of the present invention. See PCT Patent Application Publication WO2018 / 213631.

[0273] The first mode of treatment involves combining a TLR prodrug with another treatment (e.g., another formulated prodrug that inhibits TLRs (e.g., TLR1 / 2, TLR4, TLR7, TLR8, and / or TLR7 / 8), a chemotherapeutic agent (e.g., an ICD-inducing chemotherapeutic agent), etc.) into a single liposome that enables systemic (or local) intracellular distribution and drug delivery to tumor sites. The dual delivery method achieved synergistic enhancement of adaptive and innate immunity, significantly improving animal survival time. In certain embodiments, the nanocarrier comprises vesicles (i.e., lipid bilayers that encapsulate fluids).

[0274] A second mode of treatment involves local delivery to the tumor or peritumoral region of a drug that inhibits TLRs, combined with a lipid (e.g., liposomes) containing inhibitors of TLRs (e.g., TLR1 / 2, TLR4, TLR7, TLR8, and / or TLR7 / 8). Such local delivery of TLR inhibitors combined with TLR prodrugs has been demonstrated to induce cytotoxic tumor killing and tumor reduction at the local site. These adaptive immune responses involve a boost to the innate immune system reflected by CRT expression and activation of a DC population particularly well-suited to produce a cytotoxic T cell response.

[0275] A third treatment modality involves vaccination using dying cancer cells (e.g., KPC cells), where TLR inhibition is induced ex vivo. Such vaccination has been found to induce a systemic immune response that can disrupt tumor growth at distant sites and enable adoption into non-immune animals. Those skilled in the art will recognize and implement the methods of the treatment modality provided herein. VII.) Liposomes

[0276] In one embodiment, the subject matter of the present disclosure is based on a method for providing a prodrug of the present disclosure (see the section titled "Prodrug") that is suitable for incorporating into a nanocarrier including a lipid coating layer to enhance the delivery of the corresponding prodrug and to provide a combination therapy including the prodrug. Advantages of using the prodrug of the present invention include enhanced control of formulation into LNPs (e.g., liposomes) of the present disclosure. This allows the prodrug to be maintained in an inactive form during systemic circulation, thereby enabling the liposome to release the activator after phagocytosis by cells, for example, within a tumor.

[0277] In certain embodiments, one or more TLR prodrugs (e.g., any one or more TLR prodrug inhibitors taught by formula I or formula II, and / or TR3, TR5, TR5(A), TR5(B), TR6, TR6(A)) (see the section titled “Prodrug”) are formulated as a lipid moiety that can form vesicle (e.g., liposome) structures in aqueous solution or as components of a lipid bilayer containing liposomes.

[0278] In certain embodiments, one or more TLR lipid moieties (e.g., formula III or formula IV, and / or any one or more TLR lipid moieties taught in TR8, TR11) (see the section titled “Lipids”) can be formulated and / or co-formulated in a vesicle (e.g., liposome) structure in aqueous solution, or form components of a lipid bilayer containing liposomes.

[0279] Liposomes can be used directly or provided as components in combined formulations (e.g., combinations with other drug or lipid portions or therapeutic modes disclosed herein).

[0280] In certain embodiments, the liposomes formulated with the TLR prodrug contain lipids, PHGP, vitamin E, cholesterol, and / or fatty acids.

[0281] In a particular embodiment, the formulated liposomes include a lipid moiety containing TR8.

[0282] In a particular embodiment, the formulated liposomes include a lipid moiety containing TR11.

[0283] In a particular embodiment, the formulated liposomes include a lipid moiety comprising formula III.

[0284] In a particular embodiment, the formulated liposome comprises a lipid moiety containing formula IV.

[0285] In certain embodiments, the formulated liposomes include a lipid moiety containing alpha-galactosylceramide (α-GalCer).

[0286] In one embodiment, the liposomes contain cholesterol.

[0287] In one embodiment, the liposome contains DPPG.

[0288] In one embodiment, the liposome contains DMPG.

[0289] In one embodiment, the liposome is Lyso PC.

[0290] In one embodiment, the liposome is (Δ9-Cis)PG.

[0291] In one embodiment, the liposomes contain Soy Lyso PC.

[0292] In one embodiment, the liposome contains PG.

[0293] In one embodiment, the liposome contains PA-PEG3-mannose.

[0294] In one embodiment, the liposomes contain C16 PEG2000 ceramide.

[0295] In one embodiment, the liposome contains MPLA.

[0296] In one embodiment, the liposome contains 3-deacyl MPLA.

[0297] In one embodiment, the liposomes contain CHEMS.

[0298] In one embodiment, the liposome contains stearic acid.

[0299] In one embodiment, the liposomes contain the phospholipids listed in Table III.

[0300] In one embodiment, the liposome comprises TR6, further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0301] In one embodiment, the liposome comprises TR6, further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0302] In one embodiment, the liposome comprises TR6, further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0303] In one embodiment, the liposome comprises TR6, further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0304] In one embodiment, the liposome contains TR5.

[0305] In one embodiment, the liposome comprises TR5, further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0306] In one embodiment, the liposome comprises TR5, further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0307] In one embodiment, the liposome comprises TR5, further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0308] In one embodiment, the liposome comprises TR5, further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0309] In one embodiment, the liposome comprises TR5(B), further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0310] In one embodiment, the liposome comprises TR5(B), further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0311] In one embodiment, the liposome comprises TR5(B), further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0312] In one embodiment, the liposome comprises TR5(B), further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0313] In one embodiment, the liposome comprises TR3, further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0314] In one embodiment, the liposome comprises TR3, further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker.

[0315] In one embodiment, the liposome comprises TR3, further comprising CHEMS, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0316] In one embodiment, the liposome comprises TR3, further comprising stearic acid, and further comprising LU, where LU is a hydromethylcarbamate linker, and further comprising the helper lipids listed in Table II.

[0317] In one embodiment, the liposomes of the present disclosure comprise a TLR prodrug co-formulated with one or more additional immunomodulators, wherein the immunomodulators include, but are not limited to, immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, sting agonists, CTLA4 inhibitors, PD-1 inhibitors, and / or their prodrugs.

[0318] In one embodiment, the liposomes of the present disclosure comprise a TLR prodrug co-formulated with one or more additional immunomodulators, wherein the immunomodulators include, but are not limited to, neurokinin 1 (NK1) antagonists and / or their prodrugs.

[0319] In one embodiment, the liposomes of the present disclosure comprise a TLR prodrug co-formulated with one or more additional immunomodulators, wherein the immunomodulators include, but are not limited to, A2aR antagonists and / or their prodrugs.

[0320] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with an ICD-inducing chemotherapeutic agent.

[0321] In a preferred embodiment, the liposome comprises a TLR prodrug co-formulated with an ICD-inducing chemotherapeutic agent selected from the list of doxorubicin (DOX), mitoxantrone (MTO), oxaliplatin (OXA), cyclophosphamide (CP), bortezomib, carfilzomib, or paclitaxel.

[0322] In a preferred embodiment, the liposome contains a TLR prodrug co-formulated with a Toll receptor TLR agonist / prodrug.

[0323] In a preferred embodiment, the liposome contains a TLR prodrug co-formulated with a Toll receptor (TLR) agonist / prodrug selected from the list of rexiquimod (R848), gardikimod, 852A, DSR6434, telratolimod, CU-T12-9, monophosphoryllipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD®.

[0324] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with a PD-1 inhibitor / prodrug.

[0325] In a preferred embodiment, the liposome comprises a TLR prodrug co-formulated with a PD-1 inhibitor / prodrug selected from AUNP12, CA-170, or BMS-986189 or a list of its prodrugs.

[0326] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with doxorubicin (DOX).

[0327] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with mitoxantrone (MTO).

[0328] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with doxorubicin (DOX) and a PD-1 prodrug.

[0329] In a preferred embodiment, the liposomes include a TLR prodrug co-formulated with mitoxantrone (MTO) and a PD-1 prodrug.

[0330] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with doxorubicin (DOX) and an IDO antagonist / prodrug.

[0331] In a preferred embodiment, the liposomes include a TLR prodrug co-formulated with mitoxantrone (MTO) and an IDO antagonist / prodrug.

[0332] In preferred embodiments, the liposomes contain doxorubicin (DOX) and a TLR prodrug co-formulated with a PD-1 prodrug and an IDO antagonist / prodrug.

[0333] In preferred embodiments, the liposomes include mitoxantrone (MTO) and a TLR prodrug co-formulated with a PD-1 prodrug and an IDO antagonist / prodrug.

[0334] In a preferred embodiment, the liposome comprises a TLR prodrug co-formulated with an IDO antagonist / prodrug.

[0335] In a preferred embodiment, the liposome comprises a TLR prodrug co-formulated with an IDO antagonist / prodrug.

[0336] In a preferred embodiment, the liposomes include a TLR prodrug co-formulated with an IDO antagonist / prodrug and a PD-1 prodrug.

[0337] In a preferred embodiment, the liposomes include a TLR prodrug co-formulated with an IDO antagonist / prodrug and a PD-1 prodrug.

[0338] In a preferred embodiment, the liposomes contain TR6 co-formulated with doxorubicin (DOX).

[0339] In a preferred embodiment, the liposomes contain TR6 co-formulated with mitoxantrone (MTO).

[0340] In a preferred embodiment, the liposome comprises TR6 co-formulated with doxorubicin (DOX) and / or an IDO prodrug and / or an IDO antagonist / prodrug.

[0341] In a preferred embodiment, the liposome comprises TR6 co-formulated with mitoxantrone (MTO) and / or an IDO prodrug and / or an IDO antagonist / prodrug.

[0342] In a preferred embodiment, the liposomes contain TR6 co-formulated with NK1.

[0343] In a preferred embodiment, the liposome comprises TR6 co-formulated with MTO and TR8.

[0344] In a preferred embodiment, the liposome comprises TR6 co-formulated with DOX and the A2aR prodrug.

[0345] In a preferred embodiment, the liposomes contain TR5 co-formulated with DOX.

[0346] In a preferred embodiment, the liposomes contain TR5 co-formulated with the A2aR prodrug.

[0347] In a preferred embodiment, the liposome comprises TR5 co-formulated with DOX and the A2aR prodrug.

[0348] In a preferred embodiment, the liposome comprises TR5 co-formulated with the A2aR prodrug and an anti-PD1 antibody.

[0349] In a preferred embodiment, the liposome comprises TR5 co-formulated with the A2aR prodrug and the IDO prodrug.

[0350] In a preferred embodiment, the liposomes contain TR5(B) co-formulated with doxorubicin (DOX).

[0351] In a preferred embodiment, the liposomes contain TR5(B) co-formulated with mitoxantrone (MTO).

[0352] In a preferred embodiment, the liposome comprises TR5(B) co-formulated with doxorubicin (DOX) and / or an IDO prodrug and / or an IDO antagonist / prodrug.

[0353] In a preferred embodiment, the liposome comprises TR5(B) co-formulated with mitoxantrone (MTO) and / or an IDO prodrug and / or an IDO antagonist / prodrug.

[0354] In a preferred embodiment, the liposomes contain TR8 co-formulated with doxorubicin (DOX).

[0355] In a preferred embodiment, the liposomes contain TR8 co-formulated with mitoxantrone (MTO).

[0356] In a preferred embodiment, the liposome comprises TR8 co-formulated with doxorubicin (DOX) and / or a TLR prodrug and / or an IDO antagonist / prodrug.

[0357] In a preferred embodiment, the liposome comprises TR8 co-formulated with mitoxantrone (MTO) and / or a TLR prodrug and / or an IDO antagonist / prodrug.

[0358] In a preferred embodiment, the liposomes contain TR11 co-formulated with doxorubicin (DOX).

[0359] In a preferred embodiment, the liposomes contain TR11 co-formulated with mitoxantrone (MTO).

[0360] In a preferred embodiment, the liposome comprises TR11 co-formulated with doxorubicin (DOX) and / or a TLR prodrug and / or an IDO antagonist / prodrug.

[0361] In a preferred embodiment, the liposome comprises TR11 co-formulated with mitoxantrone (MTO) and / or a TLR prodrug and / or an IDO antagonist / prodrug.

[0362] In another preferred embodiment, the liposomes comprise solid lipid nanoparticles (SLNPs) that include liposomes containing a TLR prodrug.

[0363] Those skilled in the art will recognize and understand that solubility is one of the most common problems they face in the drug development process. Chemical conjugations of drugs / anticancer agents via lipid molecules (i.e., lipid-based prodrugs) provide a platform for solving problems in formulating drugs into aqueous suspensions. The main advantage of delivering drugs using lipid conjugations (lipid-based prodrugs) lies in their ability to improve pharmacokinetics / half-life and targeted delivery.

[0364] Lipid-based prodrugs(s) can be integrated / formulated into liposomal formulations using techniques known in the art, by appropriately selecting lipid molecules, thereby offering many advantages over conventional drug delivery systems. (KOHLI, et. al., J. Control Release, 0:pp 274-287 (Sept. 28, 2014) and GARCIA-PINEL, et. al., Nanomaterials 9:638 (2019)). The advantages of combining lipid-prodrugs with liposomes are doubled because (i) liposomes containing lipid-prodrugs not only increase the solubility of the drug / prodrug itself, but also (ii) they have the ability to encapsulate multiple drugs (both hydrophilic and lipophilic) (see the section titled Nanocarriers).

[0365] For the purposes of this disclosure, the main advantages of liposomal formulations are as follows: i) The liposome formulation must be biocompatible / biodegradable and have no general toxicity. ii) The flexibility and manipulability of the size and surface charge may vary depending on the required purpose. For the purposes of this disclosure, liposome formulations may have a size range of 40 to 150 nm in diameter and a surface charge range of -40 to +40 mV. Furthermore iii) The liposomes of the present invention have one or more lipid-prodrugs as the lipid portion that constitutes the liposome(s). In addition, multiple drugs having different solubility profiles (hydrophilic or lipophilic) (e.g., acting by different mechanisms of action) can be formulated into these liposomes (either in the lipid bilayer or the hydrophilic core).

[0366] As is recognized by those skilled in the art, all methods for producing liposomes involve the following four basic steps: (i) The step of drying the lipids from the organic solvent, (ii) The step of dispersing lipids in an aqueous solution, (iii) the step of purifying the obtained liposomes, and (iv) The final product is analyzed. See AKBARZADEH, et. al., Nanoscale Research Letters, 8:102 (2013).

[0367] Another aspect of the present invention discloses liposome encapsulation technology (LET), a delivery technology used to permeate drugs. LET is a method for creating submicroscopic foamy structures called liposomes that encapsulate a number of materials. These “liposomes” form a barrier around their contents that is resistant to oral and gastric enzymes, alkaline solutions, digestive fluids, bile salts, as well as the intestinal flora and free radicals produced in the human body. Thus, the contents of the liposome are protected from oxidation and degradation. This protective phospholipid shield or barrier remains intact until the contents of the liposome are delivered to a precise target gland, organ, or system where they are utilized (see the section titled Nanocarriers).

[0368] In one embodiment, the liposome(s) of the Disclosure are synthesized using several different ratios of TLR prodrugs, TLR lipid moieties, lipids, and / or lipid-prodrugs. As disclosed herein, the TLR prodrug may include the helper lipids disclosed herein (see, for example, Table II).

[0369] In one embodiment, the liposome(s) of this disclosure are synthesized using several different ratios of TLR prodrugs, TLR lipid moieties, lipids, and / or lipid-prodrugs. As disclosed herein, the TLR prodrug may further comprise DSPE-PEG.

[0370] In a preferred embodiment, the liposome of the present invention comprises a composition having the following ratios. [Table A]

[0371] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios. [Table B]

[0372] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios. [Table C-1] [Table C-2] Here, lipid 1 includes TR5 and CHEMS.

[0373] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios. [Table D] Here, lipid 1 includes TR5 and stearic acid.

[0374] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios. [Table E] Here, lipid 1 includes TR6 and CHEMS.

[0375] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios. [Table F] Here, lipid 1 includes TR6 and stearic acid.

[0376] In a more preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios. [Table G] Here, lipid 1 includes TR3 and stearic acid.

[0377] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure. VIII.) Pharmaceutical Formulation

[0378] As used herein, the term “drug” is synonymous with “pharmaceutical.” In certain embodiments, the liposomes of this disclosure are processed into encapsulated dosage forms and administered to a patient for the treatment of a disease.

[0379] Generally speaking, pharmaceutical formulation is the process of combining various chemical substances with a pure active pharmaceutical ingredient (API) to produce the final drug product. Formulation research involves developing drug preparations that are stable and tolerable for patients. For orally administered drugs, this typically involves incorporating the drug into tablets or capsules. It is important to fully understand that dosage forms contain various other substances besides the drug itself, and research must be conducted to ensure that the drug is compatible with these other substances.

[0380] Excipients are inert substances used as carriers for the active ingredient of a drug product, in this case as liposomes containing the TLR prodrug. In addition, excipients can be used to assist in the process by which the drug product is manufactured. The active ingredient is then dissolved or mixed with the excipient. Excipients are also sometimes used to increase the volume of formulations containing very potent active ingredients, enabling convenient and precise dosage. Once an active ingredient has been purified, it cannot remain in its purified form for extended periods. Often, the active ingredient denatures, separates from the solution, or adheres to the sides of the container.

[0381] Excipients are added to stabilize the active ingredient, ensuring that the active ingredient remains active and stable for a sufficiently long period to make the product competitive against other products and safe for the end user. Examples of excipients include, but are not limited to, anti-adhesives, binders, coatings, disintegrants, fillers, diluents, flavorings, colorants, lubricants, and preservatives. The final formulation contains the active ingredient and excipients, which are then encapsulated in a pharmaceutical dosage form.

[0382] Pre-formulation involves characterizing the physical, chemical, and mechanical properties of a drug in order to select which other components should be used in the preparation. Next, formulation studies consider factors such as stability, particle size, polymorphism, pH, and solubility, as all of these can affect bioavailability and therefore the activity of the drug. The drug must be combined with inert additives in a manner that ensures the amount of drug present is consistent in each dose unit (e.g., each vial). The doses should have a uniform appearance.

[0383] These studies are unlikely to be completed by the start of clinical trials. This means that simpler preparations will be developed early on for use in Phase I clinical trials. These typically consist of vials or hand-filled capsules containing small amounts of the drug and diluent. Evidence of long-term stability of these formulations is not necessary because they will only be used (tested) for a few days. However, long-term stability is crucial in supply chain management because the time from when the final formulation is packaged until it reaches patients can be months or even years. Something called drug load (i.e., the ratio of active drug to the total content of the dose) must be considered. A low drug load can cause homogeneity problems. A high drug load can lead to flow problems or require larger capsules if the bulk density of the compound is low. By the time Phase III clinical trials are reached, the drug formulation should be developed to be close to the final preparation used on the market.

[0384] Knowledge of stability is essential up to this stage, and conditions must be developed to ensure that the drug is stable in the preparation. If the drug is found to be unstable, the results of clinical trials will be invalid, as it would be impossible to know what the actual dose was. Stability studies are conducted to test whether temperature, humidity, oxidation, or photodegradation (ultraviolet or visible light) has any effect, and the preparation is analyzed to see if any degradation products have been formed. It is also important to check for any undesirable interactions between the preparation and the container. If a plastic container is used, tests are conducted to see if any of the components adsorb onto the plastic, and whether any plasticizers, lubricants, pigments, or stabilizers leach from the plastic into the preparation. The adhesive for the container label also needs to be tested to ensure that it does not leach from the plastic container into the preparation. The way the drug is formulated can avoid some of the problems associated with oral administration. Drugs are usually taken orally as tablets or capsules. The drug (active substance) itself needs to dissolve in an aqueous solution at a controlled rate. Factors such as particle size and crystal morphology can significantly affect dissolution. Rapid dissolution is not always ideal. For example, a slow dissolution rate can prolong the duration of action or avoid initial high plasma levels.

[0385] In some embodiments, nanocarriers (e.g., liposomes containing the TLR prodrug) and / or liposomes co-formulated with an immunomodulatory agent, including a TLR prodrug, are administered alone or in mixtures with a physiologically acceptable carrier (e.g., saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practices. For example, when used as an injectable, the nanocarrier can be formulated as a sterile suspension, dispersion, or emulsion using a pharmaceutically acceptable carrier. In certain embodiments, normal saline can be used as the pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, 5% glucose, etc., including glycoproteins to enhance stability, such as albumin, lipoprotein, or globulin. In compositions containing a carrier with saline or other salts, the carrier is preferably added after nanocarrier formation. Thus, after the nanocarrier is formed and loaded with the appropriate drug(s), the nanocarrier can be diluted with a pharmaceutically acceptable carrier, such as normal saline. Similarly, TLR prodrug liposomes can be introduced into carriers that facilitate the suspension (e.g., emulsification, dilution, etc.) of nanomaterials.

[0386] The pharmaceutical composition may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions, suspensions, dispersions, emulsions, etc., may be packaged for use or filtered under sterile conditions. In certain embodiments, drug delivery nanocarriers (e.g., nanoparticles coated with LB) are lyophilized, and the lyophilized preparation is mixed with a sterile aqueous solution before administration. The composition may also contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, osmotic pressure adjusters, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0387] In addition, in certain embodiments, the pharmaceutical formulation may include a lipid protectant that protects lipids from free radical and lipid peroxidation damage during storage. Suitable and intended herein are lipophilic free radical quenchers, such as alpha-tocopherol, and water-soluble iron-specific chelating agents, such as ferrioxamine. The concentration of nanocarriers (e.g., liposomes containing TLR prodrugs) in the pharmaceutical formulation can vary widely, for example, from less than approximately 0.05%, typically from at least approximately 2-5% to as much as 10-50%, or up to 40%, or up to 30% by weight, and is selected mainly by fluid volume, viscosity, etc., according to a chosen specific administration method. For example, the concentration may be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients with atherosclerosis-related congestive heart failure or severe hypertension. Alternatively, nanocarriers composed of irritating lipids may be diluted to low concentrations to reduce inflammation at the administration site. The amount of nanocarrier administered depends on the specific drug used, the medical condition being treated, and the clinician's judgment, but is generally between approximately 0.01 mg and 50 mg per kilogram of body weight, preferably between approximately 0.1 mg and 5 mg per kilogram of body weight.

[0388] Those skilled in the art will recognize that the precise dosage varies depending on the specific TLR prodrug and any co-formulated immunomodulator, as well as the desired medical effect, and patient factors such as age, sex, and overall condition. Those skilled in the art can take these factors into account and use them to easily establish effective therapeutic concentrations without excessive experimentation.

[0389] For the administration of the drugs to humans (or non-human mammals) in the curative, remission, retardative, or prophylactic treatment of the diseases described herein, the prescribing physician will ultimately determine the appropriate dosage of the drug for a given human (or non-human) subject, which can be expected to vary according to the individual's age, weight, and response, as well as the nature and severity of the patient's disease. In certain embodiments, the dosage of the drug delivered by the nanocarrier may be approximately the same as that used for the free drug. However, as stated above, the nanocarriers described herein can significantly reduce the toxicity of the drug(s) administered thereby and significantly increase the therapeutic range. Therefore, in some cases, a dosage exceeding that prescribed for the free drug(s) may be used.

[0390] Those skilled in the art will recognize and be able to make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention as disclosed herein. Such changes and modifications are intended to be within the scope of this disclosure. IX.) Combination Therapy

[0391] As is recognized and understood by those skilled in the art, the growth and survival of cancer cells may be influenced by multiple signaling pathways. Therefore, to treat such conditions, it is useful to combine various enzyme / protein / receptor inhibitors that exhibit different priorities to targets and modulate the activity of the targets. By targeting one or more signaling pathways (or one or more biological molecules involved in a given signaling pathway), the likelihood of drug resistance developing in a cell population and / or the toxicity of the treatment can be reduced.

[0392] Accordingly, liposomes comprising the TLR prodrugs of this disclosure can be used in combination with one or more other enzyme / protein / receptor inhibitors or one or more therapies for the treatment of diseases, such as cancer or infections. Examples of diseases and indications that can be treated with combination therapy are described herein. Examples of cancers include, but are not limited to, solid tumors and humoral tumors, such as hematological malignancies. Examples of infections include viral infections, bacterial infections, fungal infections or parasitic infections.

[0393] For example, liposomes containing the TLR prodrugs of this disclosure can be combined with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, PI3K (alpha, beta, gamma, delta), CSFIR, KIT, FLK-II, KDR / FLK-1, FLK-4, flt- 1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, TAM kinase (Axl, Mer, Tyro3), FLT3, VEGFR / Flt2 , Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf.

[0394] In further embodiments, liposomes comprising the TLR prodrug of this disclosure may be combined with one or more of the following inhibitors for the treatment of cancer or infectious diseases. Non-limiting examples of inhibitors that can be combined with the compounds of this disclosure for the treatment of cancer and infectious diseases include: FGFR inhibitors (FGFR1, FGFR2, FGFR3 or FGFR4, e.g., INCB54828, INCB62079 and INCB63904), JAK inhibitors (JAK1 and / or JAK2, e.g., ruxolitinib, baricitinib or INCB39110), TLR inhibitors (e.g., epacadostat, NLG919 or BMS-986205), LSD1 inhibitors (e.g., INCB59872 and INCB60003), TDO inhibitors, PI3K-delta inhibitors (e.g., INCB50797 and INCB50465), PI3K-gamma inhibitors, e.g., PI3K-gamma selective inhibitors, Pim inhibitors (e.g., INCB53914), CSF1R inhibitors, Examples include TAM receptor tyrosine kinases (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.

[0395] In further embodiments, liposomes comprising the TLR prodrugs of the present disclosure may be combined with one or more activators of invariant natural killer T (iNKT) cells, including, but not limited to, α-galactosylceramide (α-GalCer) and its analogues, including C8-galactosyl(α)ceramide, C16-galactosyl(α)ceramide, and C24:1-galactosyl(α)ceramide (Avanti Polar Lipids, Alabaster, Alabama).

[0396] In addition, liposomes comprising the TLR prodrugs of this disclosure can be used in combination with other methods of treating cancer, such as chemotherapy, radiotherapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, or surgery.

[0397] 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.

[0398] Liposomes containing TLR prodrugs can be used in combination with one or more anticancer drugs, such as chemotherapeutic agents. Examples of chemotherapeutic agents include avalerix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezombi, bortezomib, intravenous busulfan, oral busulfan, carsterone, capecitabine, carboplatin, Carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin difutitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, e Stramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alpha-2a, irino Tecan, lapatinib ditosylate, lenalTLRmide, letrozole, leucovorin, leuprolide acetate, rebamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenylpropionatePhenpropionate, Nelarabine, Nofetumomab, Olaparib, Oxaliplatin, Paclitaxel, Pamidronate, Panitumumab, Pegaspargase, Pegfilgrastim, Pemetrexed disodium, Pentostatin, Pipobroman, Plicamycin, Procarbazine, Quinacrine, Rasburicase, Rituximab, Ruxolitinib, Rucaparib, Sorafenib, Streptozocin, Sunitinib, Mare Examples include sunitinib iodide, tamoxifen, temozolomide, teniposide, testactone, thalidomide (thalTLRmide), thioguanine, thiotepa, topotecan, toremifene, tocitumomab, trastuzumab, tretinoin, uracil mustard, barrubicin, vinblastine, vincristine, vinorelbine, vorinostat, niraparib, beriparib, talazoparib, and zoledronate.

[0399] Other anticancer drugs (multiple options may be used) include antibody therapies such as trastuzumab (Herceptin), costimulatory molecules such as antibodies against CTLA-4 (e.g., ipilimumab), antibodies against 4-1BB (e.g., urelumab, utomirumab), antibodies against PD-1 and PD-L1 / L2, or antibodies against cytokines (IL-10, TGF-beta, etc.).

[0400] Examples of antibodies against PD-1 and / or PD-L1 / L2 that can be combined with the compounds of this disclosure for the treatment of cancer or infectious diseases, such as viral, bacterial, fungal, and parasitic infections, include, but are not limited to, nivolumab, pembrolizumab, MPDL3280A, MEDI-4736, and SHR-1210.

[0401] In addition, liposomes comprising the TLR prodrugs of this disclosure can be used in combination with one or more immune checkpoint inhibitors for the treatment of diseases, such as cancer or infections. Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules, such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, PD-1, PD-L1, and PD-L2.

[0402] 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, TLR, KIR, LAG3, PD-1, TIM3, and VISTA. In further embodiments, liposomes containing the TLR prodrug provided herein can be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGFR beta inhibitors. X.) Method for delivering liposomes containing a TLR prodrug to Toll-like receptor ("TLR") expressing cells.

[0403] As is well 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 the context of cancer, a typical method involves administering a biologically effective amount of the TLR prodrug of the Disclosure and / or liposomes of the Disclosure containing the TLR prodrug to a mammal having a tumor.

[0404] A typical embodiment is a method for delivering a therapeutic agent to cells expressing TLR1 / 2, TLR4, TLR7, TLR8, and / or TLR7 / 8, comprising forming a TLR prodrug by conjugating the drug portion of the Disclosure to a lipid of the Disclosure via a linking unit, and exposing the cells to the TLR prodrug.

[0405] In one embodiment, the TLR prodrug comprises a drug moiety of formula I and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0406] In one embodiment, the TLR prodrug comprises the drug moiety of formula I and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0407] In one embodiment, the TLR prodrug comprises a drug moiety of formula II and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0408] In one embodiment, the TLR prodrug comprises the drug moiety of formula II and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0409] In one embodiment, the TLR prodrug comprises TR3 and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0410] In one embodiment, the TLR prodrug comprises TR3 and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0411] In one embodiment, the TLR prodrug comprises TR6 and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0412] In one embodiment, the TLR prodrug comprises TR6 and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0413] In one embodiment, the TLR prodrug comprises TR5(B) and CHEMS conjugated via a LU containing a hydromethylcarbamate linker.

[0414] In one embodiment, the TLR prodrug comprises TR5(B) and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0415] 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 a TLR prodrug, which is produced by conjugating a drug portion with a lipid of the Disclosure via a linking unit, thereby exposing cells to the TLR prodrug.

[0416] In one embodiment, the TLR prodrug comprises a drug moiety of formula I and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0417] In one embodiment, the TLR prodrug comprises the drug moiety of formula I and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0418] In one embodiment, the TLR prodrug comprises a drug moiety of formula II and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0419] In one embodiment, the TLR prodrug comprises the drug moiety of formula II and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0420] In one embodiment, the TLR prodrug comprises TR3 and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0421] In one embodiment, the TLR prodrug comprises TR3 and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0422] In one embodiment, the TLR prodrug comprises TR6 and CHEMS conjugated via an LU containing a hydromethylcarbamate linker.

[0423] In one embodiment, the TLR prodrug comprises TR6 and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0424] In one embodiment, the TLR prodrug comprises TR5(B) and CHEMS conjugated via a LU containing a hydromethylcarbamate linker.

[0425] In one embodiment, the TLR prodrug comprises TR5(B) and stearic acid, conjugated via an LU containing a hydromethylcarbamate linker.

[0426] The TLR prodrugs, liposomes, and co-formulated liposomes of this disclosure inhibit the activity / protein interactions of TLR proteins and are therefore useful for treating diseases and disorders associated with TLR activity.

[0427] In further embodiments of the present disclosure, TLR prodrugs, liposomes, or pharmaceutically acceptable salts or stereoisomers thereof are useful for therapeutic administration to enhance, stimulate, and / or increase immunity in cancer, chronic infection, or sepsis, including enhancing the response to vaccination.

[0428] In further embodiments, the Disclosure provides a method for inhibiting T cell function of TLRs (e.g., TLR1 / 2, TLR4, TLR7, TLR8, and / or TLR7 / 8). The method comprises administering to an individual or patient a TLR prodrug, liposome, and / or any of the formulas described herein (e.g., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), or a TLR prodrug, liposome, and nanoencapsulated TLR inhibitor prodrug listed in any of the claims and described herein, or a pharmaceutically acceptable salt or stereoisomer thereof. The TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs of the Disclosure may be used alone, in combination with other agents or therapies, or as adjuvants or neoadjuvants to treat diseases or disorders, including cancer and other diseases. Any of the TLR prodrugs, liposomes, and nanoencapsulated TLR prodrugs of this disclosure, including any of the embodiments thereof, may be used for the uses and methods described herein.

[0429] In addition, the TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs of this disclosure inhibit TLR function and consequently block the TLR pathway.

[0430] In further embodiments, the disclosure provides in vivo treatments of an individual or patient using TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs or salts or stereoisomers thereof to inhibit the growth of cancerous tumors.

[0431] TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs, or any of the formulas described herein (e.g., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), or any of those listed in any of the claims, or any of the TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs, or their salts or stereoisomers, can be used to inhibit the growth of cancerous tumors.

[0432] Alternatively, the TLR prodrugs, liposomes, and nanoencapsulated TLR prodrugs of this disclosure, or any of the formulas described herein, or any of the compounds listed in any of the claims and described herein (e.g., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), or their salts or stereoisomers, may be used in conjunction with other agents or standard cancer treatments as described herein.

[0433] In further embodiments, the Disclosure provides a method for inhibiting tumor cell growth in vitro. The method involves contacting tumor cells in vitro with any of the TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs of the Disclosure, or any of the formulas described herein (e.g., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), or any of the TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs listed in any of the claims, or salts or stereoisomers thereof.

[0434] In further embodiments, the Disclosure provides a method for inhibiting the growth of tumor cells in a patient. The method comprises contacting tumor cells with any of the TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs of the Disclosure, or any of the formulas described herein (e.g., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), or any of the TLR prodrugs, liposomes, and nanoencapsulated TLR inhibitor prodrugs listed in any of the claims, or salts or stereoisomers thereof. XI.) Methods for treating cancer and other immunological disorders

[0435] Another embodiment of the present disclosure is a method for treating cancer. The method comprises administering to a patient a therapeutically effective amount of liposomes, compounds listed in any of the claims and described herein, or salts thereof, containing TLR prodrugs of the present (i.e., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)). Examples of cancer include cancers whose growth can be inhibited using TLR inhibitors and TLR prodrugs of the present disclosure, as well as cancers that are typically responsive to immunotherapy.

[0436] In some embodiments, the present disclosure provides a method for enhancing, stimulating, and / or increasing a patient's immune response. The method involves administering to a patient a therapeutically effective amount of a liposome containing a TLR prodrug and / or the TLR prodrug (i.e., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), a compound listed in any of the claims and described herein, or a salt thereof.

[0437] Non-limiting examples of cancers treatable with liposomes containing the TLR prodrugs of this disclosure, TLR prodrugs, and co-formulated liposomes include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, and genital cancer. Examples of cancers that may be included, but not limited to, include pedicle cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic or acute lymphoblastic leukemia, chronic or acute leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or urethral cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, and combinations of the above cancers. The compounds of this disclosure are also useful for treating metastatic cancers, particularly metastatic cancers that express TLRs.

[0438] In some embodiments, cancers treatable with the liposomes or TLR prodrugs of this disclosure include melanoma (e.g., metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), head and neck squamous cell carcinoma, urothelial carcinoma (e.g., bladder cancer), and high-frequency microsatellite instability (MSI). high This includes cancer. In addition, this disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the liposomes or TLR prodrugs or co-formulated liposomes of this disclosure.

[0439] In additional embodiments, cancers treatable with the formulations and / or co-formulated liposomes or TLR prodrugs of the Disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., lymphoma, leukemia, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin lymphoma, or multiple myeloma) and combinations of the said cancers.

[0440] In further embodiments, cancers treatable with the formulations and / or co-formulated liposomes or TLR prodrugs of this disclosure include, but are not limited to, cholangiocarcinoma, triple-negative breast cancer, rhabdomyosarcoma, small cell lung cancer, leiomyosarcoma, hepatocellular carcinoma, Ewing's sarcoma, brain cancer, brain tumors, astrocytoma, neuroblastoma, neurofibroma, basal cell carcinoma, chondrosarcoma, epithelioid sarcoma, ocular cancer, fallopian tube cancer, gastrointestinal cancer, gastrointestinal stromal tumors, hairy cell leukemia, intestinal cancer, islet cell carcinoma, oral cancer, throat cancer, laryngeal cancer, lip cancer, mesothelioma, cervical cancer, nasal cavity cancer, eye cancer, intraocular melanoma, pelvic cancer, rectal cancer, renal cell carcinoma, salivary gland cancer, paranasal sinus cancer, spinal cord cancer, tongue cancer, tubular cancer, urethral cancer, and ureteral cancer.

[0441] In addition, in some embodiments, the formulations and / or co-formulated liposomes or TLR prodrugs of the present disclosure can be used to treat sickle cell disease and sickle cell anemia.

[0442] Furthermore, in some embodiments, diseases and indications treatable with the formulations and / or co-formulated liposomes or TLR 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.

[0443] 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).

[0444] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, hamartoma, and teratoma.

[0445] Examples of lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell, anaplastic small cell, anaplastic large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatoid hamartoma, and mesothelioma.

[0446] Similar types of gastrointestinal cancers include esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastric cancer (cancer, lymphoma, leiomyosarcoma), pancreatic cancer (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), colorectal cancer (adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma, leiomyoma), and colorectal cancer.

[0447] Examples of genitourinary cancers include kidney cancer (adenocarcinoma, Wilms' tumor [nephroblastoma]), bladder and urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), and testicular cancer (seminocarcinoma, teratoma, embryonic carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma).

[0448] Examples of liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0449] Examples of bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulosarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor.

[0450] Examples of neurological cancers include skull cancer (osteoma, hemangioma, granuloma, xanthomas, osteoosteitis), meningeal cancer (meningioma, meningiosarcoma, gliomas), brain cancer (astrocytoma, meduoblastoma, glioma, ependymoma, germ cell tumor (pineal glandoma), glioblastoma, glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), and spinal cord cancer (neurofibroma, meningioma, glioma, sarcoma), as well as neuroblastoma and Lhermitt-Dukuro disease.

[0451] Examples of gynecological cancers include uterine cancer (endometrial cancer), cervical cancer (cervical cancer, pre-tumor cervical dysplasia), ovarian cancer (ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified cancer), granulosa-theca cell tumor, Sertoli-Leydig cell tumor, undifferentiated germ cell tumor, malignant teratoma), vulvar cancer (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, staphylosarcoma (embryonic rhabdomyosarcoma)), and fallopian tube cancer.

[0452] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic mole, lipoma, angioma, dermatofibroma, and keloid. In some embodiments, diseases and indications treatable with the compounds of this disclosure include, but are not limited to, sickle cell disease (e.g., sickle cell anemia), triple-negative breast cancer (TNBC), myelodysplastic syndrome, testicular cancer, cholangiocarcinoma, esophageal cancer, and urothelial carcinoma.

[0453] In addition, the formulations and / or co-formulated liposomes of the present disclosure, or the blockade of the TLR and / or kynurenine pathway using TLR prodrugs, can also be used to treat infectious diseases, such as viral, bacterial, fungal, and parasitic infections.

[0454] This disclosure provides a method for treating infectious diseases, such as viral infections. The method comprises administering to a patient a therapeutically effective dose of any of the formulated and / or co-formulated liposomes or TLR prodrugs listed in any of the claims and described herein, or a salt thereof, of any of the formulas described herein (i.e., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)).

[0455] 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.

[0456] In addition, this disclosure provides a method for treating a bacterial infection. The method comprises administering to a patient a therapeutically effective dose of any of the formulated and / or co-formulated liposomes or TLR prodrugs listed in any of the claims and described herein, or any of the formulas described herein (i.e., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), or a salt thereof.

[0457] Examples of pathogens that cause infectious diseases treatable by the methods of this disclosure include, but are not limited to, Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus pneumoniae, Neisseria meningitidis and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria.

[0458] In addition, this disclosure provides a method for treating fungal infections. The method comprises administering to a patient a therapeutically effective dose of any of the formulated and / or co-formulated liposomes or TLR prodrugs listed in any of the claims and described herein, or any of the formulas described herein (i.e., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), or a salt thereof.

[0459] Examples of pathogenic fungi that cause infections treatable by the methods of this disclosure include, but are not limited to, Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales (Mucor, absidia, rhizophus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0460] In addition, this disclosure provides a method for treating parasitic infections. The method comprises administering to a patient a therapeutically effective dose of any of the formulated and / or co-formulated liposomes or TLR prodrugs listed in any of the claims and described herein, or any of the formulas described herein (i.e., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)), or a salt thereof.

[0461] 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.

[0462] In a further set of embodiments within the scope of this disclosure, any formulation and / or co-formulated liposome, or TLR prodrug, or any of the formulas described herein (i.e., TR3, TR6, TR6(A), TR5, TR5(A), and / or TR5(B)) is useful in preventing or reducing the risk of developing any of the diseases referred to herein, for example, in an individual who may be predisposed to a disease, condition, or disorder but has not yet experienced or shown any of the pathologies or overall symptoms of the disease. XII.) Kits / Manufactured Products

[0463] Kits for use in laboratory, prognostic, preventive, diagnostic, and therapeutic applications as described herein are within the scope of the present invention. Such a kit may include a transport container, package, or container compartmentated to receive one or more containers, such as vials, tubes, etc., each container containing one of the separate elements used in the method, together with a label or insert containing instructions for use, for example, as described herein. For example, a container(s) may contain formulated and / or co-formulated liposomes that are detectably labeled or can be detectably labeled and / or loaded with the TLR 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 TLR prodrug.

[0464] The kit of the present invention typically comprises the above-mentioned container and one or more other associated containers containing materials desirable from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, transport containers, packages, containers, vials, and / or tube labels and / or instructions for use listing the contents, as well as accompanying documentation containing instructions for use.

[0465] Labels may be present on or with the container to indicate that the composition is used for a specific therapeutic or non-therapeutic application, such as prognosis, prevention, diagnosis, or laboratory application, and may also indicate how to use it in vivo or in vitro, such as the methods of use described herein. Directions and other information may also be included on inserts or labels included with or on the kit. Labels may be on the container or associated with the container. Labels may be on the container if the letters, numbers, or other symbols forming the label are molded or etched onto the container itself. Labels may also be associated with the container, for example as an accompanying document, if they are also present in a container or transporter that holds the container. Labels may indicate that the composition is used for the diagnosis, treatment, prevention, or prognosis of a condition, such as cancer or other immunological disorders.

[0466] The terms "kit" and "manufactured product" can be used as synonyms.

[0467] In another embodiment of the present invention, articles(s) containing compositions, such as formulated and / or co-formulated liposomes and / or TLR prodrugs, are within the scope of the present disclosure. The articles typically comprise at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, such as glass, metal, or plastic. Containers can hold formulated and / or co-formulated liposomes loaded with TLR prodrugs.

[0468] The container may, as an alternative, hold a composition effective for the treatment, diagnosis, prognosis, or prevention of a condition and may have a sterile access port (for example, the container may be an intravenous infusion bag or vial with a stopper that can be punctured by a subcutaneous injection needle). The activator in the composition may be a TLR prodrug and / or a formulation and / or co-formulated liposome loaded with a TLR prodrug disclosed herein.

[0469] The product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The product may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes, and / or accompanying documentation containing indications and / or instructions for use. Exemplary Embodiments

[0470] The embodiments provided include the following: 1) (i) Drug portion, (ii) lipid portion, and (iii) Linked unit ("LU") A TLR prodrug composition comprising a drug portion containing a TLR agonist, wherein the LU conjugates the drug portion with the lipid portion.

[0471] 2) The TLR prodrug of claim 1, further comprising the chemical structure described in formula I.

[0472] 3) The TLR prodrug of claim 1, further comprising the chemical structure described in Formula II.

[0473] 4) The TLR prodrug of claim 1, wherein the drug portion comprises the chemical structure described as TR5.

[0474] 5) The TLR prodrug of claim 1, wherein the drug portion comprises the chemical structure described as TR5(A).

[0475] 6) The TLR prodrug of claim 1, wherein the drug portion comprises the chemical structure described as TR6.

[0476] 7) The TLR prodrug of claim 1, wherein the drug portion comprises the chemical structure described as TR6(A).

[0477] 8) The TLR prodrug of claim 1, wherein the drug portion comprises the chemical structure described as TR3.

[0478] 9) The TLR prodrug of claim 1, wherein LU is a hydromethylcarbamate linker.

[0479] 10) The TLR prodrug of claim 1, wherein the lipid portion comprises the lipids listed in Table I.

[0480] 11) The TLR prodrug of claim 1, wherein the lipid portion comprises lipids listed in Table III.

[0481] 12) The TLR prodrug of claim 1, wherein the lipid portion comprises CHEMS.

[0482] 13) The TLR prodrug of claim 1, wherein the lipid portion contains stearic acid.

[0483] 14)(i) Drug portion containing TR5 (ii) Lipid portion containing CHEMS, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0484] 15)(i) Drug portion containing TR5 (ii) The lipid portion containing stearic acid, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0485] 16)(i) Drug portion containing TR5(A) (ii) Lipid portion containing CHEMS, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0486] 17)(i) Drug portion containing TR5(A) (ii) The lipid portion containing stearic acid, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0487] 18)(i) Drug portion containing TR6 (ii) Lipid portion containing CHEMS, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0488] 19)(i) Drug portion containing TR6 (ii) The lipid portion containing stearic acid, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0489] 20)(i) Drug portion containing TR6(A) (ii) Lipid portion containing CHEMS, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0490] 21)(i) Drug portion containing TR6(A) (ii) The lipid portion containing stearic acid, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0491] 22)(i) Drug portion containing TR3 (ii) The lipid portion containing stearic acid, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above.

[0492] 23) Liposomes containing a TLR prodrug, which release an active TLR inhibitor after cleavage of the LU.

[0493] 24) The liposome of claim 23, wherein LU is a hydromethylcarbamate linker.

[0494] 25) The liposome of claim 23, further comprising a helper lipid, wherein the helper lipid is as listed in Table II.

[0495] 26) The liposome of claim 23, wherein the TLR prodrug comprises TR3, TR5, TR5(A), and / or TR5(B).

[0496] 27) The liposome of claim 23, wherein the TLR prodrug comprises TR6 and / or TR6(A).

[0497] 28) The liposome of claim 23, further co-formulated with iNTK activator.

[0498] 29) The liposome of claim 28, wherein the iNKT activator is alpha-galactosylceramide (α-GalCer).

[0499] 30) The liposome of claim 23, further co-formulated with an immunomodulator, wherein the immunomodulator is selected from the group consisting of other TLR agonists and / or prodrugs, immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors, and / or their prodrugs.

[0500] 31) The liposome of claim 23, further co-formulated with an ICD-inducing chemotherapeutic agent, wherein the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzimib, or paclitaxel.

[0501] 32) The liposome of claim 23, further comprising DOX.

[0502] 33) The liposome of claim 23, further comprising MTO.

[0503] 34) The liposome of claim 23, further comprising DOX.

[0504] 35) The liposome of claim 23, further comprising MTO.

[0505] 36) A kit comprising the liposomes of claim 23.

[0506] 37) A kit comprising the liposomes of claim 26.

[0507] 38) A kit comprising the liposomes of claim 27.

[0508] 39) A liposome comprising a TLR lipid moiety, wherein the TLR lipid moiety comprises the chemical structure described in Formula III.

[0509] 40) A liposome comprising a TLR lipid moiety, wherein the TLR lipid moiety comprises the chemical structure described in formula IV.

[0510] 41) The liposome of claim 39, further comprising a helper lipid, wherein the helper lipid is listed in Table II.

[0511] 42) The liposome of claim 39, further comprising a helper lipid, wherein the helper lipid is listed in Table II.

[0512] 43) The liposome of claim 38, wherein the TLR prodrug comprises TR8.

[0513] 44) The liposome of claim 40, wherein the TLR prodrug comprises TR11.

[0514] 45) The liposome of claim 38, further co-formulated with iNTK activator.

[0515] 46) The liposome of claim 45, wherein the iNKT activator is alpha-galactosylceramide (α-GalCer).

[0516] 47) The liposome of claim 39, further co-formulated with iNTK activator.

[0517] 48) The liposome of claim 47, wherein the iNKT activator is alpha-galactosylceramide (α-GalCer).

[0518] 49) The liposome of claim 44, further co-formulated with iNTK activator.

[0519] 50) The liposome of claim 49, wherein the iNKT activator is alpha-galactosylceramide (α-GalCer).

[0520] 51) The liposome of claim 43, further co-formulated with iNTK activator.

[0521] 52) The liposome of claim 51, wherein the iNKT activator is alpha-galactosylceramide (α-GalCer).

[0522] 53) The liposome of claim 39, further co-formulated with an immunomodulator, wherein the immunomodulator is selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors and / or their prodrugs.

[0523] 54) The liposome of claim 39, further co-formulated with an ICD-inducing chemotherapeutic agent, wherein the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, or paclitaxel.

[0524] 55) The liposome of claim 40, further co-formulated with an immunomodulator, wherein the immunomodulator is selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors and / or their prodrugs.

[0525] 56) The liposome of claim 40, further co-formulated with an ICD-inducing chemotherapeutic agent, wherein the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, or paclitaxel.

[0526] 57) A method for treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of liposomes to subjects requiring such treatment, wherein the liposomes contain a TLR prodrug, (ii) a pharmaceutically acceptable salt thereof, Methods that include...

[0527] 58) The method of claim 57, wherein the TLR prodrug comprises TR3, TR5, TR5(A), and / or TR5(B).

[0528] 59) The method of claim 57, wherein the liposome comprises TR3, TR5, TR5(A), and / or TR5(B) further co-formulated with an ICD-inducing chemotherapeutic agent.

[0529] 60) The method of claim 57, wherein the liposome comprises TR3, TR5, TR5(A), and / or TR5(B) further co-formulated with an immunomodulator.

[0530] 61) The method of claim 57, wherein the liposome comprises TR3, TR5, TR5(A), and / or TR5(B) further co-formulated with an iNTK activator.

[0531] 62) The method of claim 58, wherein the iNTK activator is alpha-galactosylceramide (α-GalCer).

[0532] 63) A method for treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of liposomes to subjects requiring such treatment, wherein the liposomes contain a TLR prodrug, (ii) a pharmaceutically acceptable salt thereof, Methods that include...

[0533] 64) The method of claim 63, wherein the TLR prodrug comprises TR6 and / or TR6(A).

[0534] 65) The method of claim 63, wherein the liposome comprises TR6 and / or TR6(A) further co-formulated with an ICD-inducing chemotherapeutic agent.

[0535] 66) The method of claim 63, wherein the liposome comprises TR6 and / or TR6(A) further co-formulated with an immunomodulator.

[0536] 67) The method of claim 63, wherein the liposome comprises TR6 and / or TR6(A) further co-formulated with an iNTK activator.

[0537] 68) The method of claim 67, wherein the iNTK activator is alpha-galactosylceramide (α-GalCer).

[0538] 69) A method for treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of liposomes to subjects requiring such treatment, wherein the liposomes contain a TLR lipid portion, (ii) a pharmaceutically acceptable salt thereof, Methods that include...

[0539] 70) The method of claim 69, wherein the TLR lipid portion comprises TR8.

[0540] 71) The method of claim 69, wherein the liposome comprises TR8 further co-formulated with an ICD-inducing chemotherapeutic agent.

[0541] 72) The method of claim 69, wherein the liposome comprises TR8 further co-formulated with an immunomodulator.

[0542] 73) The method of claim 69, wherein the liposome comprises TR8 further co-formulated with iNTK activator.

[0543] 74) The method of claim 73, wherein the iNTK activator is alpha-galactosylceramide (α-GalCer).

[0544] 75) The method of claim 69, wherein the liposome comprises ID3 and TR8 further co-formulated.

[0545] 76) A method for treating a subject who has cancer or has been diagnosed with cancer, (i) Administering an effective amount of liposomes to subjects requiring such treatment, wherein the liposomes contain a TLR lipid portion, (ii) a pharmaceutically acceptable salt thereof, Methods that include...

[0546] 77) The method of claim 76, wherein the TLR lipid portion comprises TR11.

[0547] 78) The method of claim 76, wherein the liposome comprises TR11 further co-formulated with an ICD-inducing chemotherapeutic agent.

[0548] 79) The method of claim 76, wherein the liposome comprises TR11 further co-formulated with an immunomodulator.

[0549] 80) The method of claim 76, wherein the liposome comprises TR11 further co-formulated with iNTK activator.

[0550] 81) The method of claim 80, wherein the iNTK activator is alpha-galactosylceramide (α-GalCer). [Examples]

[0551] Various aspects of the present invention will be further described and illustrated by the following examples, none of which are intended to limit the scope of the invention.

[0552] (Example 1) Chemical synthesis of TR5(B) prodrugs containing various lipids

[0553] For example, the chemical synthesis of TR5(B) prodrugs containing various lipids listed in Tables I and III was performed using the following protocol. First, quinoline (1) was treated with valeric acid anhydride to obtain intermediate 2. Next, 4-aminobutanol (3) was treated with benzyl chloroformate (4) to obtain intermediate 5. Next, intermediate 5 was treated with N-hydroxyphthalimide, triphenylphosphine, and DIAD, followed by treatment with hydrazine to obtain intermediate 6. Next, intermediates 6 and 2 were heated with triethylamine to obtain intermediate 7. Next, intermediate 7 was sequentially treated with mCPBA, ammonium hydroxide, and benzenesulfonyl chloride, and then heated with HCl to obtain intermediate 8. Finally, intermediate 8 was treated with various lipid carboxylic acids to obtain the final product represented by TR5(B) (Figure 1).

[0554] (Example 2) Chemical synthesis of TR6 prodrugs containing various lipids

[0555] For example, the chemical synthesis of TR6 prodrugs containing various lipids listed in Tables I and III was performed using the following protocol. First, fluoronitroaniline (1) was treated with methylamine to obtain compound 2. Next, compound 2 was treated with trimethyl orthoformate to obtain benzimidazole (3). Next, benzimidazole (3) was treated with compound 4 to obtain compound 5. Next, compound 5 was treated with ammonium acetate to obtain compound 6. Next, compound 6 was treated with chloromethyl chloroformate (7) to obtain compound 8. Finally, compound 8 was treated with various lipid acids (9) to obtain the final product represented by TR6 (Figure 2).

[0556] (Example 3) Chemical synthesis of TR6 prodrug intermediates

[0557] In another experiment, the chemical synthesis of the TR6 prodrug intermediate was carried out using the following method: To a solution of Cu-T12-9 (5.50 g, 15.1 mmol, 1.00 equivalent) in THF (75.0 mL), LiHMDS (1 M, 18.2 mL, 1.20 equivalent) was added at -70°C, and the reaction mixture was stirred at -70°C for 0.5 hours. Next, a solution of compound 2a (2.94 g, 22.7 mmol, 2.02 mL, 1.50 equivalent) in THF (10 mL) was added to the mixture at -70°C, and the mixture was stirred for a further 1.5 hours at -70°C. LC-MS showed that Cu-T12-9 (RT=0.934 min) was not completely consumed, and the desired mass (RT=0.966 min) was detected. The reaction mixture was poured into a saturated citric acid solution (100 mL) and extracted with ethyl acetate (150 mL x 3). The organic layer was washed with brine (150 mL x 2), dried over Na2SO4, filtered, and concentrated to obtain the crude product (confirmed by LC-MS and HPLC). The crude product was purified by preparative HPLC (column: Phenomenex luna C18 250 × 80 mm × 10 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 38ACN%~68ACN%, 21 min), and directly extracted with ethyl acetate (200 mL x 2). The combined organic layers were washed with brine (200 mL x 3), dried over Na2SO4, filtered, and concentrated to obtain compound 2 (2.50 g, 5.50 mmol, yield 36.2%) as a yellow solid. The obtained compound is shown in Figure 6.

[0558] (Example 4) Chemical synthesis of TR6 prodrugs containing hemysuccinate cholesterol ("CHEMS").

[0559] In another experiment, a TR6 prodrug containing cholesteryl hemisuccinate ("CHEMS") was synthesized in the following manner. Briefly, to a solution of cholesteryl hemisuccinate (2.68 g, 5.50 mmol, 1.00 equivalent) in DMF (150 mL) was added Ag2CO3 (2.27 g, 8.25 mmol, 374 uL, 1.50 equivalents) at 25 °C. The reaction mixture was stirred at 25 °C for 0.5 h. Next, compound 2 (2.50 g, 5.50 mmol, 1.00 equivalent) and NaI (1.24 g, 8.25 mmol, 1.50 equivalents) were added to the mixture. After addition, the reaction mixture was stirred at 80 °C for 12 h. LCMS indicated that the reaction was complete and the desired mass (RT = 1.465 min) was detected. The reaction mixture was cooled to 25 °C, filtered through a celite pad, and washed with DMF (200 mL). The filtrate was concentrated at 50 °C to give a crude product, which was purified by reverse-phase MPLC (TFA conditions) and then concentrated under reduced pressure to give a crude product. The crude product was combined with EW15710-150-P1 (ca. 700 mg) and purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 5:1 to 2:1, R f = 0.6), which was detected by TLC (petroleum ether:ethyl acetate = 1:1, R f = 0.6, PMA). Target 1 (1.80 g, 1.97 mmol, yield 29.8%, purity 99.0%) was obtained as a yellow solid, which was confirmed by 1 1H NMR, 19 19F NMR, LCMS, HPLC, SFC, and DSC. The resulting compound is shown in Figure 7.

[0560] (Example 5) Chemical Synthesis of a TR3 Prodrug Containing Stearic Acid

[0561] In another experiment, a TR3 prodrug containing stearic acid was synthesized using the following method. Briefly, m-CPBA (4.86 g, 23.9 mmol, 85.0% purity, 1.50 equivalents) was added in several portions to a solution of compound 4 (9.86 g, 15.9 mmol, 94.7% purity, 1.00 equivalent) in CHCl3 (100 mL). The mixture was then stirred at 80°C for 5 hours. LC-MS showed that 6.01% of compound 4 remained (retention time = 1.041 min), and the desired mass (retention time = 1.149 min) was detected. Next, NH3.H2O (107 g, 920 mmol, 118 mL, 30% purity, 57.6 equivalents) was added, and the mixture was stirred at 15°C for 30 minutes. Finally, TosCl (3.65 g, 19.1 mmol, 1.20 equivalents) was added all at once. The mixture was stirred at 15°C for 12 hours. LC-MS showed that compound 4 was consumed and the desired mass (RT=1.049 min) was detected. The reaction mixture was separated by a separatory funnel. The aqueous phase was extracted with DCM (100 mL × 2). The combined organic phase was washed with brine (100 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude product combination was purified by preparative HPLC (column: Welch Ultimate XB-SiOH 250 × 50 × 10 μm; mobile phase: [hexane-EtOH (0.1% NH₃H₂O)]; B%: 1%~40%, 20 min), and further purified by MPLC (SiO₂, dichloromethane / methanol = 1 / 0~100 / 1, dichloromethane / methanol = 10 / 1, R f =0.35). Target A (485.61 mg, 767 μmol, yield 4.33%, 94.8% purity) was obtained as an off-white solid, and it was 1 The compounds were confirmed by 1H NMR, LC-MS, and HPLC. The obtained compounds are shown in Figure 8.

[0562] (Example 6) Synthesis and Characterization of LNP-TR6 Liposomes

[0563] Liposomes containing the TR6 prodrug (referred to as LNP-TR6) were synthesized and characterized using microfluidic techniques performed on a NanoAssemblr benchtop instrument (Precision NanoSystems) in the following manner. Briefly, stock solutions of each of the following lipid components, Hydro Soy PC [(HSPC: L-α-phosphatidylcholine, hydrogenated (soybean)], cholesterol, DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]), and TR6 were prepared at a concentration of 20 mg / ml in ethanol. Each of the lipid components (HSPC, CHOL, DSPE-PEG, and TR6) was mixed together in a molar ratio of 40:31:26:3 to synthesize LNP-TR6. The size of P-TR6 is determined by various parameters, such as the flow rate, temperature, and concentration of the lipid mixture. An optimized ratio of lipid mixture with a molar ratio of 40:31:26:3 was preheated to 50°C using a microfluidizer heating block attachment. An aqueous phase containing 1 mM PBS buffer was also preheated to 50°C and then passed through a microfluidic cartridge at a flow rate of 3:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed from the DI water using a dialysis membrane (Sigma Aldrich) with a cutoff size of 12 kDa for at least 24 hours. During the 24 hours, the DI water was changed at least five times to maximize solvent removal. After solvent removal, LNP-TR6 was concentrated using an Amicon centrifugal filtration device (cutoff size 10 kDa, at 3000 g) as needed.

[0564] The characteristics of LNP-TR6 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Two ml of LNP-TR6 liposomes (with a liposome concentration of 0.5–1 mg / ml) were placed in a transparent four-sided plastic cuvette and analyzed directly at 25°C. The results shown in Figure 9 indicate that the Z-mean size of the nanoparticles was approximately 83 nm and the PDI was approximately 0.164.

[0565] In addition, the zeta potential of LNP-TR6 liposomes in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-TR6 was approximately -16.2 mV (Figure 10).

[0566] (Example 7) Synthesis and Characterization of LNP-TR5 Liposomes

[0567] In another experiment, liposomes containing the TR5 prodrug (referred to as LNP-TR5) were synthesized and characterized using microfluidic techniques performed on a NanoAssemblr benchtop instrument (Precision NanoSystems) in the following manner. Briefly, stock solutions of each of the following lipid components, Hydro Soy PC [(HSPC: L-α-phosphatidylcholine, hydrogenated (soybean)], cholesterol, DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]), and TR5 were prepared at a concentration of 10 mg / ml in ethanol. Each of the lipid components (HSPC, CHOL, DSPE-PEG, and TR5) was mixed together in a molar ratio of 53:33:9:5 to synthesize LNP-TR5. -The size of TR5 is determined by various parameters, such as the flow rate, temperature, and concentration of the lipid mixture. An optimized ratio of lipid mixture with a molar ratio of 53:33:9:5 was preheated to 45°C using the heating block attachment of a microfluidizer. In addition, an aqueous phase containing 1 mM PBS buffer was preheated to 45°C and then passed through a microfluidic cartridge at a flow rate of 3.5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed from the DI water using a dialysis membrane (Sigma Aldrich) with a cutoff size of 12 kDa for at least 24 hours. During the 24 hours, the DI water was changed at least 5 times to maximize solvent removal. After solvent removal, LNP-TR6 was concentrated using an Amicon centrifugal filtration device (cutoff size 10 kDa, at 3000 g) as needed.

[0568] The characteristics of LNP-TR5 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Two ml of LNP-TR5 liposomes (with a liposome concentration of 0.5–1 mg / ml) were placed in a transparent four-sided plastic cuvette and analyzed directly at 25°C. The results shown in Figure 11 indicate that the Z-mean size of the nanoparticles was approximately 85 nm and the PDI was approximately 0.096.

[0569] In addition, the zeta potential of LNP-TR5 liposomes in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-TR5 was approximately -13.1 mV (Figure 12).

[0570] (Example 8) Synthesis and Characterization of LNP-TR3 Liposomes

[0571] In another experiment, liposomes containing the TR3 prodrug (referred to as LNP-TR3) were synthesized and characterized using microfluidic techniques performed on a NanoAssemblr benchtop instrument (Precision NanoSystems) in the following manner: Briefly, stock solutions of each of the following lipid components—HSPC, cholesterol, and DSPE-PEG—were prepared at a concentration of 20 mg / ml in ethanol. The TR3 prodrug was prepared at a concentration of 2 mg / ml in ethanol. Each of the lipid components (HSPC, CHOL, DSPE-PEG, and TR3) was mixed together in a molar ratio of 53.5:33.5:6:5, and then diluted with ethanol to obtain a lipid concentration of approximately 9 mg / ml to synthesize LNP-TR3. The size of LNP-TR3 depends on various parameters, such as the flow rate, temperature, and concentration of the lipid mixture. An optimized lipid mixture with a molar ratio of 53.5:33.5:6:5 was preheated to 45°C using a microfluidizer's heating block attachment. An aqueous phase containing 1 mM PBS buffer was also preheated to 45°C and then passed through a microfluidic cartridge at a flow rate of 3.5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed from the DI water using a 12 kDa cutoff dialysis membrane (Sigma Aldrich) for at least 24 hours. During the 24 hours, the DI water was changed at least five times to maximize solvent removal. After solvent removal, LNP-TR3 was concentrated using an Amicon centrifugal filtration device (10 kDa cutoff, 3000 g) as needed.

[0572] The characteristics of LNP-TR3 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Two ml of LNP-TR3 liposomes (with a liposome concentration of 0.5–1 mg / ml) were placed in a transparent four-sided plastic cuvette and analyzed directly at 25°C. The results shown in Figure 13 indicate that the Z-mean size of the nanoparticles was approximately 88 nm and the PDI was approximately 0.125.

[0573] In addition, the zeta potential of LNP-TR3 liposomes in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-TR3 was approximately -13.2 mV (Figure 14).

[0574] (Example 9) Synthesis and Characterization of LNP-TR8 Liposomes

[0575] In another experiment, liposomes containing the TR8 prodrug (referred to as LNP-TR8) were synthesized and characterized using microfluidic techniques performed on a NanoAssemblr benchtop instrument (Precision NanoSystems) in the following manner. Briefly, stock solutions of each of the following lipid components, HSPC, cholesterol, and DSPE-PEG, were prepared separately. A stock solution of MPLA (PHAD®) monophosphoryl lipid A (synthesized) (PHAD) was prepared in dimethyl sulfoxide (10 mg / ml). Each of the lipid components (HSPC, CHOL, DSPE-PEG, and MPLA) was mixed together in a molar ratio of 55:37.5:3 and then diluted with ethanol to obtain a lipid concentration of approximately 10 mg / ml. The size of LNP-TR8 depends on various parameters, such as the flow rate, temperature, and concentration of the lipid mixture. An optimized lipid mixture with a molar ratio of 55:37:5:3 was preheated to 45°C using a microfluidizer's heating block attachment. An aqueous phase containing 1 mM PBS buffer was also preheated to 45°C and then passed through a microfluidic cartridge at a flow rate of 5:1 (aqueous phase:organic phase, lipid mixture). The solvent was removed from the DI water using a dialysis membrane (Sigma Aldrich) with a cutoff size of 12 kDa for at least 24 hours. During the 24 hours, the DI water was changed at least 5 times to maximize solvent removal. After solvent removal, LNP-TR8 was concentrated using an Amicon centrifugal filtration device (cutoff size 10 kDa, at 3000 g) as needed.

[0576] The characteristics of LNP-TR8 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Two ml of LNP-TR8 liposomes (with a liposome concentration of 0.5–1 mg / ml) were placed in a transparent four-sided plastic cuvette and analyzed directly at 25°C. The results shown in Figure 15 indicate that the Z-mean size of the nanoparticles was approximately 89 nm and the PDI was approximately 0.240.

[0577] In addition, the zeta potential of LNP-TR8 liposomes in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-TR8 was approximately -9.8 mV (Figure 16).

[0578] (Example 10) Synthesis and Characterization of LNP-ID3-TR8 Liposomes

[0579] In another experiment, liposomes containing ID3 prodrugs and TR8 prodrugs (referred to as LNP-ID3-TR8) were synthesized and characterized using microfluidic techniques performed on a NanoAssemblr benchtop instrument (Precision NanoSystems) in the following manner. Briefly, stock solutions of each of the following lipid components—HSPC, cholesterol, and DSPE-PEG—were prepared in ethanol at a concentration of 20 mg / ml. Meanwhile, a stock solution of ID3 prodrug (20 mg / ml) was prepared in acetonitrile, and a stock solution of MPLA (10 mg / ml) was prepared in DSMO. Each of the lipid components (HSPC, CHOL, DSPE-PEG, ID3, and MPLA) was mixed together in a molar ratio of 54:28:12:3:3, and then diluted in ethanol to obtain a lipid concentration of approximately 10 mg / ml. The size of LNP-ID3-TR8 depends on various parameters, such as the flow rate, temperature, and concentration of the lipid mixture. An optimized lipid mixture with a molar ratio of 54:28:12:3:3 was preheated to 45°C using a microfluidizer's heating block attachment. An aqueous phase containing 1 mM PBS buffer was also preheated to 45°C and then passed through a microfluidic cartridge at a flow rate of 3:1 (aqueous phase:organic phase, lipid mixture). Thus, liposome formulations were synthesized in such a way that the molar ratio of ID3:TR8 maintained a 4:1 ratio. The solvent was removed from the DI water using a dialysis membrane (Sigma Aldrich) with a cutoff size of 12 kDa for at least 24 hours. During the 24 hours, the DI water was changed at least 5 times to maximize solvent removal. After solvent removal, LNP-TR8 was concentrated using an Amicon centrifugal filtration device (cutoff size 10 kDa, 3000 g) as needed.

[0580] The characteristics of LNP-ID3-TR8 liposomes were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Two ml of LNP-ID3-TR8 liposomes (with a liposome concentration of 0.5–1 mg / ml) were placed in a transparent four-sided plastic cuvette and analyzed directly at 25°C. The results shown in Figure 17 indicate that the Z-mean size of the nanoparticles was approximately 82 nm and the PDI was approximately 0.170.

[0581] In addition, the zeta potential of LNP-ID3-TR8 liposomes in an aqueous dispersion was determined using a Malvern zetasizer instrument (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, approximately 1 ml of liposomes (concentration of approximately 2 mg / ml in 20 mM NaCl) was placed in a disposable capillary zeta potential cell compatible with the zetasizer. Measurements were performed at 25°C. The results showed that the zeta potential of LNP-ID3-TR8 was approximately -11.9 mV (Figure 18).

[0582] (Example 11) In vivo tumor inhibition of the combination of LNP-TR5 and LNP-DOX using B16F10 cells.

[0583] The combination of LNP-TR5 and LNP-DOX was evaluated using the following protocol. In short, mouse melanoma carcinoma B16F10 cells (0.2 × 10⁶) 6LNP-DOX (doxorubicin in liposomal form) was subcutaneously inoculated into the right posterior ventral region of C57BL / 6 mice. Animals were treated twice weekly by IV injection with a vehicle control, 3 mg / kg LNP-DOX (doxorubicin in liposomal form), and a combination of 3 mg / kg LNP-DOX and 1 mg / kg LNP-TR5. Tumor volume was measured two-dimensionally three times 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 20.

[0584] The results indicate that treatment with LNP-DOX alone significantly inhibits tumor growth. TGI was calculated to be 66.37% compared to the vehicle-treated group (p<0.01). In addition, the combination of LNP-TR5 and LNP-DOX also produced significant antitumor activity. TGI was calculated to be 52.73% (p<0.05). (See Figure 19).

[0585] (Example 12) In vivo tumor inhibition using B16F10 cells with combinations of LNP-TR6 and LNP-NK1, and combinations of LNP-TR6, LNP-TR8, and LNP-MTO.

[0586] The combination of LNP-TR6 and LNP-NK1, and the evaluation of LNP-TR6 itself, were performed using the following protocol. In short, mouse melanoma carcinoma B16F10 cells (0.2 × 10⁶) were used. 6LNP-MTO was subcutaneously inoculated into the right posterior ventral region of C57BL / 6 mice. Animals were treated twice weekly by IV injection with the following combinations: vehicle control, 2 mg / kg LNP-MTO (mitoxantrone dihydrochloride in liposomal form), combination of 3 mg / kg LNP-TR6 and 0.03 mg / kg LNP-NK1 (KRN7000 in liposomal form), and combination of 2 mg / kg LNP-TR6, 0.6 mg / kg LNP-TR8, and 2 mg / kg LNP-MTO. Tumor volume was measured two-dimensionally three times 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 at day 18.

[0587] The results indicate that treatment with LNP-TR6+LNP-NK1 and LNP-TR6+LNP-TR8+LNP-MTO significantly inhibits tumor growth compared to the vehicle-treated group. TGI was calculated to be 49.87% and 57.02%, respectively (p<0.01). (See Figure 20).

[0588] (Example 13) In vivo tumor inhibition using B16F10 cells with combinations of LNP-TR5 and LNP-AR5, and combinations of LNP-TR6 and LNP-AR5.

[0589] The combinations of LNP-TR6 and LNP-AR5, and LNP-TR5 and LNP-AR5 were evaluated using the following protocol. In short, mouse melanoma carcinoma B16F10 cells (0.2 × 10⁶) were used. 6LNP-AR5 was subcutaneously inoculated into the right posterior ventral region of C57BL / 6 mice. Animals were treated twice weekly by IV injection with vehicle control, 3 mg / kg of LNP-AR5 (liposomal form of ZM241385-stearic acid), a combination of 3 mg / kg of LNP-AR5 and LNP-TR6, and a combination of 3 mg / kg of LNP-AR5 and LNP-TR5. Tumor volume was measured two-dimensionally three times 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.

[0590] The results indicate that treatment with LNP-AR5 + LNP-TR5 produced significant antitumor activity. TGI was calculated to be 30.72% compared to the vehicle-treated group (all p<0.05). Minimal antitumor activity was observed in the LNP-AR5 and LNP-AR5 + LNP-TR6 groups. TGI was calculated to be 14.86% and 21.52%, respectively. (See Figure 21).

[0591] (Example 14) In vivo tumor inhibition using EMT6 cells with a combination of LNP-TR5, LNP-AR5, and LNP-DOX.

[0592] The combination of LNP-TR5, LNP-AR5, and LNP-DOX was evaluated using the following protocol. In short, mouse mammary cancer EMT6 cells (0.1 × 10⁶) 6The drug was subcutaneously inoculated into the right posterior ventral region of Balb / c mice. Animals were treated by IV injection with a vehicle control, 3 mg / kg doxorubicin (LNP-DOX), and a combination of 3 mg / kg LNP-DOX + LNP-AR5 + LNP-TR5. After two administrations of LNP-DOX, the LNP-DOX treatment was replaced with the vehicle. Tumor volume was measured two-dimensionally three times 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 21.

[0593] The results showed that treatment with 3 mg / kg of LNP-DOX alone, administered twice weekly, produced significant antitumor activity. The TGI was calculated to be 68.74% compared to the vehicle-treated group (p<0.05). In addition, improved antitumor activity was observed when LNP-DOX treatment was combined with LNP-AR5 + LNP-TR5. The TGI was 87.92% (all p<0.05). (See Figure 22).

[0594] (Example 15) In vivo tumor inhibition studies of LNP-TR5 and LNP-TR6 in multiple combinations using H22 cells.

[0595] The evaluation of LNP-TR5 and LNP-TR6 in multiple combinations was performed using the following protocol. In short, hepatocellular carcinoma H22 cells (1 × 10⁶) 6The drug was subcutaneously inoculated into the right posterior ventral region of Balb / c mice. Animals were treated by IV injection with the following regimens: vehicle control, 10 mg / kg anti-PD1 antibody, a combination of 4 mg / kg LNP-AR5 and 4 mg / kg LNP-TR5 for the first two doses and a combination of 2 mg / kg for the remainder of the study, a combination of 4 mg / kg LNP-AR5 and LNP-TR6, a combination of 10 mg / kg anti-PD1 antibody and a combination of 4 mg / kg LNP-AR5 and 4 mg / kg LNP-TR5 for the first two doses and a combination of 2 mg / kg for the remainder of the study, a combination of 3 mg / kg LNP-ID3 and LNP-AR5 for the first two doses and a combination of 3.5 mg / kg for the remainder of the study, and a combination of 3 mg / kg LNP-TR5 for the first two doses and 2 mg / kg for the remainder of the study. Tumor volume was measured two-dimensionally three times using calipers, and its 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.

[0596] The results indicate that treatment with anti-PD-1 + LNP-AR5 + LNP-TR5 produced significant antitumor activity. TGI was calculated to be 74.98% compared to the vehicle-treated group (all p<0.05). Minimal antitumor activity was observed with LNP-AR5 + LNP-TR5 and LNP-ID3 + LNP-AR5 + LNP-TR5. TGI was calculated to be 55.8% and 48.83%, respectively. (See Figure 23).

[0597] (Example 16) In vivo tumor inhibition studies of LNP-TR5 and LNP-TR6 in multiple combinations in colorectal cancer cells.

[0598] The evaluation of LNP-TR5 and LNP-TR6 in multiple combinations was performed using the following protocol. In short, colorectal cancer cells (1 × 10⁶) 6The drug was subcutaneously inoculated into the right posterior ventral region of C57BL / 6 mice. Animals were divided into vehicle control, 4 mg / kg doxorubicin (LNP-DOX), 10 mg / kg anti-PD1 antibody, a combination of 4 mg / kg LNP-AR5 and 4 mg / kg LNP-TR5 for the first two doses and a combination of 2 mg / kg for the remainder of the study, a combination of 4 mg / kg LNP-AR5 and LNP-TR6, a combination of 4 mg / kg LNP-DOX, LNP-AR5 and LNP-TR6, a combination of 4 mg / kg LNP-DOX, LNP-AR5 and 4 mg / kg LNP-TR5 for the first two doses and The remaining subjects of the study were treated with a 2 mg / kg combination, the first two doses with a combination of 10 mg / kg anti-PD1 antibody, 4 mg / kg LNP-AR5, and 4 mg / kg LNP-TR5, and the remaining subjects with a 2 mg / kg combination, the first two doses with a combination of 3 mg / kg LNP-ID3 and LNP-AR5, and the remaining subjects with a 3.5 mg / kg combination, and the first two doses with 3 mg / kg LNP-TR5 and the remaining subjects with 2 mg / kg, all administered via IV injection.

[0599] The study included a dosing regimen in which a group receiving a certain dose of LNP-DOX received only LNP-DOX initially, followed by a discontinuation of LNP-DOX, and then the mice were administered other drugs.

[0600] Tumor volume was measured two-dimensionally three times 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 19.

[0601] The results show that treatment with 4 mg / kg of LNP-DOX alone produced significant antitumor activity. The TGI was calculated to be 74.39% compared to the vehicle-treated group (p<0.05). In addition, combination treatments using LNP-AR5+LNP-TR5+LNP-DOX, followed by LNP-AR5+LNP-TR6+LNP-DOX, followed by LNP-AR5+LNP-TR5+anti-PD-1+LNP-AR5+LNP-TR5, and LNP-ID3+LNP-AR5+LNP-TR5 also produced significant antitumor activity. The TGIs were calculated to be 77.35%, 90.31%, 87.67%, 91.33%, and 73.77%, respectively (all p<0.05). (See Figure 24).

[0602] (Example 17) In vitro verification of the mechanism of action of liposomal TR6 prodrug

[0603] To confirm the in vitro biological activity of LNP-TR6 and further to confirm its targeting of TLR1 / 2, the following experiments were performed using RAW-Blue® cells and the QUANTI-Blue® assay (InvivoGen, San Diego, CA). Briefly, Raw Blue® cells expressing human Toll-like receptors (TLRs) (excluding TLR5) and NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter genes were used. Stimulation of these cells with TR6 resulted in NF-κB activation via TLR1 / 2, which was measured by detecting SEAP levels. RAW-Blue® cells were incubated with various concentrations of TR6 and LNP-TR6. After incubation with the compounds for 24 hours, TLR stimulation was evaluated by measuring the level of SEAP optimal density (OD) using the QUANTI-Blue® assay. OD was normalized to the control (untreated) group. The results showed that cell treatment with TR6 and LNP-TR6 could induce stimulation of TLR1 / 2, confirming the mechanism of action of TR6 and its activity in LNP form. (See Figure 25).

[0604] (Example 18) In vitro verification of the mechanism of action of liposomal TR8 prodrug

[0605] In another experiment, to confirm the in vitro bioactivity of LNP-TR8 and its targeting of TLR4, the following experiments were performed using RAW-Blue® cells and the QUANTI-Blue® assay (InvivoGen, San Diego, CA). Briefly, Raw Blue® cells expressing human Toll-like receptors (TLRs) (excluding TLR5) and NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter genes were used. Stimulation of these cells with TR8 resulted in NF-κB activation via TLR4, which was measured by detecting SEAP levels. RAW-Blue® cells were incubated with various concentrations of TR8 and LNP-TR8. After incubation with the compounds for 24 hours, TLR stimulation was evaluated by measuring the level of SEAP optimal density (OD) using the QUANTI-Blue® assay. OD was normalized to the control (untreated) group. The results showed that cell treatment with TR8 and LNP-TR8 could induce TLR4 stimulation, confirming the mechanism of action of TR8 and its activity in LNP form. (See Figure 26).

[0606] (Example 19) In vitro verification of the mechanism of action of liposomal TR5 prodrugs.

[0607] In another experiment, the following experiments were performed using RAW-Blue® cells and the QUANTI-Blue® assay (InvivoGen, San Diego, CA) to confirm the in vitro biological activity of LNP-TR5 and its targeting of TLR7 / 8. Briefly, Raw Blue® cells expressing human Toll-like receptors (TLRs) (excluding TLR5) and NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter genes were used. Stimulation of these cells with TR5 resulted in NF-κB activation via TLR7 / 8, which was measured by detecting SEAP levels. RAW-Blue® cells were incubated with various concentrations of TR5 and LNP-TR5. After incubation with the compounds for 24 hours, TLR stimulation was evaluated by measuring the level of SEAP optimal density (OD) using the QUANTI-Blue® assay. OD was normalized to the control (untreated) group. The results showed that cell treatment with TR5 and LNP-TR5 could induce stimulation of TLR7 / 8, confirming the mechanism of action of TR5 and its activity in LNP form. (See Figure 27).

[0608] (Example 20) In vitro verification of the mechanism of action of liposomal TR3 prodrugs.

[0609] In another experiment, to confirm the in vitro bioactivity of LNP-TR3, and further to confirm its targeting of TLR7 and non-targeting of TLR8, the following experiments were performed using RAW-Blue® cells and QUANTI-Blue®, HEK-Blue®, and hTLR8 cell line assays (InvivoGen, San Diego, CA). Briefly, while Raw Blue® cells express various human Toll-like receptors (TLRs) (including TLR7 / 8 but excluding TLR5), hTLR8 cells primarily express human TLR8. In addition, an NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene was used. Stimulation of these cells with TR3 resulted in TLR8-mediated NF-κB activation, which was measured by detecting SEAP levels.

[0610] RAW-Blue® cells were incubated with various concentrations of TR5, LNP-TR5, TR3, TR3 + stearic acid, and LNP-TR3. After incubation with the compounds for 24 hours, TLR stimulation was assessed by measuring the level of SEAP optimal density (OD) using the QUANTI-Blue® assay. OD was normalized to the control (untreated) group. The results indicate that both the prodrug and liposomal forms of TR5 and TR3 stimulated TLRs in RAW-Blue® cells. (See Figure 28).

[0611] Furthermore, HEK-Blue® TLR8 cells were incubated with various concentrations of TR5, LNP-TR5, TR3, TR3 + stearic acid, and LNP-TR3. After incubation with the compounds for 24 hours, TLR stimulation was evaluated by measuring the level of SEAP optimal density (OD) using the QUANTI-Blue® assay. OD was normalized to the control (untreated) group. The results indicate that only TR5 in prodrug and liposome form was able to stimulate the cells (see Figure 29).

[0612] In summary, these results indicate that TR5 is a TLR7 / 8 agonist, and therefore both TR5 and its liposome form stimulate TLR7 and TLR8. However, TR3 can stimulate only TLR7 and not TLR8. Thus, TR3 is specific to TLR7 but not to TLR8.

[0613] (Example 21) Human clinical trials for the treatment of human cancer using formulations and / or co-formulated liposomes containing TLR prodrugs

[0614] The present invention utilizes formulated and / or co-formulated liposomes containing TLR prodrugs and / or TLR lipid moieties, which accumulate particularly in tumor cells and are used in the treatment of certain tumors as well as other immunological disorders and / or other diseases. For each of these indications, two clinical approaches are successfully explored.

[0615] I.) Adjuvant therapy: Adjuvant therapy involves treating patients with formulations and / or co-formulated liposomes containing TLR prodrugs in combination with chemotherapeutic agents or pharmaceuticals or biopharmaceuticals or combinations thereof. Primary cancer targets are treated under standard protocols by the addition of formulations and / or co-formulated liposomes containing TLR prodrugs. Protocol design addresses efficacy, as assessed by examples, including, but not limited to, reduction of tumor burden of primary or metastatic lesions, extension of progression-free survival, extension of overall survival, improvement of patient health, stabilization of disease, and the ability to reduce the usual doses of standard chemotherapy and other biopharmaceuticals. These dose reductions allow for additional treatment and / or extension of treatment by reducing dose-related toxicity of chemotherapeutic agents or biopharmaceuticals.

[0616] II.) Monotherapy: In connection with the use of formulations and / or co-formulated liposomes containing TLR prodrugs in monotherapy for tumors, formulations and / or co-formulated liposomes containing TLR prodrugs are administered to the patient without chemotherapeutic agents, pharmaceuticals, or bioagents. In one embodiment, monotherapy is clinically implemented in terminally ill cancer patients with extensive metastatic disease. The protocol design addresses efficacy, as assessed 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 patient health, stabilization of disease, and the ability to reduce the usual doses of standard chemotherapy and other bioagents. Dosage

[0617] Dosage regimens may be adjusted to provide the optimal desired response. For example, a single formulation and / or co-formulated liposome containing the TLR 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, “unit dosage form” refers to a physically distinct unit suitable as a unit dose to the mammalian subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect together with the necessary pharmaceutically acceptable carrier. The specifications of the unit dosage forms of the present invention are defined by and directly depend on (a) the unique characteristics of the formulation and / or co-formulated liposome containing the TLR prodrug, (b) the individual mechanisms of the combination compound, if any, (c) the specific therapeutic or prophylactic effect to be achieved, and (d) the inherent limitations in the formulation techniques of such compounds for treating the susceptibility of an individual. Clinical Development Program (CDP)

[0618] CDP is being developed in conjunction with treatments using formulated and / or co-formulated liposomes containing TLR prodrugs, either as adjuvant therapy or monotherapy. The trial will first demonstrate safety and then confirm the efficacy of repeated doses. The trial is open-label and will compare formulated and / or co-formulated liposomes containing TLR prodrugs with standard chemotherapy and / or current treatment standards. As understood, one non-limiting criterion that may be available in connection with patient enrollment is the expression of TLRs in tumors, as determined by standard detection methods known in the art.

[0619] Formulated and / or co-formulated liposomes, or any of the embodiments disclosed herein, are expected to have satisfactory pharmacological profiles and promising biopharmaceutical properties, such as toxicological profiles, metabolic and pharmacokinetic properties, solubility, and permeability. It will be understood that determining appropriate biopharmaceutical properties is within the knowledge of those skilled in the art, for example, determining cytotoxicity in cells or inhibition of certain targets or channels to determine potential toxicity.

[0620] The scope of the present invention is not limited by the embodiments disclosed herein, which are intended as single examples of individual aspects of the invention, and any functionally equivalent ones fall within the scope of the invention. In addition to those described herein, various variations of the models, methods, and lifecycle methodologies of the invention will become apparent to those skilled in the art from the above description and teachings, and these too are intended to fall within the scope of the invention. Such variations or other embodiments can be implemented without departing from the true scope and spirit of the invention.

[0621] [Table 1] [Table 2] [Table 3] The present invention provides, for example, the following items: (Item 1) (i) Drug portion, (ii) lipid portion, and (iii) Linking unit ("LU") A TLR prodrug composition comprising, wherein the drug portion comprises a TLR agonist, and the LU conjugates the drug portion with the lipid portion. (Item 2) A TLR prodrug as described in item 1, further comprising the chemical structure described in formula I. (Item 3) The TLR prodrug described in item 1, wherein the drug portion comprises the chemical structure described as TR6. (Item 4) The TLR prodrug described in item 1, wherein the drug portion comprises the chemical structure described as TR6(A). (Item 5) The TLR prodrug described in item 1, wherein the LU is a hydromethylcarbamate linker. (Item 6) The aforementioned lipid portion contains the lipids listed in Table I, and is a TLR prodrug as described in Item 1. (Item 7) The aforementioned lipid portion contains the lipids listed in Table III, and is a TLR prodrug as described in item 1. (Item 8) The TLR prodrug described in item 1, wherein the lipid portion contains CHEMS. (Item 9) The TLR prodrug described in item 1, wherein the lipid portion contains CHEMS and has the following chemical structure. [ka] (Item 10) (i) Drug portion containing TR6 (ii) Lipid portion containing CHEMS, and (iii) LU containing a hydromethylcarbamate linker A TLR prodrug composition containing the above. (Item 11) A TLR prodrug composition as described in item 10, comprising the following chemical structure. [ka] (Item 12) Liposomes containing a TLR prodrug, which release an active TLR inhibitor after cleavage of the LU. (Item 13) The liposome according to item 12, wherein the LU is a hydromethylcarbamate linker. (Item 14) A liposome further comprising a helper lipid, wherein the helper lipid is the liposome described in item 12 of Table II. (Item 15) The liposome according to item 12, wherein the TLR prodrug comprises TR6. (Item 16) The liposomes described in item 15, which are further co-formulated with iNTK activator. (Item 17) The liposome described in item 15, wherein the iNKT activator is alpha-galactosylceramide (α-GalCer). (Item 18) The liposome according to item 15, further co-formulated with an immunomodulator, wherein the immunomodulator is selected from the group consisting of other TLR agonists and / or prodrugs, immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors, and / or their prodrugs. (Item 19) The liposome described in item 15, which is further co-formulated with an ICD-inducing chemotherapeutic agent, wherein the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, or paclitaxel. (Item 20) Liposomes, further containing DOX, as described in item 15.

Claims

1. A composition for treating a subject who has cancer or has been diagnosed with cancer, wherein the composition comprises a nanocarrier, the nanocarrier comprising a TLR prodrug and a pharmaceutically acceptable salt thereof.

2. The composition according to claim 1, wherein the TLR prodrug comprises TR6 and / or TR6(A).

3. The TLR prodrug is defined as TR6 having the following chemical structure: 【Chemistry 39】 The composition according to claim 2, having the following characteristics.

4. The TLR prodrug is defined as TR6(A) having the following chemical structure: 【Chemistry 40】 The lipid portion comprises CHEMS, and CHEMS has the following chemical structure: 【Chemistry 41】 The composition according to claim 2, having the following characteristics.

5. The composition according to claim 1, wherein the nanocarrier comprises TR6 and / or TR6(A) further co-formulated with an ICD-inducing chemotherapeutic agent.

6. The composition according to claim 5, wherein the ICD-inducing chemotherapeutic agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, or paclitaxel.

7. The composition according to claim 1, wherein the nanocarrier comprises TR6 and / or TR6(A) further co-formulated with an immunomodulator.

8. The composition according to claim 7, wherein the nanocarrier is further co-formulated with an immunomodulator, the immunomodulator being selected from the group consisting of other TLR agonists and / or prodrugs, immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, STING agonists, CTLA-4 inhibitors, PD-1 / PD-L1 inhibitors, and / or their prodrugs.

9. The composition according to claim 1, wherein the nanocarrier comprises TR6 and / or TR6(A) further co-formulated with an iNTK activator.

10. The composition according to claim 9, wherein the iNTK activator is alpha-galactosylceramide (α-GalCer).

11. The composition according to claim 1, wherein the nanocarrier comprises liposomes.

12. The composition according to claim 11, wherein the liposome is LNP-TR6.

13. The composition according to claim 1, wherein the nanocarrier comprises solid lipid nanoparticles (SLNPs).

14. The composition according to claim 13, wherein the SLNP is SLNP-TR6.

15. The composition according to claim 1, wherein the subject is a human.

16. The composition according to claim 1, wherein the cancer is melanoma.

17. The composition according to claim 1, wherein the cancer is colorectal cancer.

18. A kit comprising the nanocarrier described in Claim 7.

19. A kit comprising the nanocarrier described in Claim 9.

Images