Formulated and / or co-formulated lipid nanocarrier compositions containing Toll-like receptor ("TLR") agonist prodrugs useful in the treatment of cancer and methods thereof

Abstract

Disclosed herein are nanocarriers formulated and / or co-formulated with TLR prodrugs and / or TLR lipid moieties, as well as methods of making the nanocarriers. The TLR prodrug compositions include a drug moiety, a lipid moiety, and a linking unit, and inhibit Toll-like receptors (e.g., TLR1 / 2, TLR4, and / or TLR7). The TLR prodrugs can be formulated and / or co-formulated into nanocarriers (e.g., LNPs or SLNPs) to provide methods of treating cancer, immunological disorders, and other diseases by utilizing targeted drug delivery vehicles.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 372,416, filed March 10, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] Submission of a paper copy and an ASCII file of the sequence listing The contents of the following submission(s) are hereby incorporated by reference in their entirety: a written copy of the Sequence Listing recorded on March 9, 2023. In addition, the contents of a computer readable form (CRF) of the Sequence Listing titled COPY 1 in an ASCII text file (Filename: 1551-20007.00-SEQ LIST-As-Filed 10-March-2023.txt, recorded on March 9, 2023, size: 4.00KB) and the contents of a computer readable form (CRF) of the Sequence Listing titled COPY 2 in an ASCII text file (Filename: 1551-20007.00-SEQ LIST-As-Filed XX-Mar-2023.txt, recorded on March XX, 2023, size: XXXKB).

[0003] STATEMENT OF RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable.

[0004] FIELD OF THEINVENTION The invention described herein relates to prodrug compositions that inhibit Toll-like receptor ("TLR")(s) after the active inhibitor is released from the prodrug, and nanoformulations containing such prodrugs. In particular, the invention relates to prodrug compositions that are formulated into nanocarriers (e.g., liposomes) and used as vehicles for cancer treatment in humans. The invention also relates to co-formulation of such prodrugs with other immunomodulators or prodrugs. The invention further relates to the treatment of cancer and other immunological disorders and diseases. [Background technology]

[0005] 2. Background of the Invention Cancer is the second leading cause of death worldwide after coronary artery disease. Millions of people die from cancer each year, with well over half a million deaths annually in the United States alone, where 1,688,780 new cases of cancer were diagnosed in 2017 (American Cancer Society). Deaths from heart disease have declined significantly, but cancer deaths in general are rising. Early in the next century, cancer is predicted to become the leading cause of death unless medical advances change current trends.

[0006] Some cancers stand out as having high mortality rates. In particular, lung cancer (18.4% of all cancer deaths), breast cancer (6.6% of all cancer deaths), colorectal cancer (9.2% of all cancer deaths), liver cancer (8.2% of all cancer deaths), and gastric cancer (8.2% of all cancer deaths) are the leading causes of cancer deaths worldwide for both men and women of all ages (GLOBOCAN 2018). These and virtually all other cancers share a common fatal feature that they metastasize to sites distal to the primary tumor, and are almost exclusively fatal metastatic disease. Furthermore, even cancer patients who survived their primary cancer early on shared a common experience of their lives being dramatically altered. Many cancer patients experience intense anxiety caused by the realization that there is a potential for recurrence or treatment failure. Many cancer patients also experience physical debilitation after treatment. Furthermore, many cancer patients experience disease recurrence.

[0007] Although cancer treatment has improved over the past decades and survival rates have increased, the heterogeneity of cancer still requires new therapeutic strategies that utilize multiple treatment modalities. This is especially true in the treatment of solid tumors in anatomically critical sites (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma), whose treatment may be limited to standard radiotherapy and / or chemotherapy. Nevertheless, adverse effects of these treatments are chemoresistance and radioresistance, which promote locoregional recurrence, distant metastases, and secondary primary tumors, in addition to severe side effects that reduce the quality of life of patients.

[0008] Toll-like receptors ("TLRs") are a family of 10 identified pattern recognition receptors that respond to pathogen-associated molecular patterns (PAMPs) and self-derived damage-associated molecular patterns (DAMPs). TLRs then activate downstream pathways that initiate innate immune responses by producing proinflammatory cytokines, type I interferons (IFNs), and other mediators.

[0009] The TLR class of proteins are single transmembrane receptors that are usually expressed on sentinel cells, such as macrophages and dendritic cells, that recognize conserved molecules derived from microorganisms. Once these microorganisms have breached a bodily barrier (e.g., the skin or intestinal mucosa), they are recognized by TLRs, which then activate immune cell responses.

[0010] Upon activation, TLRs recruit adaptor proteins (i.e., proteins that mediate other protein-protein interactions) into the cytosol of immune cells to propagate antigen-induced signaling pathways. These recruited proteins then mediate the subsequent activation of other downstream proteins, including protein kinases (IKKi, IRAK1, IRAK4, and TBK1), which further amplify the signal, ultimately resulting in the upregulation or repression of genes that orchestrate inflammatory responses and other transcriptional events.

[0011] Within the TLR family, several members are noteworthy. TLR1 and TLR2 ("TLR1 / 2") are cell surface receptors that form heterodimers that recognize 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 abrogate the immunosuppressive effects of Treg cells, and thus can enhance adaptive immunity.

[0012] In addition, TLR4 is a cell surface receptor for various bacterial and viral components, among them lipopolysaccharide (LPS). LPS, also known as endotoxin, is a cell wall component 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 (MPLA), a metabolic product of LPS, has been found to retain many of the immunostimulatory functions of LPS, yet is significantly less toxic than its parent. Thus, 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 generated and characterized as immune-stimulating adjuvants. Many of these have been used clinically as adjuvants in vaccine cocktails, including with tumor antigens to illicit anti-tumor immunity.

[0013] In addition, TLR4 is also the receptor for high mobility group box 1 (HMGB1), a protein secreted by tumor cells upon immunogenic cell death that enhances antitumor immunity by recruiting dendritic cells and stimulating antigen processing by antigen-presenting cells (APCs) and secreting inflammatory cytokines. Thus, co-delivery of TLR4 agonists and ICD-inducing chemotherapeutic drugs to tumors enhances antitumor immunity elicited by ICD chemotherapeutic drugs.

[0014] In addition, prodrugs are pharmaceuticals or compounds that are metabolized (i.e., transformed in the body) into pharmacologically active drugs after administration. Instead of directly administering a drug, the corresponding prodrug is used as an alternative to improve how the drug is absorbed, distributed, metabolized, and / or excreted. Prodrugs are often designed to improve bioavailability when the drug itself is poorly absorbed, for example, from the gastrointestinal tract. Prodrugs can be used to improve how a drug selectively interacts with cells or processes that are not its intended target. This reduces the harmful or unintended effects of the drug, which is especially important in treatments such as chemotherapy, which can have severe unintended and undesirable side effects. Thus, prodrugs can be considered as drugs that contain special non-toxic protecting groups that are used temporarily to change or eliminate undesirable properties of the parent molecule.

[0015] Finally, nanocarriers are nanomaterials that are used as a means of transporting another substance, such as a drug. There are many different types of nanocarriers. For example, nanocarriers include polymer conjugates, polymer nanoparticles, lipid-based carriers, and dendrimers, to name a few. The different types of nanomaterials used in nanocarriers allow for the delivery of hydrophobic and hydrophilic drugs throughout the body. The ability to effectively deliver hydrophobic drugs to humans is a major therapeutic benefit of nanocarriers, since the human body contains mostly water. Nanocarriers are promising in the drug delivery process because they allow drugs to be delivered to site-specific targets, allowing the drug to be delivered to certain organs and cells, and not to other organs and cells. Site specificity is a major therapeutic benefit, since it ensures that the drug being delivered is not delivered to the wrong place. In addition, nanocarriers are particularly promising for use in chemotherapy, since they can help reduce the harmful, broader-scale toxicity of chemotherapy to fast-growing healthy cells throughout the body. Chemotherapy drugs can be extremely toxic to human cells, so it is important that the chemotherapy drugs are delivered to the tumor without being released to other parts of the body.

[0016] From the above, it is easily clear to those skilled in the art that new treatment paradigms are required in the treatment of cancer and other immunological diseases.By using novel prodrugs together with modern nanocarrier formats, new disease treatments can achieve the overall goal of more effective treatment(s), reduced side effects, and higher therapeutic utility in the treatment of cancer, especially in the treatment of solid tumor cancer.

[0017] In view of the current deficiencies associated with cancer treatment, it is an object of the present invention to provide new and improved methods of treating cancer(s), immunological disorders, and other diseases utilizing prodrugs encapsulated within nanocarriers.

[0018] In this disclosure, the use of TLR agonists in conjunction with ICD-guided chemotherapeutic drugs, PD-1 antagonists, additional toll receptor agonists, STING agonists, IDO inhibitors, CTLA4 inhibitors, CD1D agonists, TGFb inhibitors, A2aR inhibitors, and / or prodrugs thereof to directly illicit immune responses against tumor cells in situ in real patients (i.e., without the need to introduce tumor antigens or remove tumor cells for ex vivo treatment). These synergistic functional agents are packaged into a single nanocarrier vehicle, ensuring co-delivery of the combination therapy and enhanced tumor selectivity. Summary of the Invention [Means for solving the problem]

[0019] 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, nanocarriers comprising the TLR prodrug(s) are formulated for use as a delivery modality to treat human diseases, such as cancer, including solid tumor cancer, and other immunological disorders. In certain embodiments, the nanocarriers comprise a lipid bilayer that can be incorporated into a drug delivery vehicle (i.e., a liposome). In further embodiments, the nanocarriers comprise solid lipid nanoparticles ("SLNPs"). In further preferred embodiments, the liposomes comprise cholesterol hemisuccinate ("CHEMS"). In further preferred embodiments, the liposomes of the present invention comprise stearic acid. In further preferred embodiments, the liposomes of the present invention comprise a stearic acid derivative that is non-cleavable.

[0020] In further embodiments, the TLR prodrugs of the present disclosure include TR12-prodrugs.

[0021] In further embodiments, the TLR prodrugs of the present disclosure include TR13-prodrugs.

[0022] In a further embodiment, the 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 invention into a nanocarrier of the invention; and (iii) administering the nanocarrier to a patient.

[0023] In another embodiment, the invention comprises a method of delivering a TLR inhibitor along with one or more additional immunomodulatory agents to a tumor comprising: (i) synthesizing a TLR prodrug; (ii) co-formulating the TLR prodrug of the invention along with one or more additional immunomodulatory agents of the invention into a nanocarrier; and (iii) administering the nanocarrier to a patient.

[0024] In another embodiment, the immunomodulatory agent comprises an agonist of other TLRs, 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, a TGFβ inhibitor, an A2aR inhibitor, and / or a prodrug thereof.

[0025] In another embodiment, the present disclosure teaches a method for synthesizing a TLR prodrug.

[0026] In another embodiment, the present disclosure teaches methods for synthesizing TR12 prodrugs.

[0027] In another embodiment, the present disclosure teaches methods for synthesizing TL13 prodrugs.

[0028] In another embodiment, the present disclosure teaches methods of formulating TLR prodrugs into nanocarriers, including but not limited to liposomes.

[0029] In another embodiment, the present disclosure teaches methods of formulating a TR12 prodrug into nanocarriers, including but not limited to liposomes.

[0030] In another embodiment, the present disclosure teaches methods of formulating TR13 prodrugs into nanocarriers, including but not limited to liposomes.

[0031] In another embodiment, the present disclosure teaches methods of formulating TR12 prodrugs into nanocarriers, including but not limited to SLNPs.

[0032] In another embodiment, the present disclosure teaches methods of formulating TR13 prodrugs into nanocarriers, including but not limited to SLNPs.

[0033] In another embodiment, the present disclosure teaches methods of using the nanocarriers of the present disclosure to treat cancer, immunological disorders, and other diseases in humans. [Brief description of the drawings]

[0034] [Figure 1] General chemical synthesis for TR12.

[0035] [Diagram 2] Chemical synthesis for TR12 and TR13 prodrug intermediate(s).

[0036] [Diagram 3] General chemical synthesis for TR13.

[0037] [Figure 4] Synthesis scheme of TLR inhibitor prodrugs using a carboxylic acid functionality.

[0038] [Diagram 5] Synthesis scheme of TLR inhibitor prodrugs using an alcohol functional group.

[0039] [Figure 6] Synthetic schemes for TLR inhibitor prodrugs with secondary amine, amide, or aniline functional groups.

[0040] [Figure 7] Characterization of SLNP-TR12 solid lipid nanocarriers.

[0041] [Figure 8] Characterization of SLNP-TR12 solid lipid nanocarriers (zeta potential).

[0042] [Figure 9] Characterization of SLNP-IC1-TR12 solid lipid nanocarriers (8:1, NTI121).

[0043] [Figure 10] Characterization of SLNP-IC1-TR12 solid lipid nanocarriers (8:1 with NTI121) (zeta potential).

[0044] [Figure 11] Characterization of SLNP-IC1-TR12 solid lipid nanocarriers (at 16:1).

[0045] [Figure 12] Characterization of SLNP-IC1-TR12 solid lipid nanocarriers (at 16:1) (zeta potential).

[0046] [Figure 13] Characterization of SLNP-AR5-TR12 solid lipid nanocarriers.

[0047] [Figure 14] Characterization of SLNP-AR5-TR12 solid lipid nanocarriers (zeta potential).

[0048] [Figure 15] Characterization of SLNP-TR12-NTI-47C solid lipid nanocarriers.

[0049] [Figure 16] Characterization of SLNP-TR12-NTI-47C solid lipid nanocarriers (zeta potential).

[0050] [Figure 17] Tumor inhibition of SLNP-TR12 as a single agent in EMT6 mouse breast cancer cells.

[0051] [Figure 18] Tumor inhibition of various doses of SLNP-TR12 compared to various doses of SLNP-TR5 in EMT6 mouse breast cancer cells.

[0052] [Figure 19] Tumor inhibition of SLNP-TR12 in combination with SLNP-IC1 in EMT6 mouse breast cancer cells.

[0053] [Figure 20] Tumor inhibition of SLNP-TR12 in combination with SLNP-IC1 in EMT6 mouse breast cancer cells.

[0054] [Figure 21] In vitro validation of the mechanism of action of TR12 prodrug in solid lipid nanoparticle ("SLNP") form.

[0055] [Figure 22] In vitro validation of the mechanism of action of SLNP-TR12 versus SLNP-TR5.

[0056] [Figure 23] In vitro verification of the specificity of SLNP-TR12 for Toll-like receptor 7. Figure 23(A) shows the activity of SLNP-TR5 and SLNP-TR12 in RAW-Blue cells. Figure 23(B) shows the specificity of SLNP-TR12 for TLR-7 in HEK-Blue cells.

[0057] [Figure 24] Ex vivo validation of SLNP-TR12 activity in mouse splenocytes.

[0058] [Diagram 25] Ex vivo validation of SLNP-TR12 activity in human PBMCs.

[0059] [Figure 26] In-vivo validation of the efficacy of SLNP-TR12 in the EMT-6 tumor model.

[0060] [Figure 27] In-vivo validation of the efficacy of SLNP-TR12 in the 4T-1 tumor model.

[0061] [Figure 28] In-vivo validation of the efficacy of multiple doses of TR12 prodrug alone and in combination with IC1 prodrug in the EMT-6 tumor model.

[0062] [Figure 29] Maximum tolerated dose (MTD) of doxorubicin prodrug alone and in combination with SLNP-TR12 in the Balb / c mouse model.

[0063] [Diagram 30] In-vivo validation of doxorubicin prodrug efficacy in the B16F10 melanoma tumor model.

[0064] [Diagram 31] In-vivo validation of doxorubicin prodrug efficacy in the MPC11 multiple myeloma tumor model.

[0065] [Diagram 32] In-vivo validation of doxorubicin prodrug efficacy in the Neuro2A neuroblastoma tumor model.

[0066] [Diagram 33] In-vivo validation of doxorubicin prodrug efficacy in the CT26 colon tumor model.

[0067] [Diagram 34] In-vivo validation of doxorubicin prodrug efficacy in the MC38 colon tumor model.

[0068] [Diagram 35] In-vivo validation of doxorubicin prodrug efficacy in the Renca kidney tumor model.

[0069] [Diagram 36] In-vivo validation of doxorubicin prodrug efficacy in the H22 liver tumor model.

[0070] [Figure 37] In-vivo validation of doxorubicin prodrug efficacy in the Hepa1-6 liver tumor model.

[0071] [Figure 38] In-vivo validation of doxorubicin prodrug efficacy in LLC1 lung tumor model.

[0072] [Figure 39] In-vivo validation of doxorubicin prodrug efficacy in the KLN205 lung tumor model.

[0073] [Diagram 40] In-vivo validation of doxorubicin prodrug efficacy in the B16BL6 melanoma tumor model.

[0074] [Diagram 41] In-vivo validation of doxorubicin prodrug efficacy in the Pan02.03 pancreatic tumor model.

[0075] [Diagram 42]In-vivo validation of doxorubicin prodrug efficacy in the RM-1 prostate tumor model.

[0076] [Diagram 43] In-vivo validation of doxorubicin prodrug efficacy in the BMT2 bladder tumor model.

[0077] [Diagram 44] In-vivo validation of doxorubicin prodrug efficacy in the clonal M-3 melanoma tumor model.

[0078] [Diagram 45] In-vivo validation of doxorubicin prodrug efficacy in the 4T1 breast orthotopic tumor model.

[0079] [Diagram 46] In-vivo validation of the efficacy of multiple TR12 prodrug(s) alone or in combination with IC1 prodrug in the CT26 tumor model.

[0080] [Figure 47] In-vivo validation of efficacy of multiple TR12 prodrug(s) alone or in combination with multiple doses of IC1 prodrug in the B16F10 tumor model.

[0081] [Figure 48] In-vivo validation of the efficacy of TR12 prodrug(s) alone or in combination with IC1 prodrug in the EMT-6 tumor model.

[0082] [Figure 49-1]Ex-vivo verification of the ability of CD47 to block cell uptake. Figure 49(A) shows lower cytokine secretion levels in PBMC treated with SLNP-TR12-47c or SLNP-TR12 / 47d. Figure 49(B) shows lower cytokine secretion levels in splenocyte treated with SLNP-TR12-47c or SLNP-TR12 / 47d. Figure 49(C) shows lower cytokine secretion levels in splenocyte treated with SLNP-TR12-47c or SLNP-TR12 / 47d. [Figure 49-2] Ex-vivo verification of the ability of CD47 to block cell uptake. Figure 49(A) shows lower cytokine secretion levels in PBMC treated with SLNP-TR12-47c or SLNP-TR12 / 47d. Figure 49(B) shows lower cytokine secretion levels in splenocyte treated with SLNP-TR12-47c or SLNP-TR12 / 47d. Figure 49(C) shows lower cytokine secretion levels in splenocyte treated with SLNP-TR12-47c or SLNP-TR12 / 47d.

[0083] [Figure 50-1] Ex-vivo validation of the immunomodulatory effect of SLNP-TR12 on tumor-infiltrating lymphocytes in Balb / C mice. Figure 50(A) shows MFI. Figure 50(B) shows the results of NK cells. Figure 50(C) shows the results of total T cells. Figure 50(D) shows the results of cytotoxic T cells. [Figure 50-2] Ex-vivo validation of the immunomodulatory effect of SLNP-TR12 on tumor-infiltrating lymphocytes in Balb / C mice. Figure 50(A) shows MFI. Figure 50(B) shows the results of NK cells. Figure 50(C) shows the results of total T cells. Figure 50(D) shows the results of cytotoxic T cells.

[0084] [Figure 51] In-vitro validation of the ability of CD47 to block cellular uptake. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] Detailed Description of the Invention Section Overview I.) Definition II.) Prodrugs III.) Drug Moiety IV.) Lipids V.) Consolidated Units (“LU”) VI.) Nanocarriers VII.) Liposomes VIII.) Pharmaceutical Formulation IX.) Combination Therapy X.) Methods for delivering liposomes containing prodrugs to cells XI.) Methods of Treating Cancer(s) and Other Immunological Disorder(s) XII.) Kits / Manufactured Products

[0086] I.) Definition Unless otherwise defined, all terms of the art, notations, and other scientific or technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise clearly indicated by context. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0087] When a trademark is used herein, reference to the trademark also refers to formulations of the product, generic drugs, and the active pharmaceutical ingredient(s) of the trademark product, unless the context dictates otherwise.

[0088] As used herein, the term "about" when referring to a value or amount of size (i.e., diameter), weight, concentration, or percentage is meant to encompass variations from the specified amount of ±20% or ±10%, in one example, ±5%, in another example, ±1%, and in yet another example, ±0.1%, as such variations are suitable for practicing the disclosed methods.

[0089] As used herein, the term "and / or," when used in the context of a list of entities, refers to the entities present either singly or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes not only A, B, C, and D individually, but also any and all combinations and subcombinations of A, B, C, and D.

[0090] As used herein, numerical ranges recited by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes, but is not limited to, 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5).

[0091] As used herein, the phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and, in addition, those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0092] The terms "advanced cancer," "locally advanced cancer," "progressive disease," and "locally advanced disease" refer to cancer that has spread through the relevant tissue capsule and are meant to include stage C disease in the American Urological Association (AUA) system, stages C1-C2 disease in the Whitmore-Jewett system, and stages T3-T4 and N+ disease in the TNM (tumor, node, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients with clinically localized (organ-confined) cancer.

[0093] As used herein, the term "alkyl" refers to any alkyl group selected from C1-C6 inclusive, including, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. 20 "Alkyl" can refer to a linear (i.e., "straight chain"), branched or cyclic, saturated or at least partially, and in some cases unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chain of 1 to 8 carbon atoms, e.g., methyl, ethyl, or propyl, attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms (i.e., C1-C8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to C1-C8 straight chain alkyl. In other embodiments, "alkyl" refers, in particular, to C1-C8 straight chain alkyl. i~8 Refers to branched chain alkyl.

[0094] An alkyl group may be optionally substituted with one or more alkyl group substituents, which may be the same or different ("substituted alkyl"). The term "alkyl group substituent" includes, but is not limited to, alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. In some embodiments, one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms may be inserted along the alkyl chain as required, and the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl"), or aryl.

[0095] Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group have been replaced with another atom or functional group, including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0096] The term "aryl" is used herein to refer to an aromatic substituent, which may be a single aromatic ring or multiple aromatic rings that are fused together, covalently linked, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group may also be a carbonyl, as in benzophenone, or an oxygen, as in diphenylether, or a nitrogen, as in diphenylamine. The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) may include, among others, phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, and benzophenone. In certain embodiments, the term "aryl" refers to cyclic aromatics containing about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, including 5- and 6-membered aromatic and heteroaromatic rings. Aryl groups can be optionally substituted with one or more aryl group substituents which can be the same or different ("substituted aryl"); "aryl group substituents" include alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R'', where R' and R'' can each independently be hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl. Illustrative examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

[0097] "Heteroaryl," as used herein, refers to an aryl group that contains one or more non-carbon atoms (e.g., O, N, S, Se, etc.) in the backbone of the ring structure. Nitrogen-containing heteroaryl moieties include, but are not limited to, pyridine, imidazole, benzimidazole, pyrazole, pyrazine, triazine, pyrimidine, etc.

[0098] The terms "anti-cancer drug," "chemotherapeutic agent," and "anti-cancer prodrug" refer to drugs (i.e., chemical compounds) or prodrugs that are known to or believed to be capable of treating cancer (i.e., killing cancer cells, inhibiting the growth of cancer cells, or treating a symptom associated with cancer). In some embodiments, the term "chemotherapeutic agent," as used herein, refers to a non-PS molecule that is used to treat cancer and / or has cytotoxic capabilities. More traditional or conventional chemotherapeutic agents can be described by mechanism of action or by class of chemical compound, and can include, but are not limited to, alkylating agents (e.g., melphalan), anthracyclines (e.g., doxorubicin), cytoskeletal disruptors (e.g., paclitaxel), epothilones, histone deacetylase inhibitors (e.g., vorinostat), inhibitors of topoisomerase I or II (e.g., irinotecan or etoposide), kinase inhibitors (e.g., bortezomib), nucleotide analogs or precursors thereof (e.g., methotrexate), peptide antibiotics (e.g., bleomycin), platinum-based agents (e.g., cisplatin or oxaliplatin), retinoids (e.g., tretinoin), and vinca alkaloids (e.g., vinblastine).

[0099] "Aralkyl" refers to an -alkyl-aryl group, in which the alkyl and / or aryl portions are optionally substituted.

[0100] "Alkylene" refers to a straight or branched chain divalent aliphatic hydrocarbon group having 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group may be straight chain, branched, or cyclic. The alkylene group may also be optionally unsaturated and / or substituted with one or more "alkyl group substituents." One or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl") may be optionally inserted along the alkylene group, where the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH-), ethylene (-CH-CH-), propylene (-(CH)-), cyclohexylene (-CH 10 -), -CH=CH-CH=CH-, -CH=CH-CH2-, -(CH2) q -N(R)-(CH2)- (wherein each q is an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl), methylenedioxyl (-O-CH2-O-), and ethylenedioxyl (-O-(CH2)2-O-). The alkylene group can have from about 2 to about 3 carbon atoms and can further have from 6 to 20 carbons.

[0101] The term "arylene" refers to a divalent aromatic group, such as a divalent phenyl or naphthyl group. The arylene group can be optionally substituted with one or more aryl group substituents and / or can contain one or more heteroatoms.

[0102] The term "amino" refers to the -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" can refer to the -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 the -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).

[0103] "Bioreactive nanomaterial" refers to engineered biomaterials that induce or catalyze biological responses. In certain embodiments, the nanomaterials catalyze biological responses at various nano / bio interfaces by inducing a response through one or more properties selected from the group consisting of composition, size, shape, aspect ratio, dissolution, electronic, redox, surface presentation, surface coating, hydrophobicity, hydrophilicity, atomically thin nanosheets, or functionalized surface groups. In certain embodiments, the bioreactive nanomaterials have the ability to inhibit TLR-1 biological responses in cells (e.g., tumor cells) and / or activate the innate immune system through "danger signal" sending and adjuvant effects.

[0104] "Bulk" (aka drug substance) means a drug substance or drug product that has not been filled into a final container for distribution. Final formulated bulk generally refers to a drug product that has been formulated and stored or held prior to filling. Drug substances may be stored or held as "bulk" or "concentrated bulk" prior to formulation into a drug product.

[0105] The terms "carboxylate" and "carboxylic acid" refer to -C(=O)O, respectively. -The term "carboxyl" can also refer to the group -C(=O)OH.

[0106] The terms "conjugate" and "conjugated" as used herein can refer to the binding (e.g., covalent bonding) of two or more components (e.g., chemical compounds, polymers, biomolecules, particles, etc.) to one another. In some embodiments, a conjugate can include monovalent moieties derived from two different chemical compounds covalently linked via a divalent linker moiety (e.g., an optionally substituted alkylene or arylene). In some embodiments, the linker can contain one or more biodegradable bonds, such that one or more bonds of the linker can be broken when the prodrug is exposed to certain physiological environments or enzymes (e.g., esterases).

[0107] The term "compound" refers to and includes the chemical compound (e.g., prodrug) itself, as well as the following, whether or not expressly stated, unless the context makes it clear that the following is excluded: amorphous and crystalline forms of the compound, including polymorphic forms, where these forms may be part of a mixture or may be isolated; free acid and free base forms of the compound, which are typically those forms depicted in the structures provided herein; isomers of the compound, which refers to optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers, as well as mixtures of optical isomers, including racemic and non-racemic mixtures, where an isomer may be in isolated form or may be a mixture with one or more other isomers; isomers of the compound, which refers to optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers, as well as mixtures of optical isomers, including racemic mixtures and non-racemic mixtures, and isomers may be in isolated form or may be a mixture with one or more other isomers; topologies, including deuterium- and tritium-containing compounds, including compounds containing radioisotopes, including therapeutically and diagnostically effective radioisotopes; multimeric forms of the compounds, including dimeric, trimeric, etc. forms; salts of the compounds, preferably pharma- ceutical acceptable salts, including acid addition salts and base addition salts, including salts with organic and inorganic counterions, including zwitterionic forms, where if the compound is associated with two or more counterions, the two or more counterions may be the same or different; and solvates of the compounds, including hemisolvates, monosolvates, disolvates, etc., including organic solvates and inorganic solvates, where the inorganic solvates include hydrates, where if the compound is associated with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some cases, reference herein to the compounds of the invention includes an explicit reference to one or of the above forms, e.g., salts and / or solvates, but this reference is merely for emphasis and should not be construed as excluding any form other than those specified above.

[0108] "Drug product" generally refers to a final formulation containing an active drug component, although not necessarily in association with an inactive component (i.e., a liposome containing a TLR inhibitor prodrug). The term also includes final dosage forms that do not contain an active component but are intended to be used as a placebo.

[0109] The term "disulfide" can refer to a -SS- group.

[0110] The term "empty vesicles" refers to unloaded lipid vesicles, per se.

[0111] The term "ester", as used herein, means a chemical compound derived from an acid (organic or inorganic) in which at least one -OH hydroxy group is replaced by an -O-alkyl (alkoxy) or O-aryl (aryloxy) group.

[0112] The term "esterase" as used herein is a hydrolytic enzyme that breaks down esters into acids and alcohols.

[0113] "Excipient" means an inert substance used as a carrier for active ingredients in drugs, such as vaccines. Excipients are also sometimes used to bulk up formulations containing highly potent active ingredients, allowing for convenient and accurate dosing. Examples of excipients include, but are not limited to, anti-adherents, binders, coatings, disintegrants, fillers, diluents, flavors, colors, lubricants, and preservatives.

[0114] The terms "halo," "halide," or "halogen," as used herein, refer to fluoro, chloro, bromo, and iodo groups.

[0115] The terms "hydroxyl" and "hydroxy" refer to the --OH group.

[0116] The terms "inhibit" or "inhibition of," as used herein, mean to reduce by a measurable amount or to prevent completely.

[0117] The terms "individual" or "patient" when used in the context of this disclosure can be used interchangeably.

[0118] As used herein, the term "ligand" generally refers to a species, e.g., a molecule or ion, that interacts with, e.g., binds to, another species in some manner. See MARTELL, AE, and HANCOCK, RP, Metal Complexes in Aqueous Solutions, Plenum: New York (1996), which is incorporated herein by reference in its entirety.

[0119] The term "lipid" as used herein refers to a class of naturally occurring (organic) compounds that are insoluble in polar solvents. In the context of this disclosure, lipids refer to conventional lipids, phospholipids, cholesterol, lipids chemically functionalized for attachment of PEG and ligands, and the like.

[0120] The term "lipid bilayer" or "LB" refers to any bilayer of oriented amphiphilic lipid molecules with their hydrocarbon tails pointing inward to form a continuous non-polar phase.

[0121] The terms "liposome" or "lipid vesicle" or "vesicle" are used interchangeably to refer to an aqueous compartment enclosed by a lipid bilayer as conventionally defined (see STRYER (1981) Biochemistry, 2d Edition, WH Freeman & Co., p. 213).

[0122] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0123] The term "mercapto" or "thiol" refers to a -SH group.

[0124] The terms "metastatic cancer" and "metastatic disease" are meant to mean spread to regional lymph nodes or distant sites and include stage D disease on the AUA system and stage TxNxM+ on the TNM system.

[0125] 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, a nanocarrier comprises a lipid bilayer that encases (or surrounds or covers) a porous particle core. In certain embodiments, a nanocarrier is a liposome, lipid nanoparticle ("LNP"), or solid lipid nanoparticle ("SLNP").

[0126] The terms "nanoscale particle," "nanomaterial," "nanocarrier," and "nanoparticle" refer to a structure having at least one region with a dimension (e.g., length, width, diameter, etc.) of less than about 1,000 nm. In some embodiments, the dimension is smaller (e.g., less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, or even less than about 20 nm). In some embodiments, the dimensions are between about 20 nm and about 250 nm (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm).

[0127] The term "nanovesicle" refers to a "lipid vesicle" (or a population of vesicles having an average diameter) having a diameter ranging from about 20 nm, or from about 30 nm, or from about 40 nm, or from about 50 nm, or from about 500 nm, or from about 400 nm, or from about 300 nm, or from about 200 nm, or from about 150 nm, or from about 100 nm, or from about 80 nm. In certain embodiments, the nanovesicle has a diameter ranging from about 40 nm to about 80 nm, or from about 50 nm to about 70 nm.

[0128] "Pharmaceutically acceptable" refers to a composition that is non-toxic, inert, and / or physiologically compatible with humans or other mammals.

[0129] "Pharmaceutical formulation" refers to the process of combining various chemicals with pure drug substances to produce a final drug product.

[0130] The term "phosphonate" refers to the group -P(=O)(OR)2, where each R can be independently H, alkyl, aralkyl, aryl, or negative charge (i.e., there are substantially no R groups attached to the oxygen atom, resulting in the presence of an unshared electron pair on the oxygen atom). Thus, in other words, each R can be present or absent, and when present, is selected from H, alkyl, aralkyl, or aryl.

[0131] The term "phosphate" refers to the group -OP(=O)(OR')2, where R' is H or a negative charge.

[0132] The term "prodrug" refers to a pharmaceutical agent or compound that is metabolized into a pharmacologically active drug after administration. For purposes of this disclosure, the prodrugs of the present invention include three components: (i) a drug moiety, (ii) a lipid moiety, and (iii) a linking unit ("LU").

[0133] The term "TLR prodrug" refers to a prodrug of the present invention, wherein the drug moiety comprises a TLR inhibitor.

[0134] The term "pyrolipid" refers to a conjugate of a lipid with a porphyrin, a porphyrin derivative, or a porphyrin analog. In some embodiments, a pyrolipid can include a lipid conjugate in which a porphyrin or a derivative or analog thereof is covalently attached to a lipid side chain. See, for example, U.S. Patent Application Publication No. 2014 / 0127763.

[0135] As used herein, the terms "specific," "specifically binds," and "specifically binds to" refer to selective binding of the nanocarriers of the present invention to the target TLR-1 or related family members.

[0136] The term "supported lipid bilayer" refers to a lipid bilayer that encapsulates a porous particle core. This definition is so indicated 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 can have a thickness ranging from about 6 nm to about 7 nm, including a 3-4 nm thick hydrophobic core, plus a layer of hydrated hydrophilic head groups (about 0.9 nm each), plus two partially hydrated regions of about 0.3 nm each. In various embodiments, the lipid bilayer surrounding the liposome comprises a continuous or substantially continuous bilayer, which effectively covers and seals the TLR inhibitor.

[0137] The term "thioalkyl" can refer to the group -SR, where R is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl. Similarly, the terms "thioaralkyl" and "thioaryl" refer to the group -SR, where R is aralkyl and aryl, respectively.

[0138] As used herein, "treating" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as extending survival, reducing morbidity, and / or reducing side effects that are a by-product of alternative treatment modalities; as will be readily recognized in the art, complete eradication of the disease, although preferred, is not a requirement of the act of treatment.

[0139] The term "therapeutically effective amount" refers to that amount of active prodrug, nanoencapsulated prodrug, or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal, individual or human.

[0140] The term "unsupported lipid bilayer" refers to the uncoated lipid bilayer in a lipid vesicle or liposome.

[0141] II.) Prodrugs As shown in this disclosure, and for the purposes of the present invention, suitable prodrugs are formed by conjugating a drug moiety of the present invention (see the section entitled Drug Moiety) to a lipid moiety of the present invention (see the section entitled Lipid) via a LU of the present disclosure (see the section entitled Linking Unit). For the purposes of the present disclosure, the formation of TLR prodrugs can utilize several strategies. (See, for example, Figures 4, 5, and 6).

[0142] Thus, in some embodiments, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor of the present disclosure.

[0143] In a further embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor of the present disclosure, where the TLR inhibitor inhibits TLR1 / 2.

[0144] In a further embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor of the present disclosure, where the TLR inhibitor inhibits TLR4.

[0145] In a further embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor of the present disclosure, where the TLR inhibitor inhibits TLR7.

[0146] In a further embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor of the present disclosure, where the TLR inhibitor inhibits TLR8.

[0147] In a further embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor of the present disclosure, where the TLR inhibitor inhibits TLR7 / 8.

[0148] In a further embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor of the present disclosure, where the TLR inhibitor inhibits TLR1 / 2 and the prodrug comprises a prodrug of Formula I.

[0149] In one embodiment, the prodrug has the following chemical structure as shown in Formula I: [ka] In an exemplary embodiment of Formula I, A= [ka] [ka] [is].

[0150] Thus, in one embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor of formula I.

[0151] In one embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor as depicted in FIG.

[0152] In one embodiment, the prodrug is a drug-lipid moiety that comprises a TLR inhibitor as depicted in FIG.

[0153] In a further embodiment, the TLR prodrug is a drug-lipid moiety that comprises a lipid of the present disclosure.

[0154] In a further embodiment, the TLR prodrug is a drug-lipid moiety, where the lipid is a CHEMS.

[0155] In a further embodiment, the TLR prodrug is a drug-lipid moiety, where the lipid is stearic acid.

[0156] In a further embodiment, the TLR prodrug is a drug-lipid moiety, where the lipid is a stearic acid derivative that is non-cleavable.

[0157] In a further embodiment, the TLR prodrug is a drug-lipid moiety comprising a LU of the present disclosure.

[0158] In a further embodiment, the TLR prodrug is a drug-lipid moiety, where LU is a hydromethylcarbamate linker.

[0159] In a further embodiment, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present invention, where the TLR inhibitor comprises the chemical compositions TR12 and / or TR13.

[0160] In a further embodiment, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the present invention, where the TLR inhibitor comprises TR12 and / or TR13, and further comprises CHEMS.

[0161] In a further embodiment, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the invention, where the TLR inhibitor comprises TR12 and / or TR13 and further comprises stearic acid.

[0162] In a further embodiment, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the invention, where the TLR inhibitor comprises TR13 and further comprises a non-cleavable stearic acid derivative.

[0163] In a further embodiment, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the invention, where the TLR inhibitor comprises TR12 and further comprises CHEMS, and LU is a hydromethylcarbamate linker.

[0164] In a further embodiment, the prodrug is a drug-lipid moiety comprising a TLR inhibitor of the invention, where the TLR inhibitor comprises TR12 and further comprises stearic acid, and LU is a hydromethylcarbamate linker.

[0165] In a further embodiment, the prodrug has the following structure: [ka] wherein the TLR inhibitor comprises TR12 and further comprises stearic acid.

[0166] In a further embodiment, the prodrug has the following structure: [ka] wherein the TLR inhibitor comprises TR13 and further comprises a non-cleavable stearic acid derivative.

[0167] In a further embodiment, the prodrug has the following formula: [ka] ("TR12-prodrug") wherein the TLR inhibitor comprises TR12 and further comprises a lipid of the disclosure.

[0168] In an additional embodiment of the present disclosure, the subject matter provides a TLR inhibitor prodrug comprising a parent drug of a therapeutic agent conjugated to a lipid. In some embodiments, the prodrug comprises (a) a monovalent drug moiety, (b) a monovalent lipid moiety, and (c) a bivalent 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 linked). The monovalent drug moiety and the monovalent lipid moiety can be monovalent derivatives of chemical compounds and lipids, respectively. For example, the monovalent derivatives can be deprotonated derivatives of chemical compounds or lipids that comprise hydroxyl, thiol, amino, or carboxylic acid groups.

[0169] In a further embodiment of the present disclosure, the subject matter provides a TLR inhibitor prodrug comprising a parent drug of a therapeutic agent conjugated to a lipid. In some embodiments, the prodrug comprises (a) a bivalent drug moiety, (b) a bivalent lipid moiety, and (c) a bivalent linker moiety comprising an in vivo degradable linkage, where the bivalent drug moiety and the bivalent lipid moiety are linked via a linker (e.g., covalently linked). The bivalent drug moiety and the bivalent lipid moiety can be bivalent derivatives of chemical compounds and lipids, respectively. For example, the bivalent derivatives can be deprotonated derivatives of chemical compounds or lipids that comprise hydroxyl, thiol, amino, or carboxylic acid groups.

[0170] Those skilled in the art will recognize and can make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention disclosed herein, and such changes and modifications are intended to be within the scope of the present disclosure.

[0171] III.) Drug Moiety Another aspect of the present invention provides novel TLR prodrug compound(s) having the following formula, designated TR12 and TR13:

[0172] Those skilled in the art will appreciate that the compounds are useful as TLR inhibitors (e.g., inhibiting TLRs, such as TLR1 / 2, TLR4, TLR7, TLR8 and / or TLR7 / 8). By way of brief background, TLR-1 (CD281) recognizes pathogen-associated molecular patterns with specificity for gram-positive bacteria. TLR-1 is found on the epithelial cell layer lining the small and large intestine and is a key 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. TLR1 has been demonstrated to cooperate with TLR2 (as a heterodimer) to recognize peptidoglycan and (triacyl)lipopeptides and to interact with TLR2. See FARHAT, et. al., J. Leukoc. Biol. 83(3): 692-701 (2007) and JIN, et. al., Cell. 130(6): 1071-1082 (2007).

[0173] TLR2 (CD282) is a protein that is encoded by the TLR2 gene in humans. TLR2 is a membrane protein expressed on the surface of certain cells that recognizes foreign bodies and passes appropriate signals to immune system cells. TLR2 is most abundantly expressed in peripheral blood leukocytes and mediates the host response to gram-positive bacteria and yeast through stimulation of NF-κB. See BARRELLO, et. al., Int. J. Immun. & Pharm. 24(3): 549-556 (2011). TLR2 resides on the plasma membrane where it responds to lipid-containing PAMPs, such as lipoteichoic acid, and lipopeptides containing di- and tri-acylated cysteines. TLR2 does this by forming a dimeric complex with either TLR1 or TLR6 on the plasma membrane. See BOTOS, et. al., Structure 19(4): 447-459 (2011).

[0174] TLR4 (CD284) is another member of the TLR family. Its activation leads to the intracellular signaling pathway NF-κB and inflammatory cytokine production, involved in the activation of the innate immune system. TLR4 is best known to recognize lipopolysaccharide (LPS), a component present in many Gram-negative bacteria (e.g., Neisseria species) and select 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 with the LBP protein. Conformational changes in TLR4 induce the recruitment of intracellular adaptor proteins, which contain essential TIR domains to activate downstream signaling pathways. LU, et. al., Cytokine 42(2): 145-151 (2008). TLR4 can activate the MAPK and NF-κB pathways, suggesting a possible direct role of cell-autonomous TLR4 signaling in regulating carcinogenesis, particularly via increased proliferation, inhibition of apoptosis, and metastasis of tumor cells.

[0175] 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 the 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. Id. However, the presence of GU-rich sequences in single-stranded RNA is not sufficient to stimulate TLR7. TLR7 has been shown to play an important role in the pathogenesis of autoimmune disorders, such as systemic lupus erythematosus (SLE), and in regulating antiviral immunity. In addition, TLR7 agonists have been investigated for cancer immunotherapy due to their ability to induce robust production of anti-cancer cytokines, such as interleukin-12.

[0176] TLR8 is another member of the family, a protein designated CD288. TLR8 is primarily expressed in lung and peripheral blood leukocytes and is in close proximity to another family member, TLR7. TLR8 is an endosomal receptor that recognizes single-stranded RNA (ssRNA) and can recognize ssRNA viruses such as influenza, Sendai, and Coxsackie B viruses. TLR8 binding to viral RNA recruits MyD88, leading to activation of the transcription factor NF-kB and an antiviral response. See ZHANG, et. al., Sci. Rep., 6, 29447; doi: 10.1038 / srep29447 (2016).

[0177] Based on the above, the present disclosure describes a class of TLR inhibitors.

[0178] In one embodiment, the class of TLR inhibitors inhibits TLR1 / 2.

[0179] In one embodiment, the class of TLR inhibitors inhibits TLR7.

[0180] In one embodiment, the drug moiety of the disclosure has the following chemical structure (denoted TR12): [ka] The compounds include those having the formula:

[0181] Those skilled in the art will recognize and can make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention disclosed herein, and such changes and modifications are intended to be within the scope of the present disclosure.

[0182] IV.) Lipids Generally speaking, and for the purposes of this disclosure, the term "lipid" is used in its broadest sense and includes several sub-categories of lipids, including, but not limited to, phospholipids / fatty acids. As recognized by those skilled in the art, phospholipids refer to a class of lipids that are the main components of all cell membranes. Phospholipids are capable of forming lipid bilayers due to their amphipathic characteristics. The structure of a phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group that can be modified with simple organic molecules such as choline, ethanolamine, or serine. These two components are usually linked together by a glycerol molecule. A representative list of phospholipid / fatty acid(s) of the present invention is set forth in Table III.

[0183] As a brief background, at the most fundamental level, liposome properties depend on subtle physicochemical interactions among the various lipid species in their composition. Individual lipids can be combined to form a wide variety of superstructures, including bilayers, and the bilayer properties can be tailored to modulate drug release and membrane stability. In a simplified bilayer model, acyl chain length defines the bilayer thickness and phase transition temperature (Tm), acyl chain saturation controls bilayer fluidity, and head group interactions affect inter- and intra-lipid forces. Liposome behavior can be tuned by incorporating synthetic lipids, such as lipid prodrugs, fusogenic lipids, and functionalizable lipids, into the bilayer. See KOHLI, et. al., J. Control Release, 0: pp. 274-287 (Sept. 28, 2014).

[0184] In one embodiment of the present disclosure, the TLR prodrug comprises a monovalent lipid moiety.

[0185] In one embodiment, the TLR prodrug comprises a divalent lipid moiety.

[0186] In one embodiment, the lipid has the following chemical structure: [ka] Cholesterol having the formula:

[0187] In one embodiment, the lipid has the following chemical structure: [ka] The DPPG has the formula:

[0188] In one embodiment, the lipid has the following chemical structure: [ka] The compound includes DMPG having the formula:

[0189] In one embodiment, the lipid has the following chemical structure: [ka] The Lyso PC having the formula:

[0190] In one embodiment, the lipid comprises (Δ9-Cis)PG.

[0191] In one embodiment, the lipid has the following chemical structure: [ka] Soy Lyso PC having the formula:

[0192] In one embodiment, the lipid has the following chemical structure: [ka] The PG has the formula:

[0193] In one embodiment, the lipid has the following chemical structure: [ka] C16 PEG2000 ceramide (Ceramde) having the formula:

[0194] In one embodiment, the lipid has the following chemical structure: [ka] and cholesterol hemisuccinate ("CHEMS"), having the formula:

[0195] In one embodiment, the lipid has the following chemical structure as shown in Formula II: [ka] It includes a class of lipids having the formula:

[0196] In an exemplary embodiment of Formula II, X= [ka] and Y= [ka] and Z= [ka] [is].

[0197] In one embodiment, the lipid has the following chemical structure as shown in Formula III: [ka] It includes a class of lipids having the formula: In an exemplary embodiment of Formula III, X= [ka] [is].

[0198] In further embodiments, the lipid moiety of the present disclosure comprises a class of invariant natural killer T (iNKT) cells.

[0199] In a further embodiment, the lipid moiety of the present disclosure comprises alpha-galactosylceramide (α-GalCer).

[0200] For reference, a further list of the chemical formulas and abbreviation(s) of the lipids disclosed herein is set forth in Table I.

[0201] In additional embodiments, the lipid comprises a phospholipid / fatty acid disclosed herein and set forth in Table III.

[0202] In a further embodiment, the lipid comprises stearic acid.

[0203] In a further embodiment, the lipid comprises a non-cleavable stearic acid derivative.

[0204] In addition, the TLR prodrug and / or liposome(s) of the present disclosure may include one or more helper lipids, also referred to herein as "helper lipid components". The helper lipid components are preferably selected from the group including phospholipids and steroids. The phospholipids are preferably di- and monoesters of phosphoric acid. Preferred members of the phospholipids are phosphoglycerides and sphingolipids. Steroids, as used herein, are naturally occurring and synthetic compounds based on partially hydrogenated cyclopenta[a]phenanthrene. Preferably, the steroids contain 21-30 C atoms. A particularly preferred steroid is cholesterol.

[0205] Without wishing to be bound by any theory, it should be noted that due to the specific molar percentage of helper lipid(s) contained in the lipid composition according to the invention, which may be either a PEG-free helper lipid or in particular a PEG-containing helper lipid, surprising effects may be realized, more particularly when the content of any of this type of helper lipid is encompassed within the concentration ranges specified herein.

[0206] In a further aspect of the present invention, the lipid composition, preferably present as a lipoplex or liposome, preferably exhibits a neutral or overall anionic charge. The anionic lipid is preferably any neutral or anionic lipid as described herein. The lipid composition, in a preferred embodiment, comprises any helper lipid or combination of helper lipids and any TLR inhibitor as described herein. In a further embodiment, the composition according to the present invention containing nucleic acid(s) forms a lipoplex. In a preferred embodiment, the term lipoplex as used herein refers to a composition composed of neutral or anionic lipid, neutral helper lipid and TLR inhibitor of the present invention. For references to the use of helper lipids in the art, see, e.g., 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).

[0207] In a preferred embodiment, the helper lipids of the present invention comprise the helper lipids listed in Table II.

[0208] In one embodiment, the TLR prodrug comprises a lipid of the invention, where the lipid is CHEMS and the drug moiety is TR12.

[0209] In one embodiment, the TLR prodrug comprises a lipid of the invention, where the lipid is CHEMS, the drug moiety is TR12, and further comprises LU, where LU is a hydromethylcarbamate linker.

[0210] In one embodiment, the TLR prodrug comprises a lipid of the invention, where the lipid is CHEMS, the drug moiety is TR12, and further comprises LU, which is a hydromethylcarbamate linker, and further comprises a helper lipid moiety, which comprises a helper lipid of Table II.

[0211] In one embodiment, the TLR prodrug comprises a lipid of the invention, where the lipid is CHEMS, the drug moiety is TR12, and CHEMS is monovalent.

[0212] In one embodiment, the TLR prodrug comprises a lipid of the invention, where the lipid is stearic acid and the drug moiety is TR12.

[0213] In one embodiment, the TLR prodrug comprises a lipid of the invention, where the lipid is stearic acid, the drug moiety is TR12, and the stearic acid is monovalent.

[0214] In one embodiment, the TLR prodrug comprises a lipid of the invention, where the lipid is stearic acid, the drug moiety is TR12, and further comprises LU, where LU is a hydromethylcarbamate linker.

[0215] In one embodiment, the TLR prodrug comprises a lipid of the present invention, wherein the lipid is stearic acid, the chemical composition is TR12, and further comprises LU, wherein LU is a hydromethylcarbamate linker, and further comprises a helper lipid component, wherein the helper lipid component comprises a helper lipid of Table II.

[0216] Those skilled in the art will recognize and can make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention disclosed herein, and such changes and modifications are intended to be within the scope of the present disclosure.

[0217] V.) Consolidated Units (“LU”) In some embodiments, the presently disclosed subject matter provides prodrugs, including drug-lipid conjugates that include biodegradable linkers, such as esters, thioesters, and other linkers known in the art.

[0218] Exemplary embodiments of ester chemistry are described herein: [ka]

[0219] In some embodiments, the prodrug is a drug-lipid conjugate, where the drug-lipid conjugate is cleaved by an esterase.

[0220] In one embodiment, the prodrugs of the present invention may be represented by the following scheme: [ka] to include LU via a secondary amine, amide, or aniline.

[0221] An exemplary synthesis is as follows: [ka] As stated above. Cleavage of prodrug structures containing secondary amines, amides, or anilines can be achieved using the following exemplary syntheses: [ka] via esterase hydrolysis of secondary amine, amide, or aniline prodrugs under [In the formula, R1 and R2 can be and molecule that binds to N via C).

[0222] In one embodiment, the secondary amide nitrogen of the TR12 drug moiety is conjugated to CHEMS via a hydromethylcarbamate linker.

[0223] In one embodiment, the secondary amide nitrogen of the TR12 drug moiety is conjugated to stearic acid via a hydromethylcarbamate linker.

[0224] Those skilled in the art will recognize and can make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention disclosed herein, and such changes and modifications are intended to be within the scope of the present disclosure.

[0225] VI.) Nanocarriers Generally speaking, and for purposes of this disclosure, nanocarrier(s) are within the scope of the present invention. Nanocarriers are nanomaterials that are used as transport modules for another substance, such as a drug. Commonly used nanocarriers include micelles, polymers, carbon-based materials, liposomes, solid lipid nanoparticles, and other materials. Nanocarriers can deliver drugs to otherwise inaccessible sites in the body due to their small size. Nanocarriers may include polymer conjugates, polymer nanoparticles, lipid-based carriers, dendrimers, carbon nanotubes, and gold nanoparticles. Lipid-based carriers include both liposomes and micelles. In certain embodiments, the nanocarrier is a liposome, lipid nanoparticle ("LNP"), or solid lipid nanoparticle ("SLNP").

[0226] In addition, nanocarriers are useful in the drug delivery process because they can deliver drugs to site-specific targets, delivering drugs to certain organs or cells, and not elsewhere. Site specificity has a great therapeutic benefit, as it ensures that the drug being delivered is not delivered to the wrong place. In addition, nanocarriers are promising for use in chemotherapy, as they can help reduce the harmful broader-scale toxicity of chemotherapy to fast-growing healthy cells in the body. Chemotherapy drugs can be highly toxic to human cells, so it is important that they are delivered to the tumor without being released to other parts of the body.

[0227] Generally speaking, there are four ways in which nanocarriers can deliver drugs, including passive targeting, active targeting, pH specificity, and temperature specificity.

[0228] Passive targeting refers to the ability of nanocarriers to travel through the tumor vasculature, be captured, and accumulate in the tumor. This accumulation is caused by the enhanced permeability and retention effect. The leaky vasculature of tumors is a vascular network formed in tumors, which contains many small holes. These holes contain many bends that can not only allow nanocarriers to enter, but also trap the nanocarriers. As more nanocarriers are trapped, the drug accumulates at the tumor site. This accumulation delivers a large dose of drug directly to the tumor site.

[0229] Active targeting involves incorporating targeting modules, such as ligands or antibodies, onto the surface of the nanocarrier that are specific for a particular type of cell in the body. Generally, nanocarriers have a high surface area to volume ratio and can incorporate multiple ligands on their surface.

[0230] In addition, certain nanocarriers release the drugs they contain only in a specific pH range. pH specificity also allows nanocarriers to deliver drugs directly to tumor sites. This is due to the fact that tumors are generally more acidic than normal human cells, with a pH of about 6.8. Normal tissues have a pH of about 7.4. Thus, nanocarriers that release drugs only in a certain pH range can therefore be used to release drugs only in the acidic tumor environment. A highly acidic environment releases the drug as the acidic environment breaks down the structure of the nanocarrier. In general, these nanocarriers do not release drugs in neutral or basic environments, effectively targeting the acidic environment of tumors while leaving normal body cells alone. This pH sensitivity can also be achieved in micellar systems by adding copolymer chains to the micelles that have been 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, releasing most of the drug in one go, rather than gradually as with other drug treatments.

[0231] In addition, some nanocarriers have been shown to deliver drugs more effectively at certain temperatures. Because tumor temperatures are generally around 40°C higher than the temperature throughout the rest of the body, this temperature gradient helps act as a safeguard for tumor-specific site delivery. See REZAEI, et. al., Polymer, 53(16): 3485-3497 (2012).

[0232] As disclosed herein, lipid-based nanocarriers, such as liposomes, are within the scope of the present invention. Lipid-based nanoparticles (LBNP or LNP), such as liposomes, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), can transport hydrophobic and hydrophilic molecules, exhibit very low or no toxicity, and increase drug action time by using drug half-life extension and controlled release. Lipid nanoparticles can 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 promote drug release in acidic environments and can be associated with small molecules or antibodies that recognize tumor cells or their receptors (e.g., folic acid (FoA)). Nanodrugs can also be used in combination with other therapeutic strategies to improve patient response. See GARCIA-PINEL, et. al., Nanomaterials 9(639) (2019).

[0233] In various embodiments, the silicasome drug carriers described herein comprise porous silica (or other material) nanoparticles (e.g., silica bodies having a surface and defining a plurality of pores suitable for receiving molecules therein) coated with a lipid bilayer. The fact that the nanoparticles are referred to as silica nanoparticles does not exclude that materials other than silica are also incorporated within the silica nanoparticles. In some embodiments, the silica nanoparticles may be substantially spherical, with a plurality of pore openings at the surface that provide 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 ovoid, rod-shaped, substantially regular polygonal, irregular polygonal, etc.

[0234] Generally, silica nanoparticles include a silica body defining an exterior surface and sidewalls within the pores between pore openings. The pores can extend throughout the silica body to another pore opening, or the pores can extend only partially throughout the silica body such that the pores have a base defined by the silica body.

[0235] In some embodiments, the silica body is mesoporous. In other embodiments, the silica body is microporous. As used herein, "mesoporous" means having pores with a diameter 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, are large enough to contain one or more therapeutic compounds therein. In such embodiments, the pores allow small molecules, such as therapeutic compounds, e.g., anti-cancer compounds, to be attached or bound to the inner surface of the pores and released from the silica body when used for therapeutic purposes. In some embodiments, the pores are substantially cylindrical.

[0236] In certain embodiments, the nanoparticles comprise a plurality of pores having a pore size between about 1 nm and about 10 nm or between about 2 nm and about 8 nm in diameter. In certain embodiments, the nanoparticles comprise a plurality of pores having a pore size between about 1 nm and about 6 nm or between about 2 nm and about 5 nm. Other embodiments include particles having a pore size of less than 2.5 nm.

[0237] In other embodiments, the pore size is between 1.5 and 2.5 nm. Silica nanoparticles with other pore sizes can be prepared, for example, by using different surfactants or swelling agents during preparation of the silica nanoparticles. In various embodiments, the nanoparticles can include particles as large as about 1000 nm (e.g., mean or median diameter (or another characteristic dimension). In various embodiments, however, the nanoparticles are typically less than 500 nm or less than about 300 nm, since particles larger than 300 nm may generally not be effective for entering living cells or vascular fenestrations. In certain embodiments, the nanoparticles range in size from about 40 nm, or from about 50 nm, or from about 60 nm to about 100 nm, or to about 90 nm, or to about 80 nm, or to about 70 nm. In certain embodiments, the nanoparticles range in size from about 60 nm to about 70 nm. Some embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 1000 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 500 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 200 nm.

[0238] 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, nanoparticle size refers to the average or median size of the primary particles as measured by transmission electron microscopy (TEM) or similar visualization techniques known in the art. Further examples of mesoporous silica nanoparticles include, but are not limited to, MCM-41, MCM-48, and SBA-15. See KATIYARE, et. al., J. Chromotog. 1122(1-2): 13-20 (2006).

[0239] The method of making porous silica nanoparticles is well known to those skilled in the art. In a particular embodiment, mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate (TEOS) with a template made from micellar rods. The result is a collection of nano-sized spheres or rods filled with regularly arranged pores. The template can then be removed by washing with a solvent adjusted to an appropriate pH (see, for example, TREWYN et al. (2007) Chem. Eng. J. 137(1): 23-29).

[0240] In certain embodiments, mesoporous particles can also be synthesized using a simple sol-gel method (see, for example, NANDIYANTO, et al. (2009) Microporous and Mesoporous Mat. 120(3): 447-453). In certain embodiments, tetraethyl orthosilicate can also be used with an additional polymer monomer as a template. In certain embodiments, 3-mercaptopropyl)trimethoxysilane (MPTMS) is used instead of TEOS.

[0241] In certain embodiments, mesoporous silica nanoparticles are cores are synthesized by a modification of the sol / gel procedure described by MENG et. al. (2015) ACS Nemo, 9(4): 3540-3557.

[0242] Although the method described herein is demonstrated with respect to porous silica nanoparticles (e.g., mesoporous silica), those skilled in the art will recognize that similar methods can be used with other porous nanoparticles.Many other mesoporous materials that can be used in drug delivery nanoparticles are known to those skilled in the art.For example, in certain embodiments, mesoporous carbon nanoparticles can be utilized.

[0243] 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.).

[0244] Similarly, in certain embodiments, mesoporous polymer particles can be utilized. The synthesis of highly ordered mesoporous polymers and carbon frameworks from organic-organic assemblies of triblock copolymers and soluble low molecular weight phenolic resin precursors (resoles) via an evaporation-induced self-assembly strategy was reported by MENG, et. al. (2006) Chem. Mat. 6(18): 4447-4464.

[0245] The nanoparticles described herein are exemplary and non-limiting, and numerous other lipid bilayer coated nanoparticles will be available to those of skill in the art using the teachings provided herein.

[0246] In one embodiment, the present invention teaches a nanocarrier comprising a TLR prodrug.

[0247] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises CHEMS.

[0248] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid.

[0249] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises a non-cleavable stearic acid derivative.

[0250] In one embodiment, the present invention teaches a nanocarrier comprising a TLR prodrug, where the TLR prodrug comprises TR12.

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

[0252] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises CHEMS, and the liposome further comprises TR12.

[0253] In one embodiment, the invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises a TLR inhibitor.

[0254] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TR12.

[0255] In one embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TR12 (denoted as LNP-TR12).

[0256] In a further embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TR12, and the liposome is co-formulated with an A2aR antagonist, wherein the A2aR antagonist comprises an A2aR antagonist designated AR5 (denoted LNP-TR12-AR5).

[0257] In a further embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TR12, and the liposome is co-formulated with a TGFb inhibitor, wherein the TGFb inhibitor comprises a TGFb inhibitor designated TB4 (designated LNP-TR12-TB4).

[0258] In a further embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TR12, and the liposome is co-formulated with a PD-1 antagonist, wherein the PD-1 antagonist comprises a PD-1 antagonist denoted as PD3 (denoted as LNP-TR12-PD3).

[0259] In a further embodiment, the present invention teaches a nanocarrier comprising a liposome, wherein the lipid comprises stearic acid, and the liposome further comprises TR12, and the liposome is co-formulated with an IDO inhibitor, the IDO inhibitor comprising an IDO inhibitor designated ID3 (designated LNP-TR12-ID3).

[0260] In a preferred embodiment, the lipid particle comprises a solid lipid nanoparticle (SLNP) that comprises a liposome containing a TLR prodrug.

[0261] In a preferred embodiment, the lipid particle comprises a solid lipid nanoparticle (SLNP) comprising a liposome containing a TLR prodrug, where the TLR prodrug comprises TR12.

[0262] In a preferred embodiment, the lipid particle comprises a solid lipid nanoparticle (SLNP) comprising a liposome containing a TLR prodrug, where the TLR prodrug comprises TR13.

[0263] In one embodiment, the present invention teaches a nanocarrier comprising a solid lipid nanoparticle ("SLNP"), where the solid lipid nanoparticle comprises stearic acid, and the solid lipid nanoparticle further comprises TR12 (denoted as SLNP-TR12).

[0264] In a further embodiment, the present invention teaches a nanocarrier comprising SLNPs, wherein the lipid comprises stearic acid, and the SLNPs further comprise TR12, and the SLNPs are co-formulated with an A2aR antagonist, wherein the A2aR antagonist comprises an A2aR antagonist designated AR5 (denoted SLNP-TR12-AR5).

[0265] In a further embodiment, the present invention teaches a nanocarrier comprising SLNPs, where the lipid comprises stearic acid, and the SLNPs further comprise TR12, and the SLNPs are co-formulated with an immunogenic cell death ("ICD")-inducing prodrug, the ICD-inducing prodrug comprising an ICD-inducing prodrug denoted as IC1 (denoted as SLNP-TR12-IC1).

[0266] In a further embodiment, the present invention teaches a nanocarrier comprising SLNPs, wherein the lipid comprises stearic acid, and the SLNPs further comprise TR12, and the SLNPs are co-formulated with an immunogenic cell death ("ICD")-inducing prodrug, the ICD-inducing prodrug comprising an ICD-inducing prodrug designated IC1, and the ratio designated as 8:1 (designated as SLNP-TR12-IC1 and / or NTI-121).

[0267] In a further embodiment, the present invention teaches a nanocarrier comprising SLNPs, wherein the lipid comprises stearic acid, and the SLNPs further comprise TR12, and the SLNPs are co-formulated with an immunogenic cell death ("ICD")-inducing prodrug, the ICD-inducing prodrug comprising an ICD-inducing prodrug designated IC1, the ratio being designated 16:1 (designated SLNP-TR12-IC1).

[0268] In a further embodiment, the present invention teaches a nanocarrier comprising SLNPs, where the lipid comprises stearic acid, and the SLNPs further comprise TR12, where the SLNPs are co-formulated with a custom peptide (GSGCERVIGTGWVRC) (SEQ ID NO: 1) conjugated to palmitoyl (denoted SLNP-TR12-NTI-47C).

[0269] By using this peptide (CERVIGTGWVRC) (SEQ ID NO:2) in this type of SLNP, it is contemplated that the function-blocking peptide structurally mimics an epitope on CD47 and binds to SIRPα.

[0270] Furthermore, it is understood that the CD47 molecule is known as a widely expressed cell surface receptor that activates the transmission of "don't-eat-me" signals, which may reduce the desired uptake of nanoparticles by macrophages and have the potential to remain in the blood circulation for a long time.

[0271] In a further embodiment, the present invention teaches a nanocarrier comprising SLNPs, wherein the lipid comprises stearic acid, and the SLNPs further comprise TR12, and the SLNPs are co-formulated with a TGFb inhibitor, wherein the TGFb inhibitor comprises a TGFb inhibitor denoted as TB4 (denoted as SLNP-TR12-TB4).

[0272] In a further embodiment, the present invention teaches a nanocarrier comprising SLNPs, where the lipid comprises stearic acid, and the SLNPs further comprise TR12, and the SLNPs are co-formulated with a PD-1 antagonist, where the PD-1 antagonist comprises a PD-1 antagonist denoted PD3 (denoted SLNP-TR12-PD3).

[0273] In a further embodiment, the present invention teaches a nanocarrier comprising SLNPs, wherein the lipid comprises stearic acid, and the SLNPs further comprise TR12, and the SLNPs are co-formulated with an IDO inhibitor, wherein the IDO inhibitor comprises an IDO inhibitor denoted as ID3 (denoted as SLNP-TR12-ID3).

[0274] In a further preferred embodiment, the solid lipid nanoparticles of the present invention comprise a composition having the following ratio(s): [Table 4]

[0275] In a further preferred embodiment, the solid lipid nanoparticles of the present invention comprise a composition having the following ratio(s): [Table 5] wherein lipid 1 comprises a TR12-prodrug, the lipid moiety comprises stearic acid, the helper lipid is a helper lipid described in Table II, and the stabilizer is polyvinyl alcohol (e.g., Moliwol 488), poloxamer (e.g., Pluronic® F127), Tween® 80, PEG400, and Kolliphor RH 40, where lipid 2 and lipid 3 (lipid prodrug) comprise a lipid prodrug of the present disclosure or a lipid prodrug selected from the group consisting of ID3, PD3, AR5, IC1, NTI-47C, and / or TB4 inhibitors (e.g., ID3-STEA, PD3-STEA, AR5-STEA, TB4-STEA, IC1-STEA, NTI-47C-STEA, etc.), MPLA, and telluratomod, and the CD47 mimetic protein comprises a peptide having the following sequence GSGCERVIGTGWVRC (SEQ ID NO: 1) conjugated to palmitoly and / or stearyl group(s), etc. Functionally, the use of this peptide CERVIGTGWVRC (SEQ ID NO: 2) in this novel SLNP is that the CD47 mimetic protein structurally mimics an epitope on CD47 and binds to SIRP alpha.

[0276] Those skilled in the art will recognize and can make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention disclosed herein, and such changes and modifications are intended to be within the scope of the present disclosure.

[0277] The scope of the present disclosure teaches three non-limiting possible treatment modalities using the formulated prodrugs of the present invention. See PCT Patent Application Publication WO2018 / 213631.

[0278] The first treatment modality involves combining a TLR prodrug in combination with another therapy (e.g., another formulated prodrug that inhibits a TLR (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 allows for systemic (or local) biodistribution and drug delivery to the tumor site. The dual delivery approach achieved synergistic enhancement of adaptive and innate immunity to significantly improve animal survival. In certain embodiments, the nanocarrier comprises a vesicle (i.e., a lipid bilayer that encapsulates fluid).

[0279] The second treatment modality involves the local delivery of drugs that inhibit TLRs in combination with lipids (e.g., liposomes) that contain inhibitors of TLRs (e.g., TLR1 / 2, TLR4, TLR7, TLR8, and / or TLR7 / 8) to tumors or the surrounding area. Such local delivery of TLR inhibitors in combination with TLR prodrugs has been demonstrated to induce cytotoxic tumor killing and tumor shrinkage at the local site. These adaptive immune responses are accompanied by a boost of the innate immune system, reflected by CRT expression, and the activation of DC populations that are particularly well suited to generate cytotoxic T cell responses.

[0280] The third treatment modality involves vaccination with dying cancer cells (e.g., KPC cells), where TLR inhibition is induced ex vivo.It has been discovered that such vaccination can generate a systemic immune response that can prevent tumor growth at distant sites, and allows for adoptive transfer to non-immunized animals.Those skilled in the art can recognize and implement the methods of the treatment modalities provided herein.

[0281] VII.) Liposomes In one aspect, the subject matter of the present disclosure is based on the approach to provide the prodrugs of the present disclosure (see the section entitled "Prodrugs") that are suitable for incorporating into nanocarriers that include lipid coating layers to enhance the delivery of the corresponding prodrugs and provide combination therapy that includes the prodrugs.The advantages of using the prodrugs of the present disclosure include facilitating the controlled formulation into the LNPs (e.g., liposomes) of the present disclosure.This allows the prodrugs to remain in an inactive form during systemic circulation, thereby allowing the liposomes to release the active agent after phagocytosis by cells, for example, in tumors.

[0282] In certain embodiments, one or more TLR prodrugs (e.g., any one or more of the TLR prodrug inhibitors taught in Formula I, and / or TR12 or TR13) (see the section entitled "Prodrugs") are formulated with a lipid moiety capable of forming a vesicular (e.g., liposomal) structure in aqueous solution or forming a component of the lipid bilayer that comprises a liposome.

[0283] In certain embodiments, one or more TLR lipid moieties (e.g., any one or more of the TLR lipid moieties taught in Formula III) (see the section entitled "Lipids") can be formulated and / or co-formulated into vesicular (e.g., liposomal) structures in aqueous solution, or form components of lipid bilayers, including liposomes.

[0284] The liposomes can be used directly or provided as a component in a combined formulation (eg, in combination with another drug moiety, or lipid moiety, or therapeutic modality disclosed herein).

[0285] In certain embodiments, the liposomes formulated with the TLR prodrugs include lipids, PHGP, vitamin E, cholesterol, and / or fatty acids.

[0286] In certain embodiments, the liposomes that are formulated include a lipid moiety that includes TR12.

[0287] In certain embodiments, the liposomes that are formulated include a lipid moiety that includes TR13.

[0288] In certain embodiments, the liposomes that are formulated include a lipid moiety comprising Formula III.

[0289] In certain embodiments, the liposomes formulated include a lipid moiety that includes alpha-galactosylceramide (α-GalCer).

[0290] In one embodiment, the liposome comprises cholesterol.

[0291] In one embodiment, the liposome comprises DPPG.

[0292] In one embodiment, the liposome comprises DMPG.

[0293] In one embodiment, the liposome is Lyso PC.

[0294] In one embodiment, the liposome is (Δ9-Cis)PG.

[0295] In one embodiment, the liposome comprises Soy Lyso PC.

[0296] In one embodiment, the liposome comprises PG.

[0297] In one embodiment, the liposome comprises PA-PEG3-mannose.

[0298] In one embodiment, the liposome comprises C16 PEG2000 ceramide.

[0299] In one embodiment, the liposome comprises MPLA.

[0300] In one embodiment, the liposome comprises 3-Deacly MPLA.

[0301] In one embodiment, the liposomes comprise CHEMS.

[0302] In one embodiment, the liposomes comprise stearic acid.

[0303] In one embodiment, the liposomes comprise a phospholipid as set forth in Table III.

[0304] In one embodiment, the liposome comprises TR12, further comprises CHEMS, and further comprises LU, wherein said LU is a hydromethylcarbamate linker.

[0305] In one embodiment, the liposome comprises TR12, further comprises stearic acid, and further comprises LU, wherein said LU is a hydromethylcarbamate linker.

[0306] In one embodiment, the liposome comprises TR12, further comprises CHEMS, and further comprises LU, wherein said LU is a hydromethylcarbamate linker, and further comprises a helper lipid as set forth in Table II.

[0307] In one embodiment, the liposome comprises TR12, further comprises stearic acid, and further comprises LU, wherein said LU is a hydromethylcarbamate linker, and further comprises a helper lipid as set forth in Table II.

[0308] In one embodiment, the liposome comprises TR12.

[0309] In one embodiment, the liposome comprises TR13.

[0310] In one embodiment, the liposomes of the present disclosure comprise a TLR prodrug co-formulated with one or more additional immunomodulatory agents, including but not limited to immunogenic cell death-inducing chemotherapeutic agents, IDO antagonists, sting agonists, CTLA4 inhibitors, PD-1 inhibitors, and / or prodrugs thereof.

[0311] In one embodiment, the liposomes of the present disclosure comprise a TLR prodrug co-formulated with one or more additional immunomodulatory agents, including but not limited to, neurokinin 1 (NK1) antagonists, and / or prodrugs thereof.

[0312] In one embodiment, the liposomes of the present disclosure comprise a TLR prodrug co-formulated with one or more additional immunomodulatory agents, including but not limited to A2aR antagonists and / or prodrugs thereof.

[0313] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with an ICD-inducing chemotherapeutic agent.

[0314] In a preferred embodiment, the liposome comprises a TLR prodrug co-formulated with an ICD-inducing chemotherapy drug selected from the following list: doxorubicin (DOX), mitoxantrone (MTO), oxaliplatin (OXA), cyclophosphamide (CP), bortezomib, carfilzimib, or paclitaxel.

[0315] In a preferred embodiment, the liposome comprises a TLR prodrug co-formulated with a Toll receptor TLR agonist / prodrug.

[0316] In a preferred embodiment, the liposome comprises a Toll receptor (TLR) prodrug co-formulated with a TLR agonist / prodrug selected from the following list: Resiquimod (R848), Guardiquimod, 852A, DSR6434, Telratolimod, CU-T12-9, Monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD®.

[0317] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with a PD-1 inhibitor / prodrug.

[0318] In a preferred embodiment, the liposome comprises a TLR prodrug co-formulated with a PD-1 inhibitor / prodrug selected from the list of AUNP12, CA-170, or BMS-986189 or prodrugs thereof.

[0319] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with doxorubicin (DOX).

[0320] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with mitoxantrone (MTO).

[0321] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with doxorubicin (DOX) and a PD-1 prodrug.

[0322] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with mitoxantrone (MTO) and a PD-1 prodrug.

[0323] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with doxorubicin (DOX) and an IDO antagonist / prodrug.

[0324] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with mitoxantrone (MTO) and an IDO antagonist / prodrug.

[0325] In a preferred embodiment, the liposomes contain doxorubicin (DOX) and a TLR prodrug co-formulated with a PD-1 prodrug and an IDO antagonist / prodrug.

[0326] In a preferred embodiment, the liposomes contain mitoxantrone (MTO) and a TLR prodrug co-formulated with a PD-1 prodrug and an IDO antagonist / prodrug.

[0327] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with an IDO antagonist / prodrug.

[0328] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with an IDO antagonist / prodrug.

[0329] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with an IDO antagonist / prodrug and a PD-1 prodrug.

[0330] In a preferred embodiment, the liposomes contain a TLR prodrug co-formulated with an IDO antagonist / prodrug and a PD-1 prodrug.

[0331] In a preferred embodiment, the liposomes contain TR12 co-formulated with doxorubicin (DOX).

[0332] In a preferred embodiment, the liposomes contain TR12 co-formulated with mitoxantrone (MTO).

[0333] In a preferred embodiment, the liposomes contain TR12 co-formulated with doxorubicin (DOX) and / or IDO prodrugs and / or IDO antagonists / prodrugs.

[0334] In a preferred embodiment, the liposomes contain TR12 co-formulated with mitoxantrone (MTO) and / or IDO prodrugs and / or IDO antagonists / prodrugs.

[0335] In a preferred embodiment, the liposomes comprise TR12 co-formulated with NK1.

[0336] In a preferred embodiment, the liposomes contain TR12 co-formulated with MTO.

[0337] In a preferred embodiment, the liposomes contain TR12 co-formulated with DOX and an A2aR prodrug.

[0338] In another preferred embodiment, the liposome comprises a solid lipid nanoparticle (SLNP), which comprises a liposome containing a TLR prodrug.

[0339] Those skilled in the art will recognize and understand that solubility is one of the most common problems that those skilled in the art face in the drug development process.The chemical conjugation of drugs / anticancer drugs through lipid molecules (i.e., lipid-based prodrugs) provides a platform to solve the problem of formulating drugs into aqueous suspensions.The main advantage of using lipid conjugation (lipid-based prodrugs) to deliver drug(s) is its ability to improve pharmacokinetics / half-life and targeted delivery.

[0340] Lipid-based prodrug(s) can be integrated / formulated into liposomal formulations using techniques known in the art by appropriate selection of lipid molecules, which provides many advantages over traditional 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 advantage of combining lipid-prodrugs with liposomes is two-fold, since (i) lipid-prodrug-containing liposomes not only increase the solubility of the drug / prodrug itself, but also (ii) have the ability to encapsulate multiple drugs, both hydrophilic and lipophilic (see the section titled Nanocarriers).

[0341] For the purposes of this disclosure, the primary advantages of liposomal formulations are: i) the liposomal formulation is biocompatible / biodegradable and has no general toxicity; ii) flexibility and manipulation of size and surface charge depending on the desired objective. The liposome formulation(s), for purposes of this disclosure, can have a size range of 40-150 nm in diameter and a surface charge ranging from -40 to +40 mV; And iii) The liposomes of the present invention have either single or multiple lipid-prodrugs as the constituent lipid moieties of the liposome(s). In addition, multiple drugs (e.g., acting by different mechanisms of action) with different solubility profiles (hydrophilicity or lipophilicity) can be formulated in these liposomes (either in the lipid bilayer or in the hydrophilic core).

[0342] As will be appreciated by those skilled in the art, all methods of making liposomes include four basic steps: (i) drying the lipid from the organic solvent; (ii) dispersing lipids in an aqueous solution; (iii) purifying the resulting liposomes; and (iv) Analysing the final product. See AKBARZADEH, et. al., Nanoscale Research Letters, 8:102 (2013).

[0343] Another aspect of the present invention discloses liposome encapsulation technology (LET), which is a delivery technology used to deliver drugs. LET is a method of making submicroscopic foams 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, and intestinal flora, as well as 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 the correct target gland, organ, or system, where the contents are utilized (see the section titled Nanocarriers).

[0344] In one embodiment, the liposome(s) of the present disclosure are synthesized using multiple different ratios of TLR prodrug, TLR lipid moiety, lipid, and / or lipid-prodrug. As disclosed herein, the TLR prodrug can include a helper lipid disclosed herein (see, e.g., Table II).

[0345] In one embodiment, the liposome(s) of the present disclosure are synthesized using multiple different ratios of TLR prodrug, TLR lipid moiety, lipid, and / or lipid-prodrug. As disclosed herein, the TLR prodrug may further comprise DSPE-PEG.

[0346] In a preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 6]

[0347] In a further preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 7]

[0348] In a further preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 8] Here, lipid 1 includes TR12 and CHEMS.

[0349] In a further preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 9] Here, lipid 1 comprises TR12 and stearic acid.

[0350] In a further preferred embodiment, the liposomes of the present invention comprise a composition having the following ratios: [Table 10] Here, lipid 1 comprises TR12 and a non-cleavable stearic acid derivative.

[0351] Those skilled in the art will recognize and can make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention disclosed herein, and such changes and modifications are intended to be within the scope of the present disclosure.

[0352] VIII.) Pharmaceutical Formulation As used herein, the term "drug" is synonymous with "medicinal drug." In certain embodiments, the liposomes of the present disclosure are processed into an encapsulated dosage form and given to a patient for the treatment of a disease.

[0353] Generally speaking, pharmaceutical formulation is the process of combining various chemicals into pure drug substances to produce the final drug product. Formulation studies involve developing preparations of drugs that are stable and acceptable to patients. For drugs taken orally, this usually involves incorporating the drug into a tablet or capsule. It is important to appreciate that dosage forms contain a variety of other substances apart from the drug itself, and studies must be conducted to ensure that the drug is compatible with these other substances.

[0354] Excipients are inert substances used as carriers for the active ingredient of the drug product, in this case liposomes containing a TLR prodrug. In addition, excipients can be used to aid in the process by which the drug product is manufactured. The active ingredient is then dissolved or mixed with the excipient. Excipients are also sometimes used to bulk up formulations containing very potent active ingredients, allowing for convenient and accurate dosing. Once an active ingredient is purified, it cannot remain in purified form for long periods of time. Often, the active ingredient denatures, separates from the solution, or sticks to the sides of the container.

[0355] Excipients are added to stabilize the active ingredient to ensure that the active ingredient remains active and stable for a long enough period of time so that the shelf life of the product makes it competitive with other products and safe for the end user. Examples of excipients include, but are not limited to, anti-adherents, binders, coatings, disintegrants, fillers, diluents, flavors, colorants, lubricants, and preservatives. The final formulation includes the active ingredient and excipients, which are then encapsulated into a pharmaceutical dosage form.

[0356] Pre-formulation involves characterizing the physical, chemical, and mechanical properties of the drug to select which other ingredients should be used in the preparation. Formulation studies then consider factors such as stability, particle size, polymorphism, pH, and solubility, as these can all affect bioavailability and therefore the activity of the drug. The drug must be combined with the inert additives in a manner that ensures that the amount of drug present is consistent in each dosage unit (e.g., each vial). The dosage should have a uniform appearance.

[0357] These studies are unlikely to be completed by the time clinical trials begin. This means that simple preparations are developed early on for use in Phase I clinical trials. These typically consist of vials, hand-filled capsules, containing small amounts of drug and diluents. Evidence of the long-term stability of these preparations is not necessary, as they are used (tested) in just a few days. However, the time from when the final preparation is packaged to when it reaches the patient can be months or years, so long-term stability is very important in managing the supply chain. What is called drug loading (i.e., the ratio of active drug to the total content of the dose) must be considered. Low drug loading can cause homogeneity problems. High drug loading can result in flow problems or require large capsules if the bulk density of the compound is low. By the time Phase III clinical trials are reached, the drug's formulation should have been developed to be close to the preparation that will ultimately be used in the market.

[0358] Knowledge of stability is essential up to this stage, and conditions must be developed to ensure that the drug is stable in the formulation. If the drug proves unstable, the results of the clinical trial will be invalid, since it would be impossible to know what the administered dose actually was. Stability studies are performed to test if temperature, humidity, oxidation, or photolysis (ultraviolet or visible light) have any effect, and the formulation is analyzed to see if any degradation products have been formed. It is also important to check if there are any undesirable interactions between the formulation and the container. If plastic containers are used, tests are done to see if any of the ingredients have adsorbed onto the plastic, and if any plasticizers, lubricants, pigments, or stabilizers have leached from the plastic into the formulation. The adhesive for the container label also needs to be tested to ensure that it does not leach from the plastic container into the formulation. The way drugs are 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 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 slower dissolution rate can extend the duration of action or avoid initial high plasma levels.

[0359] In some embodiments, nanocarriers (e.g., SLNPs or liposomes containing TLR prodrugs) and / or liposomes co-formulated with immunomodulatory agents are administered alone or in admixture with a physiologically acceptable carrier (e.g., saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. For example, nanocarriers, when used as injections, can be formulated as a sterile suspension, dispersion, or emulsion using a pharma- ceutically acceptable carrier. In certain embodiments, normal saline can be used as a pharma- ceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, 5% glucose, and the like, including glycoproteins, such as albumin, lipoproteins, globulins, and the like, to enhance stability. In compositions that include a carrier containing saline or other salts, the carrier is preferably added after nanocarrier formation. Thus, after the nanocarriers are formed and loaded with the appropriate drug(s), the nanocarriers can be diluted with a pharma- ceutically acceptable carrier, such as normal saline. Similarly, the TLR prodrug liposomes can be incorporated into a carrier that facilitates suspension (eg, emulsification, dilution, etc.) of the nanomaterial.

[0360] The pharmaceutical composition can be sterilized by conventional well-known sterilization techniques. The resulting aqueous solutions, suspensions, dispersions, emulsions, etc. can be packaged for use or filtered under aseptic conditions. In certain embodiments, the drug delivery nanocarrier (e.g., nanoparticles coated with LNP or SLNP) is lyophilized, and the lyophilized preparation is mixed with a sterile aqueous solution before administration. The composition can also contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, osmotic pressure adjusting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0361] In addition, in certain embodiments, the pharmaceutical formulation may include lipid protective agents that protect lipids against free radical and lipid peroxidation damage in storage. Lipophilic free radical quenchers, such as alpha-tocopherol, and water-soluble iron-specific chelators, such as ferrioxamine, are suitable and contemplated herein. The concentration of nanocarriers (e.g., SLNPs or liposomes containing TLR prodrugs) in the pharmaceutical formulation may vary widely, for example, less than about 0.05%, usually at least about 2-5% to as much as 10-50%, or up to 40%, or up to 30% by weight, and is selected primarily by fluid volume, viscosity, etc., according to the particular administration method selected. 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 will depend on the particular drug used, the condition being treated, and the judgment of the clinician, but will generally be between about 0.01 mg and about 50 mg per kilogram of body weight, preferably between about 0.1 mg and about 5 mg per kilogram of body weight.

[0362] One of skill in the art will recognize that the exact dosage will vary depending on the particular TLR prodrug and any co-formulated immunomodulatory agent, as well as the desired medical effect, and patient factors such as age, sex, overall condition, etc. Taking these factors into consideration, one of skill in the art can readily establish effective therapeutic concentrations without undue experimentation.

[0363] For administration to humans (or non-human mammals) in the curative, ameliorative, retardative, or preventative treatment of the diseases described herein, the prescribing physician will ultimately determine the dosage of the drug appropriate for a given human (or non-human) subject, which can be expected to vary according to the age, weight, and response of the individual, as well as the nature and severity of the patient's disease. In certain embodiments, the dosage of the drug provided by the nanocarrier may be approximately the same as that used for the free drug. However, as noted above, the nanocarriers described herein can significantly reduce the toxicity and significantly increase the therapeutic window of the drug(s) administered therewith. Thus, in some cases, dosages that exceed those prescribed for the free drug(s) are utilized.

[0364] Those skilled in the art will recognize and can make changes and modifications to the disclosed embodiments without altering the function and purpose of the invention disclosed herein, and such changes and modifications are intended to be within the scope of the present disclosure.

[0365] IX.) Combination Therapy As will be recognized and understood by those skilled in the art, the growth and survival of cancer cells may be influenced by multiple signaling pathways.Therefore, it is useful to combine different enzyme / protein / receptor inhibitors that show different priorities for targets and modulate the activity of targets to treat such conditions.Targeting one or more signaling pathways (or one or more biological molecules involved in a given signaling pathway) can reduce the likelihood of drug resistance occurring in cell populations and / or reduce the toxicity of treatment.

[0366] Therefore, the liposome or SLNP containing the TLR prodrug of the present disclosure can be used in combination with one or more other enzyme / protein / receptor inhibitors or one or more therapies for the treatment of disease, such as cancer or infectious disease. Examples of diseases and indications treatable with combination therapy include those described in this disclosure. Examples of cancer include, but are not limited to, solid tumors and liquid tumors, such as blood cancers. Examples of infectious diseases include viral infections, bacterial infections, fungal infections, or parasitic infections.

[0367] For example, liposomes or SLNPs containing the TLR prodrugs of the present disclosure can be combined with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, PI3K (alpha, beta, gamma, delta), CSFIR, KIT, FLK-II, KDR / FLK-1, FLK-4, flt- 1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, TAM kinase (Axl, Mer, Tyro3), FLT3, VEGFR / Flt2 , Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf.

[0368] In further embodiments, the liposomes or SLNPs containing the TLR prodrugs of the present disclosure can 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 the 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, These 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), such as HDAC8 inhibitors, angiogenesis inhibitors, interleukin receptor inhibitors, bromo and extra-terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors, such as INCB54329 and INCB57643), poly ADP ribose polymerase (PARP) inhibitors, such as rucaparib, olaparib, niraparib, veliparib, or talazoparib, arginase inhibitors (INCB01158), PD-1 inhibitors, PD-1 / L-1 inhibitors, PD-1 / L-2 inhibitors, and adenosine receptor antagonists, or combinations thereof.

[0369] In further embodiments, liposomes or SLNPs containing 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-galactosylceramida (α-GalCer) and its analogs, including C8 galactosyl(α)ceramide, C16 galactosyl(α)ceramide, and C24:1 galactosyl(α)ceramide (Avanti Polar Lipids, Alabaster, Alabama).

[0370] In addition, the liposomes or SLNPs containing the TLR prodrugs of the present disclosure can be further used in combination with other methods of treating cancer, for example, by chemotherapy, radiation therapy, tumor targeted therapy, adjuvant therapy, immunotherapy, or surgery.

[0371] Examples of immunotherapies include cytokine treatments (e.g., interferon, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccines, monoclonal antibodies, adoptive T cell transfer, Toll receptor agonists, STING agonists, oncolytic virotherapy, and immune modulating small molecules including thalidomide or JAK1 / 2 inhibitors.

[0372] Liposomes or SLNPs containing TLR prodrugs can be used in combination with one or more anti-cancer drugs, such as chemotherapeutic drugs, such as abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezombi, bortezomib, intravenous busulfan, oral busulfan, calsterone, capecitabine, carboplatin, Carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, Stramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alpha 2a, irinostat Tecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, 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, malefenib, The present invention may be exemplified by any of the following: sunitinib inocut, tamoxifen, temozolomide, teniposide, testolactone, thalidomide (thalTLRmide), thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, niraparib, veliparib, talazoparib, and zoledronate.

[0373] Other anti-cancer agent(s) include antibody therapeutics such as trastuzumab (Herceptin), antibodies against costimulatory molecules such as CTLA-4 (e.g., ipilimumab), antibodies against 4-1BB (e.g., urelumab, utomirumab), antibodies against PD-1 and PD-L1 / L2, or antibodies against cytokines (IL-10, TGF-beta, etc.).

[0374] Examples of antibodies against PD-1 and / or PD-L1 / L2 that can be combined with compounds of the 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.

[0375] In addition, the liposomes or SLNPs containing the TLR prodrugs of the present disclosure can be used in combination with one or more immune checkpoint inhibitors for the treatment of diseases, such as cancer or infectious diseases. Exemplary immune checkpoint inhibitors include inhibitors 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.

[0376] In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137. In further embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, TLR, KIR, LAG3, PD-1, TIM3, and VISTA. In further embodiments, the liposomes comprising the TLR prodrugs provided herein can be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGF beta ("TGFb") inhibitors.

[0377] X.) Methods for delivering liposomes containing TLR prodrugs to Toll-like receptor ("TLR") expressing cells As is known in the art, a wide variety of compositions and methods are known in the art for using prodrugs and / or nanocarriers to kill tumor cells. In the context of cancer, a typical method involves administering to a tumor-bearing mammal a biologically effective amount of a TLR prodrug of the present disclosure, and / or a nanocarrier of the present disclosure that includes a TLR prodrug.

[0378] An exemplary embodiment is a method of delivering a therapeutic agent to a cell expressing TLR1 / 2, TLR4, TLR7, TLR8, and / or TLR7 / 8, comprising conjugating a drug moiety of the present disclosure to a lipid of the present disclosure via a linking unit to form a TLR prodrug, and exposing the cell to the TLR prodrug.

[0379] In one embodiment, the TLR prodrug comprises a drug moiety of formula I and a CHEMS conjugated via a LU that comprises a hydromethylcarbamate linker.

[0380] In one embodiment, the TLR prodrug comprises a drug moiety of formula I and stearic acid conjugated via a LU that contains a hydromethylcarbamate linker.

[0381] In one embodiment, the TLR prodrug comprises a drug moiety of Formula I and a non-cleavable stearic acid derivative.

[0382] In one embodiment, the TLR prodrug comprises TR12 and CHEMS conjugated via a LU that contains a hydromethylcarbamate linker.

[0383] In one embodiment, the TLR prodrug comprises TR12 and stearic acid conjugated via a LU that contains a hydromethylcarbamate linker.

[0384] Another exemplary embodiment is a method of treating an individual suspected of suffering from metastatic cancer, comprising parenterally administering to said individual a pharmaceutical composition comprising a therapeutically effective amount of a TLR prodrug produced by conjugating a drug moiety to a lipid of the present disclosure via a linking unit, thereby exposing cells to the TLR prodrug.

[0385] In one embodiment, the TLR prodrug comprises a drug moiety of formula I and a CHEMS conjugated via a LU that comprises a hydromethylcarbamate linker.

[0386] In one embodiment, the TLR prodrug comprises a drug moiety of formula I and stearic acid conjugated via a LU that contains a hydromethylcarbamate linker.

[0387] In one embodiment, the TLR prodrug comprises a drug moiety of Formula I and a non-cleavable stearic acid derivative.

[0388] In one embodiment, the TLR prodrug comprises TR12 and CHEMS conjugated via a LU that contains a hydromethylcarbamate linker.

[0389] In one embodiment, the TLR prodrug comprises TR12 and stearic acid conjugated via a LU that contains a hydromethylcarbamate linker.

[0390] The TLR prodrugs, nanocarriers, liposomes, co-formulated nanocarriers and co-formulated liposomes of the present disclosure inhibit TLR protein activity / protein interactions and are therefore useful for treating diseases and disorders associated with TLR activity and the aforementioned diseases and disorders.

[0391] In further embodiments of the present disclosure, the TLR prodrugs, nanocarriers, or pharma- ceutically acceptable salts or stereoisomers thereof are useful for therapeutic administration to enhance, stimulate, and / or augment immunity in cancer, chronic infection, or sepsis, including enhancing responses to vaccination.

[0392] In further embodiments, the present disclosure provides a method for inhibiting T cell function of TLR (e.g., TLR1 / 2, TLR4, TLR7, TLR8, and / or TLR7 / 8). The method includes administering to an individual or patient a TLR prodrug, liposome, nanocarrier, and / or any of the formulas described herein (e.g., TR12 and / or TR13), or any of the TLR prodrugs, nanocarriers, and nanoencapsulated TLR inhibitor prodrugs listed in any of the claims and described herein, or a pharma- ceutically acceptable salt or stereoisomer thereof. The TLR prodrugs, liposomes, nanocarriers, and nanoencapsulated TLR inhibitor prodrugs of the present disclosure can be used alone, in combination with other drugs or therapies, or as adjuvants or neoadjuvants to treat diseases or disorders, including cancer and other diseases. Any of the TLR prodrugs, liposomes, nanocarriers, and nanoencapsulated TLR prodrugs of the present disclosure, including any of the embodiments thereof, can be used for the uses and methods described herein.

[0393] In addition, the TLR prodrugs, liposomes, nanocarriers, and nanoencapsulated TLR inhibitor prodrugs of the present disclosure inhibit TLR function, thereby blocking the TLR pathway.

[0394] In further embodiments, the present disclosure provides for in vivo treatment of individuals or patients using TLR prodrugs, liposomes, nanocarriers, and nanoencapsulated TLR inhibitor prodrugs or salts or stereoisomers thereof such that the growth of cancerous tumors is inhibited.

[0395] The TLR prodrugs, liposomal, and nanoencapsulated TLR inhibitor prodrugs, or any of the formulas described herein (e.g., TR12 and / or TR13), or any of the claims and described herein, or salts or stereoisomers thereof, can be used to inhibit the growth of cancerous tumors.

[0396] Alternatively, the TLR prodrugs, liposomes, nanocarriers, and nanoencapsulated TLR prodrugs of the present disclosure, or any of the formulas described herein, or any of the claims listed and described herein (e.g., TR12 and / or TR13), or salts or stereoisomers thereof, can be used in conjunction with other agents or standard cancer treatments, as described in this disclosure.

[0397] In a further embodiment, the present disclosure provides a method for inhibiting tumor cell growth in vitro, comprising contacting tumor cells in vitro with a TLR prodrug, liposome, nanocarrier, and nanoencapsulated TLR inhibitor prodrug of the present disclosure, or any of the formulas described herein (e.g., TR12 and / or TR13), or any of the claims and described herein, or a salt or stereoisomer thereof.

[0398] In a further embodiment, the present disclosure provides a method for inhibiting the growth of tumor cells in a patient, comprising contacting the tumor cells with a TLR prodrug, liposome, nanocarrier, and nanoencapsulated TLR inhibitor prodrug of the present disclosure, or any of the formulas described herein (e.g., TR12 and / or TR13), or any of the TLR prodrugs, liposome, and nanoencapsulated TLR inhibitor prodrugs listed in any of the claims and described herein, or a salt or stereoisomer thereof.

[0399] XI.) Methods of Treating Cancer and Other Immunological Disorders Another embodiment of the present disclosure is a method for treating cancer.The method comprises administering to a patient a therapeutically effective amount of a liposome containing the TLR prodrug herein (i.e., TR12 and / or TR13), a compound listed in any of the claims and described herein, or a salt thereof.Examples of cancer include cancers whose growth can be inhibited using the TLR inhibitors of the present disclosure and the TLR prodrugs of the present disclosure, and cancers that typically respond to immunotherapy.

[0400] In some embodiments, the disclosure provides a method of enhancing, stimulating, and / or increasing an immune response in a patient, comprising administering to the patient a therapeutically effective amount of a TLR prodrug and / or a nanocarrier comprising the TLR prodrug (i.e., TR12 and / or TR13), a compound recited in any of the claims and described herein, or a salt thereof.

[0401] Non-limiting examples of cancers treatable using nanocarriers comprising TLR prodrugs, TLR prodrugs and co-formulated nanocarriers of the present disclosure include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma of soft tissue, urethral cancer, The cancers that can be treated include, but are not limited to, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and other chronic or acute leukemias, childhood solid tumors, lymphocytic lymphomas, bladder cancer, kidney or urethra cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinomas, squamous cell carcinomas, T-cell lymphomas, and environmentally induced cancers, including asbestos-induced cancers, and combinations of the above cancers.The compounds of the present disclosure are also useful for treating metastatic cancers, particularly metastatic cancers that express TLR.

[0402] In some embodiments, cancers treatable with the nanocarriers or TLR prodrugs of the present disclosure include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), head and neck squamous cell carcinoma, urothelial carcinoma (e.g., bladder), and microsatellite instability high (MSI) cancer. high In addition, the present disclosure includes refractory or recurrent malignancies whose growth can be inhibited using the liposomes or TLR prodrugs or co-formulated liposomes of the present disclosure.

[0403] In additional embodiments, cancers treatable using the formulated and / or co-formulated nanocarriers or TLR prodrugs of the present disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, renal cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., lymphoma, leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma or multiple myeloma), and combinations of the foregoing cancers.

[0404] In further embodiments, cancers treatable using formulated and / or co-formulated nanocarriers or TLR prodrugs of the present disclosure include, but are not limited to, cholangiocarcinoma, bile duct carcinoma, triple negative breast cancer, rhabdomyosarcoma, small cell lung cancer, leiomyosarcoma, hepatocellular carcinoma, Ewing's sarcoma, brain cancer, brain tumor, astrocytoma, neuroblastoma, neurofibroma, basal cell carcinoma, chondrosarcoma, epithelioid sarcoma, eye cancer, fallopian tube cancer, gastrointestinal cancer, gastrointestinal stromal tumor, hairy cell leukemia, intestinal cancer, islet cell carcinoma, oral cavity cancer, cancer of the mouth, 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 cancer, tongue cancer, tubular cancer, urethral cancer, and ureteral cancer.

[0405] Additionally, in some embodiments, formulated and / or co-formulated nanocarriers of the present disclosure, or TLR prodrugs, can be used to treat sickle cell disease and sickle cell anemia.

[0406] Additionally, in some embodiments, diseases and indications treatable using the formulated and / or co-formulated nanocarriers or TLR prodrugs of the present disclosure include, but are not limited to, hematological cancers, sarcomas, lung cancer, gastrointestinal cancers, genitourinary cancers, liver cancer, bone cancer, nervous system cancers, gynecological cancers, and skin cancers.

[0407] Exemplary hematological cancers include lymphomas and leukemias, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma, myeloproliferative disorders (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocytosis (ET)), myelodysplastic syndromes (MDS), T-cell acute lymphoblastic lymphoma (T-ALL), and multiple myeloma (MM).

[0408] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, hamartoma, and teratoma.

[0409] Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell, small undifferentiated cell, large undifferentiated cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatous hamartoma, and mesothelioma.

[0410] Exemplary gastrointestinal cancers include esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastric cancer (carcinoma, lymphoma, leiomyosarcoma), pancreatic cancer (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), colon cancer (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), and colorectal cancer.

[0411] Exemplary genitourinary cancers include kidney cancer (adenocarcinoma, Wilms' tumor [nephroblastoma]), bladder and urethra cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), and testicular cancer (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma).

[0412] Exemplary liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0413] Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor.

[0414] Exemplary nervous system cancers include cranial cancer (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), meningeal cancer (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, meduoblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma, glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), and spinal cancer (neurofibroma, meningioma, glioma, sarcoma), as well as neuroblastoma and Lhermitte-Duclos disease.

[0415] Exemplary gynecological cancers include uterine cancer (endometrial cancer), cervical cancer (cervical carcinoma, pre-tumor cervical dysplasia), ovarian cancer (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulvar cancer (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), and fallopian tube cancer (carcinoma).

[0416] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevus, lipoma, angioma, dermatofibroma, and keloid. In some embodiments, diseases and indications treatable using the compounds of the present disclosure include, but are not limited to, sickle cell disease (e.g., sickle cell anemia), triple-negative breast cancer (TNBC), myelodysplastic syndrome, testicular cancer, bile duct cancer, esophageal cancer, and urothelial carcinoma.

[0417] In addition, formulated and / or co-formulated nanocarriers of the present disclosure, or blockade of the TLR and / or kynurenine pathways using TLR prodrugs, can also be used to treat infectious diseases, such as viral, bacterial, fungal, and parasitic infections.

[0418] The present disclosure provides a method for treating an infectious disease, such as a viral infection, comprising administering to a patient a therapeutically effective amount of any of the formulated and / or co-formulated nanocarriers recited in any of the claims and described herein, or any of the TLR prodrugs or formulas described herein (i.e., TR12 and / or TR13), salts thereof.

[0419] Examples of viruses causing infectious diseases treatable by the methods of the present disclosure include, but are not limited to, human immunodeficiency virus, human papilloma virus, influenza, hepatitis A, B, C or D virus, adenovirus, poxvirus, herpes simplex virus, human cytomegalovirus, severe acute respiratory syndrome virus, Ebola virus, and measles virus. In some embodiments, viruses causing infections treatable by the methods of the present disclosure include, but are not limited to, hepatitis (A, B or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0420] Additionally, the present disclosure provides a method for treating a bacterial infection, comprising administering to a patient a therapeutically effective amount of any of the formulated and / or co-formulated nanocarriers recited in any of the claims and described herein, or any of the TLR prodrugs or formulas described herein (i.e., TR12 and / or TR13), or salts thereof.

[0421] Examples of pathogens causing infections treatable by the methods of the present disclosure include, but are not limited to, chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, Salmonella, Bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria.

[0422] Additionally, the present disclosure provides a method for treating a fungal infection, comprising administering to a patient a therapeutically effective amount of any of the formulated and / or co-formulated nanocarriers recited in any of the claims and described herein, or a TLR prodrug or formula described herein (i.e., TR12 and / or TR13), or a salt thereof.

[0423] Examples of pathogenic fungi causing infections treatable by the methods of the present disclosure include, but are not limited to, Candida (such as albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (such as fumigatus and Niger), Mucorales (Mucor, absidia, rhizophus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0424] Additionally, the present disclosure provides a method for treating parasitic infections, comprising administering to a patient a therapeutically effective amount of any of the formulated and / or co-formulated nanocarriers recited in any of the claims and described herein, or TLR prodrugs or formulas described herein (i.e., TR12 and / or TR13), or salts thereof.

[0425] Examples of pathogenic parasites causing infections treatable by the methods of the present disclosure include, but are not limited to, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0426] In a further set of embodiments within the scope of the present disclosure, formulated and / or co-formulated nanocarriers, or TLR prodrugs, or any of the formulas described herein (i.e., TR12 and / or TR13), are useful for preventing or reducing the risk of developing any of the diseases mentioned in this disclosure, for example, in individuals who may be predisposed to the disease, condition or disorder but have not yet experienced or exhibited the pathology or symptomology of the disease.

[0427] In one embodiment, the methods described herein include LNP-TR12 and / or a therapeutically effective amount of LNP-TR12.

[0428] In one embodiment, the methods described herein include SLNP-TR12 and / or a therapeutically effective amount of SLNP-TR12.

[0429] In one embodiment, the methods described herein include LNP-TR13 and / or a therapeutically effective amount of LNP-TR13.

[0430] In one embodiment, the methods described herein include SLNP-TR13 and / or a therapeutically effective amount of SLNP-TR13.

[0431] In one embodiment, the methods described herein include SLNP-TR12-IC1 and / or a therapeutically effective amount of SLNP-TR12-IC1.

[0432] In one embodiment, the methods described herein include SLNP-TR12-IC1, where the ratio is shown to be 8:1.

[0433] In one embodiment, the methods described herein include SLNP-TR12-IC1, where the ratio is shown to be 16:1.

[0434] In one embodiment, the methods described herein include SLNP-TR12-AR5 and / or a therapeutically effective amount of SLNP-TR12-AR5.

[0435] In one embodiment, the methods described herein include SLNP-TR12-NTI-47C and / or a therapeutically effective amount of SLNP-TR12-NTI-47C.

[0436] XII.) Kits / Manufactured Products Kits for use in the laboratory, prognostic, preventive, diagnostic, and therapeutic applications described herein are within the scope of the present invention. Such kits can include a carrier, package, or container that is shaped to receive one or more containers, such as vials, tubes, etc., each of which includes one of the separate elements used in the method, along with a label or insert that includes instructions for use, such as those described herein. For example, the container(s) can include formulated and / or co-formulated nanocarriers that are detectably labeled or can be detectably labeled, and / or are loaded with the TLR prodrugs of the present disclosure. The kits can include a container that includes a drug unit. The kits can include all or a portion of formulated and / or co-formulated nanocarriers, liposomes, SLNPs, and / or TLR prodrugs.

[0437] Kits of the present invention typically include the container described above and one or more other containers associated therewith that contain materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, shipping containers, packages, containers, vials, and / or tube labels listing the contents and / or instructions for use, and package inserts containing the instructions for use.

[0438] A label can be present on or with the container to indicate that the composition is used for a particular therapeutic or non-therapeutic application, such as prognostic, preventive, diagnostic, or laboratory application, and can indicate a method of use, either in vivo or in vitro, such as those described herein. Directions and other information can also be included on an insert(s) or label(s) included with or on the kit. The label can be on or associated with the container. The label can be on the container if letters, numbers, or other symbols forming the label are molded or etched into the container itself. The label can be associated with the container, for example, as a package insert, if present in a receptacle or carrier that also holds the container. The label can indicate that the composition is used for the diagnosis, treatment, prevention, or prognosis of a condition, such as cancer or other immunological disorder.

[0439] The terms "kit" and "article of manufacture" may be used synonymously.

[0440] In another embodiment of the present invention, an article of manufacture(s) containing a composition, such as formulated and / or co-formulated nanocarriers, and / or TLR prodrugs, is within the scope of this disclosure. The article of manufacture typically includes at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed from a variety of materials, such as glass, metal, or plastic. The container can hold formulated and / or co-formulated nanocarriers loaded with TLR prodrugs.

[0441] The container may alternatively hold a composition effective for treating, diagnosing, prognosing or preventing a condition and may have a sterile access port (for example, the container may be an intravenous infusion bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition may be a TLR prodrug and / or formulated and / or co-formulated nanocarrier loaded with a TLR prodrug as disclosed herein.

[0442] The article of manufacture may further comprise a second container comprising a pharma- ceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The article of manufacture may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and / or package inserts containing instructions for indications and / or use.

[0443] In one embodiment, the kits described herein comprise LNP-TR12 and / or a therapeutically effective amount of LNP-TR12.

[0444] In one embodiment, the kits described herein comprise SLNP-TR12 and / or a therapeutically effective amount of SLNP-TR12.

[0445] In one embodiment, the kits described herein comprise LNP-TR13 and / or a therapeutically effective amount of LNP-TR13.

[0446] In one embodiment, the kit described herein comprises SLNP-TR13 and / or a therapeutically effective amount of SLNP-TR13.

[0447] In one embodiment, the kit described herein comprises SLNP-TR12-IC1 and / or a therapeutically effective amount of SLNP-TR12-IC1.

[0448] In one embodiment, the methods described herein include SLNP-TR12-IC1, where the ratio is shown to be 8:1.

[0449] In one embodiment, the methods described herein include SLNP-TR12-IC1, where the ratio is shown to be 16:1.

[0450] In one embodiment, the kit described herein comprises SLNP-TR12-AR5 and / or a therapeutically effective amount of SLNP-TR12-AR5.

[0451] In one embodiment, the kit described herein comprises SLNP-TR12-NTI-47C and / or a therapeutically effective amount of SLNP-TR12-NTI-47C.

[0452] Exemplary embodiments 1)(i) a drug moiety, (ii) a lipid portion, and (iii) Consolidated Unit (“LU”) wherein the drug moiety comprises a TLR agonist and the LU conjugates the drug moiety to a lipid moiety. 2) The TLR prodrug of claim 1, further comprising the chemical structure set forth in formula I. 3) The TLR prodrug of claim 1, wherein the drug moiety comprises the chemical structure set forth as TR12. 4) The TLR prodrug of claim 1, wherein the drug moiety comprises the chemical structure set forth as TR13. 5) The TLR prodrug of claim 1, wherein LU is a hydromethylcarbamate linker. 6) The TLR prodrug of claim 1, wherein the lipid moiety comprises a lipid listed in Table I. 7) The TLR prodrug of claim 1, wherein the lipid moiety comprises a lipid set forth in Table III. 8) The TLR prodrug of claim 1, wherein the lipid moiety comprises stearic acid. 9) The lipid portion comprises stearic acid and has the following chemical structure: [ka] The TLR prodrug of claim 1, having the formula: 10) The TLR prodrug of claim 4, wherein the lipid moiety comprises a non-cleavable stearic acid derivative. 11) The lipid portion comprises a non-cleavable stearic acid derivative having the following chemical structure: [ka] The TLR prodrug of claim 10, having the formula: 12) (i) a drug moiety that includes TR12; (ii) a lipid portion comprising stearic acid, and (iii) LUs containing hydromethylcarbamate linkers 23. A TLR prodrug composition comprising: 13) The following chemical structure: [ka] 13. The TLR prodrug composition of claim 12, comprising: 14) (iv) a drug moiety comprising TR12; (v) a lipid moiety containing a non-cleavable stearic acid derivative 23. A TLR prodrug composition comprising: 15) The following chemical structure: [ka] 15. The TLR prodrug composition of claim 14, comprising: 16) Liposomes containing a TLR prodrug and releasing an active TLR inhibitor after cleavage of the LU. 17) A nanocarrier that contains a TLR prodrug and releases an active TLR agonist after LU cleavage. 18) The nanocarrier of claim 17, wherein LU is a hydromethylcarbamate linker. 19) The nanocarrier of claim 17, further comprising a helper lipid, wherein the helper lipid is listed in Table II. 20) The nanocarrier of claim 17, wherein the TLR prodrug comprises TR12. 21) The nanocarrier described in claim 17, wherein the nanocarrier is a liposome. 22) The liposome of claim 21, wherein the TLR prodrug comprises TR12 and is designated LNP-TR12. 23) The liposome of claim 21, wherein the liposome is further co-formulated with one or more immunomodulatory agents or lipid-prodrugs thereof, wherein the immunomodulatory agent is selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, A2aR inhibitors, STING agonists, CTLA-4 inhibitors, IDO inhibitors, PD-1 / PD-L1 inhibitors, CD1D agonists, and / or prodrugs thereof. 24) The liposome of claim 21, wherein the liposome is further co-formulated with an ICD-inducing chemotherapy drug, and the ICD-inducing chemotherapy drug is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzimib, IC1, or paclitaxel. 25) The liposome of claim 21, further comprising DOX. 26) The liposome of claim 21, further comprising MTO. 27) The liposome of claim 24, further comprising DOX. 28) The liposome of claim 24, further comprising MTO. 29) The liposome of claim 21, wherein the liposome is further co-formulated with a toll receptor agonist or a lipid-prodrug thereof, wherein the toll receptor agonist is selected from the group consisting of resiquimod (R848), gardquimod, 852A, DSR 6434, telluritolimod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, TR5, TR6, TR3, or 3D-PHAD®. 30) The liposome of claim 21, wherein the liposome is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid-prodrug thereof, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of AUNP12, CA-170, PD3, or BMS-986189. 31) The liposome of claim 21, wherein the liposome is further co-formulated with a TGFb antagonist, the TGFb antagonist being selected from the group consisting of TB4. 32) The liposome of claim 21, wherein the liposome is further co-formulated with an IDO inhibitor, the IDO inhibitor being selected from the group consisting of ID3. 33) A kit comprising the nanocarrier described in any one of claims 1 to 21. 34) A kit comprising the liposome according to any one of claims 22 to 32. 35) The nanocarrier of claim 17, which is a solid lipid nanoparticle (SLNP). 36) The nanocarrier of claim 20, which is a solid lipid nanoparticle (SLNP). 37) The SLNP of claim 36, wherein the TLR prodrug comprises TR12 and is designated SLNP-TR12. 38) The nanocarrier described in claim 36, designated SLNP-TR12. 39) The SLNP of claim 17, wherein the SLNP is further co-formulated with one or more immunomodulatory agents or lipid-prodrugs thereof, wherein the immunomodulatory agent is selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, A2aR inhibitors, STING agonists, CTLA-4 inhibitors, IDO inhibitors, PD-1 / PD-L1 inhibitors, CD1D agonists and / or prodrugs thereof. 40) The SLNP of claim 17, wherein the SLNP is further co-formulated with an ICD-guided chemotherapy agent, and the ICD-guided chemotherapy agent is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, IC1, or paclitaxel. 41) The SLNP of claim 37, further comprising DOX. 42) The SLNP of claim 37, further comprising MTO. 43) The SLNP of claim 40, further comprising DOX. 44) The SLNP of claim 40, further comprising MTO. 45) The SLNP of claim 37, wherein the liposome is further co-formulated with a toll receptor agonist or a lipid-prodrug thereof, and the toll receptor agonist is selected from the group consisting of resiquimod (R848), gardquimod, 852A, DSR 6434, telluritolimod, CU-T12-9, monophosphoryl lipid A (MPLA), 3D(6-acyl)-PHAD®, SMU127, Pam3CSK4, or 3D-PHAD®. 46) The SLNP of claim 37, wherein the liposome is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid-prodrug thereof, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of AUNP12, CA-170, or BMS-986189. 47) The SLNP of claim 37, wherein the SLNP is further co-formulated with a PD-1 / PD-L1 antagonist or a lipid-prodrug thereof, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of AUNP12, CA-170, PD3, or BMS-986189. 48) The SLNP of claim 37, wherein the SLNP is further co-formulated with a TGFb antagonist, wherein the TGFb antagonist is selected from the group consisting of TB4. 49) The SLNP of claim 37, wherein the SLNP is further co-formulated with an IDO inhibitor, the IDO inhibitor being selected from the group consisting of ID3. 50) A kit comprising the SLNP according to any one of claims 35 to 49. 51) A method of treating a subject suffering from or diagnosed with cancer, comprising: (i) administering to a subject in need of such treatment an effective amount of a nanocarrier, where the nanocarrier comprises a TLR prodrug; (ii) a pharma- ceutically acceptable salt thereof; and A method comprising: 52) The method of claim 51, wherein the TLR prodrug comprises a TR12-prodrug. 53) The method of claim 51, wherein the nanocarrier comprises a TR12-prodrug further co-formulated with an ICD-inducing chemotherapy drug. 54) The method of claim 51, wherein the nanocarrier comprises a TR12-prodrug further co-formulated with an immunomodulatory agent. 55) The method of claim 51, wherein the nanocarrier is a liposome. 56) The method of claim 51, wherein the liposome is LNP-TR12. 57) The method of claim 51, wherein the nanocarrier is a solid lipid nanoparticle. 58) The method of claim 51, wherein the SLNP is SLNP-TR12. 59) The method of claim 56, wherein LNP-TR12 is used in combination with a PD-1 antibody, a CTLA4 antibody, or an immunogenic cell death-inducing chemotherapeutic agent (e.g., DOX or MTO). 60) The method of claim 58, wherein SLNP-TR12 is used in combination with a PD-1 antibody, a CTLA4 antibody, or an immunogenic cell death-inducing chemotherapeutic agent (e.g., DOX or MTO). 61) A method of treating a subject suffering from or diagnosed with cancer, comprising: (iii) administering to a subject in need of such treatment an effective amount of a nanocarrier, where the nanocarrier comprises a TLR prodrug; (iv) a pharma- ceutically acceptable salt thereof; and A method comprising: 62) The method of claim 61, wherein the TLR prodrug comprises a TR12-prodrug. 63) The method of claim 61, wherein the nanocarrier comprises a TR12-prodrug further co-formulated with an ICD-inducing chemotherapeutic. 64) The method of claim 61, wherein the nanocarrier comprises a TR12-prodrug further co-formulated with an immunomodulatory agent. 65) The method of claim 61, wherein the nanocarrier is a solid lipid nanoparticle ("SLNP"). 66) The method of claim 61, wherein the SLNP is SLNP-TR12. 67) The method of claim 61, wherein the nanocarrier is a liposome. 68) The method of claim 61, wherein the liposome is LNP-TR12. 69) The chemical structure of: [ka] A TR12 prodrug having the formula: 70) A liposome comprising the TR12 prodrug of claim 69. 71) A liposome comprising the TR12 prodrug of claim 69, further comprising a helper lipid. 72) The liposome of claim 71, wherein the helper lipid is listed in Table II. 73) A solid lipid nanoparticle (SLNP) comprising the TR12 prodrug of claim 69. 74) The liposome of claim 70, designated LNP-TR12. 75) The SLNP of claim 73, designated SLNP-TR12. 76) The SLNP of claim 73 co-formulated with IC1. 77) The SLNP of claim 73 co-formulated with AR5. 78) The SLNP of claim 73 co-formulated with NTI-47C. 79) The SLNP (designated NTI-121) of claim 73 co-formulated with IC1, the ratio being 8:1. 80) The SLNP of claim 73 co-formulated with IC1, the ratio being 16:1. 81) A composition comprising a solid lipid nanoparticle (SLNP), wherein the SLNP further comprises TR12 co-formulated with IC1 (denoted as SLNP-TR12-IC1). 82) A composition comprising solid lipid nanoparticles (SLNPs), wherein the SLNPs further comprise TR12 co-formulated with IC1 in an 8:1 ratio (denoted NTI-121). 83) A composition comprising a solid lipid nanoparticle (SLNP), wherein the SLNP further comprises TR12 co-formulated with IC1 in a ratio of 16:1 (denoted as SLNP-TR12-IC1). 84) A composition comprising a solid lipid nanoparticle (SLNP), wherein the SLNP further comprises TR12 co-formulated with AR5 (denoted as SLNP-TR12-AR5). 85) A composition comprising a solid lipid nanoparticle (SLNP), wherein the SLNP further comprises TR12 co-formulated with NTI-47C (denoted as SLNP-TR12-NTI-47C). 86) TR12 has the following chemical structure: [ka] The composition of any one of claims 79 to 85, comprising: 87) A composition described in any one of claims 79 to 86, used to treat cancer in an individual. 88) The composition of claim 87, used to treat cancer in an individual, wherein the cancer is selected from the group consisting of breast, colon, kidney, melanoma, myeloma, neuroblastoma, liver, lung, pancreas, prostate, and bladder. EXAMPLES

[0453] Various aspects of the present invention are further described and illustrated by the following several examples, none of which are intended to limit the scope of the invention.

[0454] Example 1 Chemical synthesis of stearic acid-containing TR12 prodrugs Chemical synthesis of TR12 containing stearic acid was synthesized using the following protocol. Briefly, a solution of compound 1 (77.0 g, 454 mmol, 1.00 equiv.), compound 1a (88.3 g, 545 mmol, 1.20 equiv.) and K2CO3 (75.3 g, 545 mmol, 1.20 equiv.) in DMF (500 mL) was stirred at 80° C. for 3 h. LCMS (EW29816-9-P1A) showed substantial desired mass (RT=0.701 min). The organic solvent was then evaporated. Water (1.00 L) was then added and the mixture was stirred at 20° C. for 0.5 h and filtered to give compound 2 (120 g, crude) as a yellow solid. Then, a mixture of compound 2 (120 g, 407 mmol, 1.00 equiv) and compound 2a (222 g, 3.04 mol, 300 mL, 7.47 equiv) in n-PrOH (900 mL) was stirred at 130° C. for 12 h in an autoclave (15 psi). LCMS (EW29816-11-P1A) showed that the desired mass was detected (RT=0.684 min). The mixture was concentrated. The residue was diluted with water (1.00 L) and EtOAc (1.00 L) was added. * The combined organics were washed with brine (1.00 L), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, dichloromethane / methanol = 100 / 1 to 10 / 1, dichloromethane / methanol = 10 / 1, R f =0.57) to give compound 3 (64.0 g, 193 mmol, 47.4% yield) as a yellow solid.

[0455] Then, to a solution of compound 3 (63.0 g, 190 mmol, 1.00 equiv) in CHCl3 (400 mL) and MeOH (8.00 mL) was added dropwise a solution of Br2 (45.5 g, 285 mmol, 14.7 mL, 1.50 equiv) in CHCl3 (80 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h. Then, the mixture was concentrated. Concentrated HCl (482 g, 4.96 mol, 473 mL, 37.5% purity, 26.1 equiv) was added to the mixture. The mixture was stirred at 100 °C for 3 h. LCMS (EW29816-14-P1B) showed substantial desired mass (RT = 0.788 min). The mixture was concentrated. The residue was adjusted to pH = 8 with 30% NH3H2O ​​and filtered to give a solid. The crude product was triturated with petroleum ether / ethyl acetate=1 / 1 (200 mL) and filtered to give compound 4 (45.0 g, 129 mmol, 68.1% yield) as a yellow solid.

[0456] A mixture of compound 4 (45.0 g, 129 mmol, 1.00 equiv) and piperazine (223 g, 2.59 mol, 20.0 equiv) was then stirred at 140° C. for 2 h. LCMS (EW29816-16-P1B) showed substantial desired mass (RT=0.668 min). HPLC (EW29816-16-P1A1) showed the main peak was detected (RT=1.237 min). The mixture was concentrated. The residue was purified by preparative HPLC (FA). The organic solvent was evaporated. The aqueous phase was adjusted to pH=8 with saturated Na2CO3 solution and filtered to give compound 5 (28.0 g, 70.4 mmol, 54.4% yield) as a white solid.

[0457] Compound 12 (9.10 g, 24.1 mmol, 1.20 equiv) was then added to a solution of compound 5 (8.00 g, 20.1 mmol, 1.00 equiv) in DMF (150 mL) at 0° C. The mixture was stirred at 20° C. for 12 h. LCMS (EW29816-35-P1A) showed substantial desired product (RT=1.112 min). The mixture was poured into water (500 mL) and filtered to obtain a solid. The residue was purified by preparative HPLC (FA) and preparative HPLC (column: Welch Ultimate XB-CN 250 * 70 *10 um; mobile phase: [heptane-EtOH (0.1% NH3H2O)]; B%: 5%-45%, 15 min) (twice) and concentrated to give TR12 (783.5 mg, 1.03 mmol, 5.13% yield, 97.2% purity) as a grey solid, which was 1 HNMR, LCMS, and HPLC confirmed the synthesis of TR12 (209.85 mg, 276.11 umol, 1.37% yield, 97.1% purity) as a white solid. 1 The synthesis shown in this example yielded the following chemical structure: [ka] To obtain a stearic acid-containing TR12-prodrug having the formula:

[0458] Example 2 Chemical Synthesis of TR12 and TR13 Prodrug Intermediate(s) In another experiment, chemical synthesis of TR12 and TR13 prodrug intermediates was carried out in the following manner. Briefly, a solution of compound 6 (51.0 g, 396 mmol, 35.2 mL, 1.00 equiv) in THF (100 mL) was mixed with compound 7 (24.2 g, 389 mmol, 28.8 mL, 9.83 equiv) in THF (100 mL). -1 Et3N (40.0 g, 396 mmol, 55.0 mL, 1.00 equiv.) was added dropwise at 0-5 °C. The mixture was stirred at 20 °C for 12 h. A white solid appeared. The organic solvent was evaporated. The crude product was distilled (-0.1 MPa, 78-84 °C) to give compound 8 (35.0 g, 226 mmol, 57.2% yield) as a yellow oil.

[0459] Next, a solution of compound 8 (17.0 g, 110 mmol, 1.00 equiv.) and NaI (24.7 g, 165 mmol, 1.50 equiv.) in acetone (170 mL) was stirred at 40° C. for 3 h. TLC (ethyl acetate / petroleum ether=0 / 1) showed that compound 8 had been consumed (R f =0.50), and major spots were detected (R f= 0.52). The mixture was filtered and the filtrate was concentrated. The residue was diluted with ethyl acetate (300 mL) and diluted with 10% aqueous Na2SO3 (200 mL). * 2), washed with brine (200 mL), dried over Na2SO4, filtered and concentrated to give compound 9 (25.0 g, crude) as a yellow oil.

[0460] Compound 9 (25.0 g, 102 mmol, 1.00 equiv.) in DCM (30 mL) was then added dropwise to a solution of compound 10a (34.7 g, 122 mmol, 41.0 mL, 1.20 equiv.), NaHCO3 (21.3 g, 254 mmol, 9.88 mL, 2.50 equiv.) and tetrabutylammonium hydrogen sulfate (41.4 g, 122 mmol, 1.20 equiv.) in DCM (150 mL) and HO (150 mL). The mixture was stirred at 20° C. for 12 h. Compound 9 was consumed (R f =0.70), and major spots were detected (R f = 0.64). The mixture was diluted with water (100 mL) and DCM (200 mL) * The mixture was extracted with 3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 5 / 1) to give compound 11 (25.0 g, 62.1 mmol, yield 61.1%) as a white solid.

[0461] Finally, SO2Cl2 (50.3 g, 373 mmol, 37.3 mL, 10 equiv.) was added dropwise to compound 11 (15.0 g, 37.3 mmol, 1.00 equiv.) at 0 °C. The mixture was stirred at 20 °C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1) confirmed that compound 11 (R f =0.80) was consumed and major spots were detected (R f =0.88). The mixture was concentrated to give compound 12 (14.0 g, crude) as a yellow oil (Figure 2).

[0462] Example 3 Chemical synthesis of non-cleavable stearic acid derivatives of TR13 prodrugs. In another experiment, the chemical synthesis of TR12 prodrug containing a non-cleavable stearic acid derivative (denoted "TR13") was synthesized using the following protocol. Briefly, to a solution of compound 10a (4.01 g, 14.1 mmol, 4.74 mL, 0.70 equiv), DIEA (7.80 g, 60.4 mmol, 10.5 mL, 3.00 equiv), HATU (9.18 g, 24.2 mmol, 1.20 equiv) in DMF (10 mL) was added compound 5 (8.00 g, 20.1 mmol, 1.00 equiv). The mixture was stirred at 20° C. for 12 h. LCMS (EW29816-26-P1A) showed substantial desired mass (RT=1.053 min). The mixture was concentrated. The residue was purified by preparative HPLC (FA) and concentrated. The crude product was diluted with MeCN (100 mL) and adjusted to pH=8 with Et3N. The mixture was filtered to give a solid. The solid was triturated with EtOAc / THF=1 / 1 (50 mL) and filtered to give TR13 (2.32 g, 3.30 mmol, 16.37% yield, 94.3% purity) as a white solid (Figure 3). The synthesis shown in this example yielded the compound with the following chemical structure: [ka] To obtain a TR12-prodrug (denoted TR13) containing a non-cleavable stearic acid derivative having the formula:

[0463] Example 4 Synthesis and characterization of SLNP-TR12 solid lipid nanoparticles In another experiment, solid lipid nanoparticles containing TR12 (denoted as SLNP-TR12) were synthesized using the following protocol. Briefly, by the solvent diffusion method, with or without stabilizers. It should be noted that examples of stabilizers that can be used in this SLNP include, but are not limited to, polyvinyl alcohol (e.g., Moliwol 488), poloxamer (e.g., Pluronic® F-68, Pluronic® F-127), Tween® 80 and 20, Kolliphor RH40, etc.

[0464] In the first step, lipid stock solutions of DSPC, CHOL, DSPE-PEG were prepared in ethanol (20 mg / ml). Separately, a TR12 prodrug stock solution was prepared in DMSO (20 mg / ml). In this example, a lipid mixture was obtained by mixing DSPC, CHOL, TR12 and DSPE-PEG in a molar ratio of 34:56:5:5 (with a lipid concentration of 20 mg / ml). This lipid mixture was then heated at 40-45 °C for 5 min. Similarly, the aqueous phase was heated (40-45 °C) using a magnetic hotplate stirrer with constant magnetic stirring (300-400 rpm).

[0465] Alternatively, as a more simplified method, SLNPS-TR12 can also be synthesized by using only DI water (without stabilizers) for the aqueous phase. In this alternative method, the lipid mixture was slowly mixed with this aqueous phase under constant stirring. Once mixing was complete, the entire mixture was sonicated for approximately 10 min using a water ultrasonic bath and then maintained on a magnetic stirrer plate again with constant stirring for approximately another 2-4 h. Finally, the solvent was removed using a dialysis membrane (Sigma Aldrich) with a cut-off size of 12 KDa against DI water for at least 8 h. During this time, the dialysis water was replaced at least three times. SLNP-TR12 was concentrated as needed using an Amicon centrifugal filter device (cut-off size of 10 KDa at 3000 g). Alternatively, a TFF (tangential flow filtration) system can be used to remove the solvent and concentrate the SLNP-TR12.

[0466] The characteristics of SLNP-TR12 were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-TR12 (concentration 1 mg / ml) was placed in a clear, four-sided plastic cuvette and directly analyzed at 25° C. The results, shown in FIG. 7, indicate that the Z-average size of the nanoparticles was approximately 105.3 nm and the PDI was approximately 0.109.

[0467] In addition, the zeta potential of SLNP-TR12 solid lipid nanoparticles in aqueous dispersion was determined using a Malvern Zetasizer instrument (Malvern Instrumentation Co, Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration approximately 3 mg / ml in DI water) was placed in a disposable capillary zeta potential cell available in the Zetasizer. Measurements were performed at 25° C. The results show that the zeta potential measurement of SLNP-TR12 was approximately −10.9 mV ( FIG. 8 ).

[0468] Example 5 Synthesis and characterization of SLNP-TR12-IC1 (NTI-121) solid lipid nanoparticles In another experiment, solid lipid nanoparticles (denoted SLNP-TR12-IC1 and / or NTI-121) containing TR12 co-formulated with an immunogenic cell death prodrug ("IC1") were synthesized. It is understood that the solvent diffusion method with or without stabilizers can be utilized. Example(s) of stabilizers that can be used are polyvinyl alcohol (e.g., Moliwol 488), poloxamer (e.g., Pluronic® F-68, Pluronic® F-127), Tween® 80 and 20, Kolliphor RH40, etc. It is understood that different types of SLNPs using different ratios of IC1:TR12 can also be prepared.

[0469] In this example, SLNP-IC1-TR12 (also denoted as NTI-121) with a ratio of IC1:TR12 of 8:1 was prepared using the solvent diffusion method described above. In the first step, lipid stock solutions of DSPC, CHOL, and DSPE-PEG were prepared in ethanol (20 mg / ml). A stock of IC1 was also prepared in ethanol with a concentration of 2.5 mg / ml. Separately, a stock solution of TR12 prodrug was prepared in DMSO (20 mg / ml). A lipid mixture was obtained by mixing DSPC, CHOL, IC1, TR12, and DSPE-PEG in a molar ratio of 33:54.56:7:0.44:5 (with a lipid concentration of approximately 20 mg / ml). This lipid mixture was then heated at 40-45 degrees. The lipid mixture was slowly mixed with this aqueous phase (DI water) under constant stirring. Once mixing was complete, the entire mixture was sonicated using a water ultrasonic bath for approximately 10 minutes and then again maintained on a magnetic stirrer plate with constant stirring for approximately an additional 2-4 hours.

[0470] Finally, the solvent was removed using a dialysis membrane (Sigma Aldrich) with a cut-off size of 12 KDa against DI water for at least 8 h. During this time, the dialysis water was exchanged at least three times. SLNP-IC1-TR12 was concentrated as needed using an Amicon centrifugal filter device (cut-off size of 10 KDa at 3000 g). Alternatively, a TFF (tangential flow filtration) system can be used to remove the solvent and concentrate the SLNP-IC1-TR12.

[0471] The characteristics of SLNP-TR12-IC1 (NTI-121) were determined using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-IC1-TR12 (concentration 1 mg / ml) was placed in a clear, four-sided plastic cuvette and directly analyzed at 25° C. The results, shown in FIG. 9, indicate that the Z-average size of the nanoparticles was approximately 93.37 nm and the PDI was approximately 0.132.

[0472] In addition, the zeta potential of SLNP-IC1-TR12 (NTI-121) solid lipid nanoparticles in aqueous dispersion was determined using a Malvern Zetasizer instrument (Malvern Instrumentation Co, Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration approximately 3 mg / ml in DI water) was placed in a disposable capillary zeta potential cell available in the Zetasizer. Measurements were performed at 25° C. The results show that the zeta potential measurement of SLNP-IC1-TR12 was approximately −12.8 mV ( FIG. 10 ).

[0473] Example 6 Synthesis and characterization of SLNP-TR12-IC1 (16:1) solid lipid nanoparticles In another experiment, solid lipid nanoparticles (designated SLNP-TR12-IC1) containing TR12 co-formulated with an immunogenic cell death prodrug ("IC1") were synthesized. It is understood that the solvent diffusion method with or without stabilizers can be utilized. Example(s) of stabilizers that can be used are polyvinyl alcohol (e.g., Moliwol 488), poloxamer (e.g., Pluronic® F-68, Pluronic® F-127), Tween® 80 and 20, Kolliphor RH40, etc. It is understood that different types of SLNPs using different ratios of IC1:TR12 can also be prepared.

[0474] In this example, SLNP-IC1-TR12 with a ratio of IC1:TR12 of 16:1 was prepared using the solvent diffusion method described above. In the first step, lipid stock solutions of DSPC, CHOL, and DSPE-PEG were prepared in ethanol (20 mg / ml). A stock of IC1 was also prepared in ethanol with a concentration of 2.5 mg / ml. Separately, a stock solution of TR12 prodrug was prepared in DMSO (20 mg / ml). A lipid mixture was obtained by mixing DSPC, CHOL, IC1, TR12, and DSPE-PEG in a molar ratio of 33:54.8:7:0.2:5 (having a lipid concentration of approximately 20 mg / ml). This lipid mixture was then heated at 40-45 degrees. The lipid mixture was slowly mixed with the aqueous phase (DI water) under constant stirring. Once mixing was complete, the entire mixture was sonicated using a water ultrasonic bath for approximately 10 minutes and then again maintained on a magnetic stirrer plate with constant stirring for approximately an additional 2-4 hours.

[0475] Finally, the solvent was removed using a dialysis membrane (Sigma Aldrich) with a cut-off size of 12 KDa against DI water for at least 8 h. During this time, the dialysis water was exchanged at least three times. SLNP-IC1-TR12 was concentrated as needed using an Amicon centrifugal filter device (cut-off size of 10 KDa at 3000 g). Alternatively, a TFF (tangential flow filtration) system can be used to remove the solvent and concentrate the SLNP-IC1-TR12 (16:1).

[0476] SLNP-TR12-IC1 was characterized using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-IC1-TR12 (concentration 1 mg / ml) was placed in a clear, four-sided plastic cuvette and directly analyzed at 25° C. The results, shown in FIG. 11, indicate that the Z-average size of the nanoparticles was approximately 94.02 nm and the PDI was approximately 0.109.

[0477] In addition, the zeta potential of SLNP-IC1-TR12 solid lipid nanoparticles in aqueous dispersion was determined using a Malvern Zetasizer instrument (Malvern Instrumentation Co, Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration approximately 3 mg / ml in DI water) was placed in a disposable capillary zeta potential cell available in the Zetasizer. Measurements were performed at 25° C. The results show that the zeta potential measurement of SLNP-IC1-TR12 was approximately −10.9 mV ( FIG. 12 ).

[0478] Example 7 Synthesis and characterization of SLNP-TR12-AR5 solid lipid nanoparticles In another experiment, solid lipid nanoparticles containing TR12 co-formulated with an A2aR inhibitor ("AR5") (denoted as SLNP-TR12-AR5). Solid lipid nanoparticles of the combination of AR5 and TR12 were prepared by the solvent diffusion method using different types of stabilizers as described above. As already mentioned, these SLNPs can also be prepared without stabilizers.

[0479] In the first step, lipid stock solutions of DSPC, CHOL, DSPE-PEG were prepared in ethanol (20 mg / ml). Separately, stock solutions of AR5 and TR12 prodrugs were prepared in DMSO (20 mg / ml). A lipid mixture was obtained by mixing DSPC, CHOL, AR5, TR12, and DSPE-PEG in a molar ratio of 26:39:28:1:5 (having a lipid concentration of 20 mg / ml). This lipid mixture was then heated at 45-50°C. Similarly, an aqueous phase containing a suitable stabilizer (e.g., 1-5% w / v Pluronic® F127) was heated using a magnetic hot plate stirrer with constant magnetic stirring (300-400 rpm). The lipid mixture was slowly mixed with this aqueous phase under constant stirring. Once mixing was complete, the entire mixture was sonicated using a water ultrasonic bath for about 10 minutes and then kept again on the magnetic stirrer plate with constant stirring for about another hour.

[0480] Finally, the solvent was removed using a dialysis membrane (Sigma Aldrich) with a cut-off size of 12 KDa against DI water for at least 8 h. During this time, the dialysis water was exchanged at least three times. SLNP-AR5-TR12 was concentrated as needed using an Amicon centrifugal filter device (cut-off size of 10 KDa at 3000 g). Alternatively, a TFF (tangential flow filtration) system can be used to remove the solvent and concentrate the SLNP.

[0481] SLNP-TR12-AR5 was characterized using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-TR12-AR5 (concentration 1 mg / ml) was placed in a clear, four-sided plastic cuvette and directly analyzed at 25° C. The results, shown in FIG. 13, indicate that the Z-average size of the nanoparticles was approximately 98 nm and the PDI was approximately 0.162.

[0482] In addition, the zeta potential of SLNP-TR12-AR5 solid lipid nanoparticles in aqueous dispersion was determined using a Malvern Zetasizer instrument (Malvern Instrumentation Co, Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration approximately 3 mg / ml in DI water) was placed in a disposable capillary zeta potential cell available in the Zetasizer. Measurements were performed at 25° C. The results show that the zeta potential measurement of SLNP-TR12-AR5 was approximately −12.0 mV ( FIG. 14 ).

[0483] Example 8 Synthesis and characterization of SLNP-TR12-NTI-47C solid lipid nanoparticles In another experiment, solid lipid nanoparticles (denoted SLNP-TR12-NTI-47C) containing TR12 co-formulated with NTI-47C, a custom peptide (GSGCERVIGTGWVRC) (SEQ ID NO: 1) conjugated with palmitoyl ("NTI-47C"), were synthesized. By using this peptide (CERVIGTGWVRC) (SEQ ID NO: 2) in this type of SLNP, it is contemplated that the function-blocking peptide (CERVIGTGWVRC) (SEQ ID NO: 2) structurally mimics the epitope on CD47 and binds to SIRPα. It is understood that the CD47 molecule is well known as a widely expressed cell surface receptor that activates the transmission of "don't eat me" signals. Therefore, this can reduce the desired uptake of nanoparticles by macrophages and has the potential to remain in the blood circulation for a long time.

[0484] SLNP-TR12-NTI-47C was synthesized using the following protocol. Briefly, SLNP-TR12-NTI-47C can be synthesized by solvent diffusion using various types of stabilizers as previously described (e.g., polyvinyl alcohol (e.g., Moliwol 488), poloxamer (e.g., Pluronic® F-68, Pluronic® F-127), Tween® 80 and 20, and Kolliphor RH40, etc.). Briefly, in the first step, lipid stock solutions of DSPC, CHOL, and NTI-47C were prepared in ethanol (20 mg / ml). Separately, a stock solution of TR12 prodrug was prepared in DMSO (20 mg / ml). A lipid mixture was obtained by mixing DSPC, CHOL, NTI-47C, TR12, and DSPE-PEG in a molar ratio of 29:35:31:5 (with a lipid concentration of 20 mg / ml). This lipid mixture was then heated at 40-45 degrees Celsius for approximately 5 minutes. Similarly, an aqueous phase containing a suitable stabilizer (e.g., 2% w / v Pluronic® F127) was heated using a magnetic hot plate stirrer with constant magnetic stirring (300-400 rpm). The lipid mixture was slowly mixed with this aqueous phase under constant stirring. Once mixing was complete, the entire mixture was sonicated using a water ultrasonic bath for approximately 10 minutes and then again kept on the magnetic stirrer plate with constant stirring for approximately another hour. Finally, the solvent was removed using a dialysis membrane (Sigma Aldrich) with a cut-off 12 KDa size against DI water for at least 6 hours. During this time, the dialysis water was exchanged at least three times. SLNP-TR12-NTI-47C was concentrated as required using Amicon centrifugal filter devices (cut-off size 10 KDa at 3000 g ).

[0485] SLNP-TR12-NTI-47C was characterized using a Malvern Zetasizer (Malvern Instrumentation Co., Westborough, MA, USA). Briefly, 2 ml of SLNP-TR12-NTI-47C (concentration 1 mg / ml) was placed in a clear, four-sided plastic cuvette and directly analyzed at 25° C. The results, shown in FIG. 15, indicate that the Z-average size of the nanoparticles was approximately 96.4 nm and the PDI was approximately 0.084.

[0486] In addition, the zeta potential of SLNP-TR12-NTI-47C solid lipid nanoparticles in aqueous dispersion was determined using a Malvern Zetasizer instrument (Malvern Instrumentation Co, Westborough, MA, USA). Briefly, approximately 1 ml of SLNP (concentration approximately 3 mg / ml in DI water) was placed in a disposable capillary zeta potential cell available in the Zetasizer. Measurements were performed at 25° C. The results show that the zeta potential measurement of SLNP-TR12-NTI-47C was approximately −13.3 mV ( FIG. 16 ).

[0487] Example 9 In vivo tumor inhibition using EMT6 cells with SLNP-TR12 as a single agent Evaluation of SLNP-TR12 as a single agent was performed using the following protocol. Briefly, mouse breast cancer EMT6 cells (0.5 × 10 6 ) was inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated with vehicle control and SLNP-TR12 at 1 mg / kg BIW by iv injection. Tumor volumes were measured bidimensionally in triplicate using calipers and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L).

[0488] The results show that treatment with 1 mg / kg SLNP-TR12 resulted in significant tumor growth inhibition compared to vehicle control (see FIG. 17).

[0489] Example 10 In vivo tumor inhibition of various doses of SLNP-TR12 compared to various doses of SLNP-TR5 using EMT6 cells Evaluation of SLNP-TR12 in comparison with various doses of SLNP-TR5 was carried out using the following protocol. Briefly, mouse breast cancer EMT6 cells (0.5×10 6 ) was inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated with vehicle control and SLNP-TR12 or SLNP-TR5 (telluratolymod in solid nanoparticle form) at 0.5 or 1 mg / kg BIW by iv injection. Tumor volumes were measured in triplicate using calipers in two dimensions and the volumes were calculated using the formula: V = (L x W x W) x 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).

[0490] The results show that treatment with either 0.5 mg / kg or 1 mg / kg SLNP-TR12 resulted in significant tumor growth inhibition. In addition, the results show that tumor growth inhibition was greater than that of the same dose of SLNP-TR5 (see FIG. 18).

[0491] Example 11 In vivo tumor inhibition of SLNP-TR12 in combination with SLNP-IC1 using EMT6 cells Evaluation of SLNP-TR12 in combination with SLNP-IC1 was carried out using the following protocol. Briefly, mouse breast cancer EMT6 cells (0.5×10 6) were inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated with vehicle control and SLNP-TR12 at 0.5 mg / kg, SLNP-IC1 (doxorubicin-HCL-stearic acid solid nanoparticle form) at 2 mg.kg, or SLNP-IC1-TR12 at 2 / 0.5 mg / kg by iv injection BIW. Tumor volumes were measured bidimensionally in triplicate using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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).

[0492] The results show that treatment with a combination of SLNP-IC1 and SLNP-TR12 induced significant tumor growth inhibition (see FIG. 19).

[0493] Example 12 In vivo tumor inhibition of SLNP-TR12 in combination with SLNP-IC1 using EMT6 cells In another experiment, evaluation of SLNP-TR12 in combination with SLNP-IC1 was performed using the following protocol. Briefly, mouse breast cancer EMT6 cells (0.5×10 6 ) were inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated with vehicle control and SLNP-TR12 at 0.5 mg / kg, SLNP-IC1 (SLNP form of doxorubicin-HCL-stearic acid) at 2 mg / kg, or SLNP-IC1-TR12 at 2 / 0.5 mg / kg BIW by iv injection. Tumor volumes were measured bidimensionally in triplicate using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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).

[0494] The results show that treatment with a combination of SLNP-IC1 and SLNP-TR12 induced significant tumor growth inhibition (see FIG. 20).

[0495] Example 13 In vitro validation of the mechanism of action of TR12 prodrug in solid lipid nanoparticle ("SLNP") form To confirm that SLNP-TR12 has a biological effect in vitro and targets TLR7, the following protocol was used. As background, RAW-Blue™ cells and QUANTI-Blue™ (InvivoGen, San Diego, CA) assay were utilized. Raw Blue™ expresses human Toll-like receptors ("TLRs") and an NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. Stimulation of these cells with TR12 results in activation of NF-κB via TLR7, which can be measured by detection of SEAP levels. Briefly, RAW-Blue™ cells were incubated with different concentrations of TR12-SA (TR12-stearic acid) and SLNP-TR12 (TR12-stearic acid in Solid Lipid Nanoparticle form). After 24 hours of incubation with compounds, TLR stimulation was assessed by measuring the level of SEAP optimal density (OD) using QUANTI-Blue™ assay. OD was normalized to the control (untreated) group. The results showed that treatment of cells with TR12 prodrug and SLNP-TR12 caused stimulation of TLR-7, confirming the mechanism of action of TR12 and the activity of TR12 in SLNP form. (See Figure 21).

[0496] Example 14 In vitro validation of the mechanism of action of SLNP-TR12 compared with SLNP-TR5 In another experiment, the in vitro biological activity of SLNP-TR12 compared to SLNP-TR5 was confirmed using the following experiment(s). As background, to confirm that SLNP-TR12 can have a biological effect in vitro, RAW-Blue™ cells and QUANTI-Blue™ (InvivoGen, San Diego, CA) assay were used. Note that Raw Blue™ expresses human TLR and NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. Stimulation of these cells with TR12 and TR5 results in activation of NF-κB via TLR7 or TLR7 / 8, which can be measured by detection of SEAP levels.

[0497] Briefly, RAW-Blue™ cells were incubated with different concentrations of TR12-SA (TR12-stearic acid), SLNP-TR12 (TR12-stearic acid in solid lipid nanoparticle form) or SLNP-TR5 (telluratorimod in solid lipid nanoparticle form). After 24 hours of incubation with the compounds, TLR stimulation was assessed by measuring the level of SEAP optimal density (OD) using QUANTI-Blue™ assay. OD was normalized to the control (untreated) group. The results showed that treatment of cells with TR12 caused stimulation of TLR7, confirming the mechanism of action of TR12 and its activity in SLNP form. In addition, SLNP-TR12 showed higher potency compared to SLNP-TR5. (See Figure 22). (See Figure 22).

[0498] Example 15 In vitro validation of the specificity of SLNP-TR12 for Toll-like receptor 7 (TLR7) In another experiment, the in vitro bioactivity of SLNP-TR12 specific for TLR7 was confirmed using the following experiment. In background, to confirm that TR12 is specific for TLR7 and does not target TLR8, RAW-Blue™ and HEK-Blue™ hTLR8 cell lines and QUANTI-Blue™ (InvivoGen, San Diego, CA) assay were used. It should be noted that HEK-Blue™ hTLR8 cells mainly express human TLR8 gene and NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene, even though Raw-Blue cells express various TLRs including TLR7 and TLR8. Therefore, the SEAP level caused by TLR8 stimulation can be easily determined by performing an assay in HEK-Blue™ detection.

[0499] Briefly, RAW-Blue™ cells were incubated with different concentrations of SLNP-TR5 or SLNP-TR12. After 24 hours of incubation with compounds, TLR stimulation was evaluated by measuring the level of SEAP optimal density (OD) using QUANTI-Blue™ assay. OD was normalized to control (untreated) group (see Figure 23(A)).

[0500] In addition, HEK-Blue TLR8 was incubated with different concentrations of SLNP-TR5 or SLNP-TR12. After 24 hours of incubation with compounds, TLR stimulation was evaluated by measuring the level of SEAP optimal density (OD) using QUANTI-Blue™ assay. OD was normalized to the control (untreated) group (see Figure 23(B)).

[0501] Taken together, the results show that both SLNP forms of TR5 and TR12 prodrugs stimulated TLR in RAW-Blue cells, and it is noteworthy that SLNP-TR12 had higher potency. The EC50 for SLNP-TR5 and SLNP-TR12 was calculated to be 339.7 and 77.47 nM, respectively.

[0502] However, when HEK-Blue hTLR8 cells were treated with SLNP-TR5 and SLNP-TR12, only SLNP-TR5 could stimulate the cells. No SEAP induction was observed in cells treated with SLNP-TR12. The potency of SLNP-TR5 in stimulating the TLR-7 pathway in HEK-Blue hTLR8 cells was lower than that in Raw-Blue cells (0.33 vs. 21.69 uM). Thus, since TR5 is a TLR7 / 8 agonist and TR12 is a TLR7 agonist, the results showed that TR5 stimulates both TLR7 and TLR8, but TR12 is specific for TLR7 and not TLR8. (See Figure 23).

[0503] (Example 16) Ex vivo validation of SLNP-TR12 activity In another experiment, the ex vivo bioactivity of SLNP-TR12 was confirmed using the following experiment. Briefly, splenocytes were isolated from Balb / c mice. Approximately 1×10 6 (1,000,000) cells (2 × 10 6 / ml) in culture medium. Then, the cells were treated with anti-CD3 / 28 cell activation beads at a cell:bead ratio of 1:1. Then, the samples were treated with different concentrations of SLNP-TR12. After 24 hours, the supernatants were collected and analyzed for TNF-alpha by ELISA. The results showed that SLNP-TR12 could induce the secretion of TNF-alpha in splenocytes isolated from mice. (See Figure 24).

[0504] (Example 17) Ex vivo validation of SLNP-TR12 activity In another experiment, the ex vivo bioactivity of SLNP-TR12 was confirmed using the following experiment. Briefly, human peripheral blood mononuclear cells (PBMCs) were treated with different concentrations of TR-12 prodrug and SLNP-TR12. After 24 hours, the supernatant was collected and analyzed for TNF-alpha by ELISA. The results showed that SLNP-TR12 could induce the secretion of TNFα in human PBMCs. (See Figure 25).

[0505] (Example 18) In vivo validation of the efficacy of SLNP-TR12 in EMT-6 tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-TR12 was confirmed using the following experiment(s). Briefly, mouse breast cancer EMT6 cells (0.5×10 6 ) was inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated biweekly with vehicle control and SLNP-TR12 at 0.25 and 0.125 mg / kg by iv injection. Tumor volumes were measured bi-dimensionally in triplicate using calipers and the volumes were calculated using the formula: V = (L x W x W) x 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).

[0506] The results show that treatment with 0.25 mg / kg SLNP-TR12 induces significant tumor growth inhibition. However, treatment(s) with 0.125 mg / kg do not induce significant tumor growth inhibition. (See FIG. 26).

[0507] (Example 19) In vivo validation of the efficacy of SLNP-TR12 in the 4T-1 tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-TR12 was confirmed using the following experiment(s). Briefly, mouse breast cancer 4T-1 cells (0.3 × 10 6) was inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated twice weekly with vehicle control and SLNP-TR12 at 0.5 mg / kg by iv injection. Tumor volumes were measured in triplicate using calipers and the volumes were calculated using the formula: V = (L x W x W) x 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).

[0508] The results show that treatment with 0.5 mg / kg SLNP-TR12 induces tumor growth inhibition in the 4T-1 model (see FIG. 27).

[0509] (Example 20) In vivo validation of the efficacy of multiple doses of TR12 prodrug (SLNP-TR12) alone and in combination with IC1 prodrug (SLNP-IC1) in the EMT-6 tumor model In separate experiments, the in vivo efficacy / activity of SLNP-TR12 as a single agent and / or in combination with SLNP-IC1 was confirmed using the following experiment(s). Briefly, mouse breast cancer EMT-6 cells (0.5×10 6 ) was inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated twice weekly with vehicle control and SLNP-TR12 at 0.25 (HD) or 0.125 (LD) mg / kg, SLNP-IC1 at 2 (HD) or 1 (LD) mg / kg, SLNP-IC1 / TR12 at 2 / 0.25 (HD) or 1 / 0.125 (LD) mg / kg by iv injection. In addition, in one group, animals were treated with SLNP-IC1 / TR12 (HD) by ip injection.

[0510] Tumor volumes were measured in three dimensions using calipers, and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L).

[0511] The results showed that combination treatment with high dose (HD) IC1 and TR12 induced the most significant tumor growth inhibition. Notably, ip injection of SLNP-IC1 / TR12 induced tumor growth inhibition, but the delivery method of iv injection was shown to be more efficient compared to the ip delivery method. (See FIG. 28).

[0512] Example 21 Maximum tolerated dose (MTD) of doxorubicin prodrug alone and in combination with SLNP-TR12 in the Balb / c mouse model In another experiment, the MTD of SLNP-TR12 as a single agent and / or in combination with SLNP-IC1 was confirmed using the following experiment(s): Briefly, Balb / c mice were treated with varying doses of SLNP-IC1 (a doxorubicin prodrug in solid lipid nanoparticle form), SLNP-TR12, and a combination of SLNP-IC1 and SLNP-TR12 by iv injection.

[0513] Animals were observed 30 minutes and 4 hours after injection for clinical signs of toxicity, including but not limited to orbital stiffness, ear position, nasal flare, lethargy, and piloerection. Animals were weighed 24, 48, and 72 hours after injection.

[0514] The results show that no immediate toxicity signs were observed in any group. SLNP-IC1 as a single agent caused mild weight loss. Notably, SLNP-TR12 as a single agent induced moderate to severe weight loss by 48 hours after injection, and after 48 hours, the weight loss was restored without intervention. The combination of SLNP-IC1 and SLNP-TR12 induced moderate weight loss, which was lower than that of the SLNP-TR12 treatment group. (See FIG. 29).

[0515] Example 22 In vivo validation of the efficacy of SLNP-IC1-TR12 in B16F10 melanoma tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse melanoma B16F10 cells (cells (0.2×10 6 ) was inoculated subcutaneously into the right hind flank region of B57 / BL mice. Animals were treated with vehicle control and SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data on day 9.

[0516] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared to the vehicle-treated group. The TGI was 63.45%. (See FIG. 30).

[0517] (Example 23) In vivo validation of the efficacy of SLNP-IC1-TR12 in the MPC11 multiple myeloma tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s): Briefly, mouse multiple myeloma MPC11 cells (0.2×10 6 ) was inoculated subcutaneously into the right flank region of Balb / c mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data on day 9.

[0518] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared with the vehicle treatment group. The TGI was 53.38%. (See FIG. 31).

[0519] Example 24 In vivo validation of the efficacy of SLNP-IC1-TR12 in the Neuro2A neuroblastoma tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse neuroblastoma Neuro2A cells (1×10 6 ) was inoculated subcutaneously into the right flank region of A / J mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 10.

[0520] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared to the vehicle treatment group. The TGI was 88.27%. (See FIG. 32).

[0521] (Example 25) In vivo validation of the efficacy of SLNP-IC1-TR12 in the CT26 colon tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse colon CT26 cells (1×10 6) was inoculated subcutaneously into the right flank region of Balb / c mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data on day 12.

[0522] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared to the vehicle treatment group. The TGI was 81.16%. (See FIG. 33).

[0523] (Example 26) In vivo validation of the efficacy of SLNP-IC1-TR12 in the MC38 colon tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse colon MC38 cells (1×10 6 ) was inoculated subcutaneously into the right flank region of C57 / bl6 mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 18.

[0524] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared with the vehicle treatment group. The TGI was 83.59%. (See FIG. 34).

[0525] Example 27 In vivo validation of the efficacy of SLNP-IC1-TR12 in the Renca kidney tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse kidney Renca cells (1×10 6 ) was inoculated subcutaneously into the right flank region of Balb / C mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data on day 13.

[0526] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared with the vehicle treatment group. The TGI was 68.18%. (See FIG. 35).

[0527] (Example 28) In vivo validation of the efficacy of SLNP-IC1-TR12 in H22 liver tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse liver H22 cells (2×10 6 ) was inoculated subcutaneously into the right flank region of Balb / C mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 21.

[0528] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared to the vehicle-treated group. The TGI was 100.96%. (See FIG. 36).

[0529] (Example 29) In vivo validation of the efficacy of SLNP-IC1-TR12 in Hepa1-6 liver tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s): Briefly, mouse liver Hepa1-6 cells (5×10 6 ) was inoculated subcutaneously into the right flank region of C57BL6 mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 16.

[0530] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared to the vehicle treatment group. TGI was 100.47%. (See FIG. 37).

[0531] (Example 30) In vivo validation of the efficacy of SLNP-IC1-TR12 in LLC1 lung tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse lung LLC1 cells (0.3×10 6) was inoculated subcutaneously into the right flank region of C57BL6 mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 16.

[0532] The results show that 6 doses of SLNP-IC1-TR12 administered for 2 weeks produced antitumor activity compared to the vehicle-treated group. The TGI was 56.65%. (See FIG. 38).

[0533] (Example 31) In vivo validation of the efficacy of SLNP-IC1-TR12 in KLN205 lung tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse lung KLN205 cells (0.8×10 6 ) was inoculated subcutaneously into the right flank region of DBA / 2 mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 21.

[0534] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared with the vehicle treatment group. TGI was 64.21%. (See FIG. 39).

[0535] Example 32 In vivo validation of the efficacy of SLNP-IC1-TR12 in a B16BL6 melanoma tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse melanoma B16B16 cells (0.2×10 6 ) was inoculated subcutaneously into the right flank region of C57BL6 mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 12.

[0536] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared with the vehicle treatment group. The TGI was 70.51%. (See FIG. 40).

[0537] (Example 33) In vivo validation of the efficacy of SLNP-IC1-TR12 in the Pan02.03 pancreatic tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse pancreatic Pan02.03 cells (3×10 6) was inoculated subcutaneously into the right flank region of C57BL6 mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice weekly. Tumor volumes were measured bidimensionally three times using calipers, and the volumes were calculated using the formula: V = (L x W x W) x 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). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data at day 62.

[0538] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared with the vehicle treatment group. The TGI was 90.48%. (See FIG. 41).

[0539] (Example 34) In vivo validation of the efficacy of SLNP-IC1-TR12 in the RM-1 prostate tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse prostate RM-1 cells (1×10 6 ) was inoculated subcutaneously into the right flank region of C57BL6 mice. Animals were treated twice weekly with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection. Tumor volumes were measured bidimensionally three times using calipers and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data of 14.

[0540] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared to the vehicle treatment group. TGI was 50.24%. (See FIG. 42).

[0541] (Example 35) In vivo validation of the efficacy of SLNP-IC1-TR12 in the BMT2 bladder tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse bladder BMT2 cells (4×10 6 ) was inoculated subcutaneously into the right flank region of C3H / He mice. Animals were treated twice weekly with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection. Tumor volumes were measured bidimensionally three times using calipers and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data of 16.

[0542] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared with the vehicle-treated group. The TGI was 67.81%. (See FIG. 43).

[0543] (Example 36) In vivo validation of the efficacy of SLNP-IC1-TR12 in the clonal M-3 melanoma tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s): Briefly, mouse melanoma clone M-3 cells (0.2×10 6) was inoculated subcutaneously into the right flank region of C57BL6 mice. Animals were treated twice weekly with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection. Tumor volumes were measured bidimensionally three times using calipers and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Body weight was measured three times weekly. Tumor growth inhibition (TGI) was calculated based on the tumor size data of 12.

[0544] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared to the vehicle treatment group. The TGI was 74.88%. (See FIG. 44).

[0545] (Example 37) In vivo validation of the efficacy of SLNP-IC1-TR12 in the 4T1 breast orthotopic tumor model In another experiment, the in vivo efficacy / activity of SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse 4T1 breast cancer cells were inoculated into the mammary fat pad of female Balb / c mice. Animals were treated with vehicle control or SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg by iv injection twice a week. Tumor volumes were measured in three dimensions using calipers, and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Body weight was measured three times a week. Tumor growth inhibition (TGI) was calculated based on the tumor size data of 26.

[0546] The results show that 6 doses of SLNP-IC1-TR12 treatment for 2 weeks produces antitumor activity compared to the vehicle treatment group. The TGI was 41.34%. (See FIG. 45).

[0547] (Example 38) In vivo validation of the efficacy of several SLNP-TR12 embodiments alone or in combination with SLNP-IC1 and / or SLNP-IC1-TR12 in the CT-26 colon tumor model In another experiment, the in vivo efficacy / activity of SLNP-TR12 alone or in combination with SLNP-IC1 and / or SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse colon cancer CT-26 cells (0.5×10 6 ) was inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated twice weekly by iv injection with vehicle control and SLNP-TR12 at 0.25 mg / kg, SLNP-IC1 at 2.0 mg / kg, and SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg. Tumor volumes were measured bidimensionally in triplicate using calipers, and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L).

[0548] The results show that combination treatment with SLNP-IC1-TR12 induces greater tumor growth inhibition compared to single agent treatment (see FIG. 46).

[0549] (Example 39) In vivo validation of the efficacy of several SLNP-TR12 embodiments alone or in combination with SLNP-IC1 and / or SLNP-IC1-TR12 in the B16F10 colon tumor model In another experiment, the in vivo efficacy / activity of SLNP-TR12 alone or in combination with SLNP-IC1 and / or SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse colon cancer B16F10 cells (0.3×10 6) was inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated twice weekly by iv injection with vehicle control and SLNP-TR12 at 0.25 mg / kg, SLNP-IC1 at 2.0 mg / kg, and SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg. Tumor volumes were measured bidimensionally in triplicate using calipers, and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L).

[0550] The results show that combination treatment with SLNP-IC1-TR12 induces greater tumor growth inhibition compared to single agent treatment (see FIG. 47).

[0551] (Example 40) In vivo validation of the efficacy of several SLNP-TR12 embodiments alone or in combination with SLNP-IC1 and / or SLNP-IC1-TR12 in the EMT-6 tumor model In a separate experiment, the in vivo efficacy / activity of SLNP-TR12 alone or in combination with SLNP-IC1 and / or SLNP-IC1-TR12 was confirmed using the following experiment(s). Briefly, mouse breast cancer EMT-6 cells (0.5×10 6 ) was inoculated subcutaneously into the right hind flank region of Balb / c mice. Animals were treated twice weekly by iv injection with vehicle control and SLNP-TR12 at 0.25 mg / kg, SLNP-IC1 at 2.0 mg / kg, and SLNP-IC1-TR12 at 2.0 / 0.25 mg / kg. Tumor volumes were measured bidimensionally in triplicate using calipers, and the volumes were calculated using the formula: V=(L×W×W)×0.5, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L).

[0552] The results show that combination treatment with SLNP-IC1-TR12 shows improved survival and TGI after the last dose of treatment compared to single agent treatment (see FIG. 48).

[0553] (Example 41) Ex vivo validation of the ability of CD47 to block cellular uptake of SLNP-TR12

[0554] In another experiment, the ex vivo verification of CD47's ability to block cell uptake was confirmed using the following experiment(s). Briefly, as background, it is understood that SLNP-TR12 induces cytokine secretion in PBMC and splenocytes. In particular, it is understood that certain cell populations in PBMC and splenocytes express ligand for CD47. Therefore, the addition of CD47 to SLNP-TR12 formulation should reduce TR12 update and cytokine secretion.

[0555] To verify the blocking effect of CD47 mimetic peptides on blocking cellular renewal, PBMCs and splenocytes were isolated from Balb / c mice. 6 / ml and treated with SLNP-TR12, SLNP-TR12-47c, and SLNP-TR12-47d for 24 h. Cultures were harvested after 24 h and TNF-a and IFN-g cytokine secretion was measured using ELISA according to standard protocols.

[0556] The results show that compared with SLNP-TR12, the groups treated with SLNP-TR12-47c or SLNP-TR12 / 47d show low secretion of TNFα and IFNγ in both PBMC (Figure 49(A)) and splenocytes (Figure 49(B) and Figure 49(C)). Collectively, these results verify the uptake blocking effect of CD47 mimetic peptide. (See Figure 49).

[0557] (Example 42) Ex vivo validation of the immunomodulatory effect of SLNP-TR12 on tumor-infiltrating lymphocytes in Balb / C mice In another experiment, the immunomodulatory effect of SLNP-TR12 was confirmed using the following experiment(s). Briefly, Balb / C mice were treated with 2.0 mg / kg vehicle control or SLNP-TR12. Then, 4 hours after injection, the spleen was removed and splenocytes were isolated. The splenocytes were stained, washed, and the percentage (%) and mean fluorescence channel (MFI) of different immune cells (Figure 50(A)) were analyzed by flow cytometry using standard methods. The data presented show the population gated from viable cells and CD45+ cells.

[0558] The results show that treatment with SLNP-TR12 increased NK cells (Figure 50(B)), total T cells (Figure 50(C)), and cytotoxic T cells (Figure 50(D)). (See Figure 50).

[0559] (Example 43) In vitro validation of the ability of CD47 to block cellular uptake of SLNP-TR12 In another experiment, the ability of CD47 to block cell uptake was confirmed using the following experiment(s). Briefly, Raw-Blue reporter cell line was used. By way of background, it is understood that RawBlue™ expresses human TLR and NF-κB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. Thus, stimulation of these cells with TR12 can result in activation of NF-κB via TLR7 or TLR7 / 8, which can be measured by detecting SEAP levels. Since these cell lines are macrophages, they express a ligand for CD45. Thus, adding CD47 to SLNP-TR12 formulation should decrease the uptake of SLNP (SLNP-TR12) and decrease the secretion of SEAP.

[0560] Therefore, Raw-Blue cells were incubated with different concentrations of SLNP-TR12, SLNP-TR12-47c, or SLNP-TR12-47d. At 24 hours of incubation, TLR stimulation was assessed by measuring the level of SEAP optical density (OD) using QUANTI-Blue™ according to standard protocols. OD was normalized to the control (untreated) group using the appropriate method.

[0561] The results showed lower secretion of SEAP in the groups treated with SLNP-TR12-47c and SLNP-TR1247-d compared to SLNP-TR12 (see FIG. 51).

[0562] (Example 44) Human clinical trials for the treatment of human cancers using formulated and / or co-formulated nanocarriers with TLR prodrugs (e.g., TR12) In accordance with the present invention, nanocarriers formulated and / or co-formulated containing TLR prodrugs and / or TLR lipid moieties are used that specifically accumulate in tumor cells and are used in the treatment of certain tumors as well as other immunological disorders and / or other diseases. For each of these indications, two clinical approaches have been successfully pursued.

[0563] I.) Adjunctive Therapy: In adjunctive therapy, patients are treated with formulated and / or co-formulated nanocarriers containing TLR prodrugs (e.g., SLNP-TR12 or SLNP-TR13) in combination with chemotherapeutic drugs or pharmaceuticals or biopharmaceuticals or combinations thereof. Primary cancer targets are treated with the addition of formulated and / or co-formulated nanocarriers containing TLR prodrugs under standard protocols. Protocol designs address efficacy, as assessed by examples including, but not limited to, the following: reduction of tumor burden in primary or metastatic lesions, prolongation of progression-free survival, prolongation of overall survival, improvement of patient health, disease stabilization, and ability to reduce regular doses of standard chemotherapy and other biological agents. These dose reductions allow additional and / or extended treatment by reducing dose-related toxicity of chemotherapeutic or biological agents.

[0564] II.) Monotherapy: In connection with the use of formulated and / or co-formulated nanocarriers containing TLR prodrugs in monotherapy of tumors, the formulated and / or co-formulated nanocarriers containing TLR prodrugs (e.g., SLNP-TR12 or SLNP-TR13) are administered to patients without chemotherapeutic or pharmaceutical or biological agents. In one embodiment, monotherapy is performed clinically in terminal cancer patients with widespread metastatic disease. The protocol design addresses efficacy as assessed by examples including, but not limited to, the following: reduction in tumor burden of primary or metastatic lesions, prolonged progression-free survival, overall survival, improved patient health, disease stabilization, and the ability to reduce regular doses of standard chemotherapy and other biological agents.

[0565] Dosage Dosage regimens can be adjusted to provide the optimal desired response. For example, a single formulated and / or co-formulated nanocarrier (e.g., SLNP-TR12 or SLNP-TR13) containing a TLR prodrug can be administered by using several divided doses over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. "Unit dosage form" as used herein refers to a physically discrete unit suitable as a unitary dosage for a mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect together with the necessary pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are determined by and directly depend on (a) the unique characteristics of the formulated and / or co-formulated nanocarrier (e.g., SLNP-TR12 or SLNP-TR13) containing a TLR prodrug, (b) the individual mechanism of the combination compound, if any, (c) the specific therapeutic or prophylactic effect to be achieved, and (d) the inherent limitations in the technology of compounding such compounds to treat susceptibility in an individual.

[0566] Clinical Development Plan (CDP) The CDP follows and develops treatments using formulated and / or co-formulated nanocarriers containing TLR prodrugs (e.g., SLNP-TR12 or SLNP-TR13) in conjunction with adjunctive or monotherapy. The trial will first demonstrate safety and then confirm efficacy of repeated administration. The trial will be open-label comparing standard chemotherapy and / or current treatment standards in addition to formulated and / or co-formulated nanocarriers containing TLR prodrugs. As will be understood, one non-limiting criterion that can be utilized in conjunction with patient enrollment is the expression of TLRs in tumors, as determined by standard detection methods known in the art.

[0567] It is believed that any of the formulated and / or co-formulated nanocarriers or embodiments disclosed herein may have a satisfactory pharmacological profile and promising biopharmaceutical properties, such as a good toxicological profile, good metabolic and pharmacokinetic properties, solubility, and permeability. It is understood that determining appropriate biopharmaceutical properties is within the knowledge of one of ordinary skill in the art (e.g., determining cytotoxicity in cells, or inhibiting certain targets or channels to determine potential toxicity).

[0568] The present invention is not limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any functional equivalents are within the scope of the invention. Various modifications of the models, methods, and lifecycle methodologies of the invention, in addition to those described herein, will be apparent to those skilled in the art from the above description and teachings, and are likewise intended to be within the scope of the invention. Such modifications or other embodiments can be made without departing from the true scope and spirit of the invention.

[0569] [Table I]

[0570] [Table II]

[0571] [Table III] [Table 11]

Claims

1. (iv) a drug moiety, (v) a lipid moiety, and (vi) Consolidated Units ("LUs") wherein the drug moiety comprises a TLR agonist, the LU conjugates the drug moiety to the lipid moiety, and the drug moiety has the following chemical structure: 【Chemical 34】 2. A TLR prodrug composition comprising:

2. 2. The TLR prodrug of claim 1, wherein the lipid moiety comprises a lipid set forth in Table I.

3. 2. The TLR prodrug of claim 1, wherein the lipid moiety comprises a lipid set forth in Table III.

4. The TLR prodrug of claim 1 , wherein the lipid moiety comprises stearic acid.

5. The lipid portion comprises stearic acid and has the following chemical structure: 【Chemistry 35】 2. The TLR prodrug of claim 1, having the formula:

6. A nanocarrier comprising a TLR prodrug, which releases an active TLR agonist after cleavage of the LU.

7. The nanocarrier of claim 6, wherein the LU is a hydromethylcarbamate linker.

8. 7. The nanocarrier of claim 6, further comprising a helper lipid, wherein the helper lipid is listed in Table II.

9. The TLR prodrug comprises TR12 and has the following chemical structure: 【Chemical 36】 The nanocarrier of claim 6, having the formula:

10. The nanocarrier of claim 9, wherein the nanocarrier is a solid lipid nanoparticle (SLNP).

11. The SLNP of claim 10, wherein the TLR prodrug comprises TR12 and is designated SLNP-TR12.

12. The SLNP of claim 11, wherein the SLNP is further co-formulated with one or more immunomodulatory agents or lipid-prodrugs thereof, wherein the immunomodulatory agent is selected from the group consisting of immunogenic cell death-inducing chemotherapeutic agents, A2aR inhibitors, STING agonists, CTLA-4 inhibitors, IDO inhibitors, PD-1 / PD-L1 inhibitors, CD1D agonists, and / or prodrugs thereof.

13. The SLNP of claim 11, wherein the SLNP is further co-formulated with an ICD-inducing chemotherapy drug, and the ICD-inducing chemotherapy drug is selected from the group consisting of DOX, MTO, OXA, CP, bortezomib, carfilzomib, IC1, or paclitaxel.

14. The SLNP of claim 13, further comprising DOX.

15. A composition comprising a solid lipid nanoparticle (SLNP), the SLNP further comprising TR12 co-formulated with IC1 (denoted as SLNP-TR12-IC1).

16. The composition of claim 15, co-formulated with IC1 in a ratio of 8:1 (designated NTI-121).

17. 16. The composition of claim 15, co-formulated with IC1 in a ratio of 16:

1.

18. A kit comprising the composition of claim 15.

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