Immunomodulatory nanoparticles

Abstract

There is provided pharmaceutical compositions comprising disulfiram (DSF) or copper di-ethyldithiocarbamate (CuET), an indirubin compound such as 6-bromo-indirubin-3'-oxime (BIO), and a stabilizer, wherein the disulfiram or CuET, the indirubin compound, and the stabilizer are associated with a lipid nanoparticle. The pharmaceutical compositions are particularly suitable for treating a cancer overexpressing p97 and NPLOC4.

Description

IMMUNOMODULATORY NANOPARTICLESCROSS-REFERENCE TO A RELATED APPLICATION

[0001] The present disclosure claims priority from U.S. provisional application number 63 / 636,082 filed on April 18, 2024 which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to cancer immunotherapy and, more particularly, to particles and nanoplexes for performing same.BACKGROUND

[0003] Cancer remains one of the leading causes of death to this date and can be broadly classified into hematological or solid malignancies. In contrast to hematological malignancies, solid malignancies (also referred to as solid tumors) can be difficult to treat in part due to the complexity of the tumor microenvironment (TME), which contains several characteristics that significantly limit the therapeutic efficacy of drug and cell therapies.

[0004] The TME is a complex collection of cells and molecular products that have profound implications in disease progression, treatment resistance, and metastasis. Typically, the TME contains multiple immune and stromal cells, such as tumor associated macrophages and cancer-associated fibroblasts, which have been significantly reprogrammed by mutated cancer cells to promote cancer survival and development. While the TME is highly heterogeneous, depending on the cancer type and anatomical location of the tumor, common TME features include the presence of an acidic microenvironment due to an overactive metabolism, chronic inflammation leading to the recruitment of immunosuppressive cells, such as myeloid-derived suppressor cells (MDSC) or regulatory T cells (Treg), as well as the upregulation of immune checkpoint receptors (e.g., PD-L1 , CTLA-4) involved in the process of immunosuppression. Additionally, in some cancers, the excessive deposition of extracellular matrix, and the secretion of proteases and immunosuppressive enzymes may limit the penetration and activity of drugs or infiltrating lymphocytes thereby reducing their therapeutic efficacy.

[0005] The aberrant vascular and lymphatic systems in the TME underline yet another aspect that may contribute to therapeutic resistance. As physiological blood circulation,and therefore tissue oxygenation, is reduced, it leads to pronounced hypoxia in poorly vascularized but highly proliferative tumor regions. Some of these alterations may result in the development of metastatic niches that promote the colonization of cancer cells in other tissues, such as the lymph nodes. The establishment of lymph node tumor metastases induces cancer immune tolerance and facilitates the dissemination of cancer cells to more distant sites for colonization of other organ systems. In some cases, tumors also exhibit an elevated interstitial pressure because of dysfunctional lymphatic drainage, further exacerbating the limited diffusion rate of drugs and reduced infiltration of anti-cancer immune cells.

[0006] The heterogeneous nature of the tumor vasculature, as well as other characteristics of the TME, such as higher acidity, hypoxia, and immunosuppression, are features that can be exploited by drug delivery systems to improve the therapeutic action of anti- neoplastic therapies. More specifically, nanotechnology has an important role to play in this context as it can address some of the limitations set forth by the TME with respect to current freely soluble drugs and cell therapies. The Enhanced Permeation and Retention (EPR) effect is a well-documented phenomenon that is due to irregular angiogenesis occurring within solid tumors, whereby larger macromolecules and nanoparticles are preferentially retained in some parts of the tumor tissue. The EPR effect is believed to occur due to the presence of gaps in the endothelium of blood vessels, allowing nano-sized particles to diffuse and accumulate in the tumor. However, recent evidence also suggests that many tumors have lower vascular permeability to nanoparticles due to the presence of a largely continuous endothelium with very few gaps. In such cases, nanoparticle transport into the tumor is generally mediated by active transport mechanisms across the endothelium, rather than passive accumulation. This can be exploited by targeted delivery of nanoparticles for active transport. Lipid-based nanoparticles are relevant to deliver therapeutic molecules to solid tumors due to their biocompatibility, versatility, and previous success in reducing the systemic toxicity of chemotherapy without affecting its efficacy.

[0007] Some nanotherapeutics are under investigation as cancer treatments. However, the limitations of the approaches to date are that they fail to address the complex interactions between nanoparticle drug carriers and the various competing tissue barriers that prevent the significant accumulation and delivery of therapeutics to tumors to generate a potent immune response. Many nanoparticles having a diameter larger than 10 nm end up being sequestered by immune cells in organs like the liver or spleen. Additionally, thesenanotherapeutics do not address the current limitations with respect to the tumor microenvironment and the complex interplay between cancer cells and immune cells.

[0008] Accordingly, there remains a need for developing improved immunotherapies that address the above limitations.SUMMARY

[0009] In one aspect, there is provided pharmaceutical composition comprising disulfiram, an indirubin compound, and a stabilizer, wherein the disulfiram and the indirubin compound are associated with a lipid nanoparticle.

[0010] In some embodiments, the indirubin compound is of formula

[0011] wherein Ri is H or CH3, each of R2, R3, and R4 are independently selected from H, Br, I, Cl and F, and Rs is selected from =0, =NOH, =NOCOCH3 or =NOCH3.

[0012] In some embodiments, the indirubin compound 6-bromo-indirubin-3’-oxime (BIO).

[0013] In some embodiments, a molar ratio of disulfiram to the indirubin compound is from 10:1 to 1:10.

[0014] In some embodiments, the composition further comprises a metal ion associated with the lipid nanoparticle.

[0015] In some embodiments, the stabilizer is of formula

[0016] wherein R is vinyl or an amine endcap and n is an integer of from 2 to 20.

[0017] In some embodiments, the stabilizer is selected from a pyrrolidone polymer, polysorbate (Tween™), sorbitan monooleate (SPAN™) and combinations thereof.

[0018] In some embodiments, the metal ion is copper.

[0019] In some embodiments, the nanoparticle is a liposome having a size of from 10 nm to 1000 nm.

[0020] In some embodiments, the lipid nanoparticle has a loading capacity of from 1 to 5 %.

[0021] In a further aspect, there is provided a pharmaceutical composition comprising metal chelate copper diethyldithiocarbamate (CuET) and an indirubin compound such as 6-bromo-indirubin-3’-oxime (BIO) in a molar ratio of from about 1:1 to about 1 :10, and a stabilizer, and wherein the CuET, the indirubin compound and the pharmaceutically acceptable excipient are associated with a lipid nanoparticle.

[0022] In some embodiments, the lipid nanoparticle has a size of from about 10 to about 1000 nm.

[0023] In some embodiments, the CuET and the indirubin compound are provided in the lipid nanoparticle at a loading capacity of from about 1 to about 5 %.

[0024] In some embodiments, the stabilizer is selected from a pyrrolidone polymer, polysorbate (Tween™), sorbitan monooleate (SPAN™) and combinations thereof.

[0025] In some embodiments, the stabilizer is of formula

[0026] wherein R is vinyl or an amine endcap and n is an integer of from 2 to 20.

[0027] In a further aspect, there is provided the use of the pharmaceutical composition as defined herein, for treating a cancer characterized by an overexpression of p97 and / or NPLOC4.

[0028] In a further aspect, there is provided a method of treating a cancer characterized by an overexpression of p97 and / or NPLOC4, the method comprising administering to a subject in need thereof the pharmaceutical composition as defined herein.

[0029] In a further aspect, there is provided the use of disulfiram and an indirubin compound for treating a cancer characterized by an overexpression of p97 and / or NPLOC4. The indirubin compound is for example 6-bromo-indirubin-3’-oxime (BIO).

[0030] In a further aspect, there is provided method of treating a cancer characterized by an overexpression of p97 and / or NPLOC4, the method comprising administering to a subject in need thereof disulfiram and an indirubin compound. The indirubin compound is for example 6-bromo-indirubin-3’-oxime (BIO).

[0031] There is provided a polymer of formula:

[0032] wherein R is vinyl or an amine endcap and n is an integer of from 2 to 20.

[0033] The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

[0034] All analyses are performed using appropriate statistical tests on groups where experiments are conducted in biological and technical triplicates, where appropriate.DESCRIPTION OF THE DRAWINGS

[0035] Reference is now made to the accompanying figures in which:

[0036] FIGs. 1A-1F show graphs of the dose-response (IC50) curves of various cancer cells treated with 6-bromo-indirubin-3’-oxime (BIO), copper diethyldithiocarbamate (CuET), or the combination of BIO and CuET (BC); data is presented as mean ± standard deviation (SD), n=4. FIG. 1A: IC50 curves of four mouse melanoma cell lines treated with varying concentrations of BIO. FIG. 1B: IC50 curves of four mouse melanoma cell lines treated with varying concentrations of BIO. FIG. 1C: IC50 curves of four mouse melanoma cell lines treated with varying concentrations of CuET. FIG. 1D: IC50 curves of four human melanoma cell lines treated with varying concentrations of CuET. FIG. 1E: IC50 curves of four mouse melanoma cell lines treated with varying concentrations of BC at a molar ratio according to Table 1. FIG. 1F: IC50 curves of four human melanoma cell lines treated with varying concentrations of BC at a specific molar ratio according to Table 1.

[0037] FIG. 2 is a graph that shows a comparison of the IC50 values in melanoma cell lines treated with BIO or BC expressed in terms of the concentration of BIO; data is presented as mean ± SD, n=4.

[0038] FIG. 3A shows a synergy map of BIO and DSF in B16F10 cells calculated according to the zero-interaction potency (ZIP) model.

[0039] FIG. 3B shows a synergy map of BIO and DSF in B16F10 cells calculated according to the highest single agent (HSA) model.

[0040] FIG. 4A shows the synergy map of BIO and DSF in COLO 205 cells calculated according to the zero-interaction potency (ZIP) model.

[0041] FIG. 4B shows the synergy map of BIO and DSF in COLO 205 cells calculated according to the highest single agent (HSA) model.

[0042] FIG. 5A shows the synergy map of BIO and DSF in PANC-1 cells calculated according to the zero-interaction potency (ZIP) model.

[0043] FIG. 5B shows the synergy map of BIO and DSF in PANC-1 cells calculated according to the highest single agent (HSA) model.

[0044] FIG. 6A shows the synergy map of BIO and DSF in A375 cells calculated according to the zero-interaction potency (ZIP) model.

[0045] FIG. 6B shows the synergy map of BIO and DSF in A375 cells calculated according to the highest single agent (HSA) model.

[0046] FIG. 7A shows the synergy map of BIO and bortezomib (BOR) in COLO 205 cells calculated according to the zero-interaction potency (ZIP) model.

[0047] FIG. 7B shows the synergy map of BIO and bortezomib (BOR) in COLO 205 cells calculated according to the highest single agent (HSA) model.

[0048] FIG. 8A shows the dose-response curves of A375 cells when treated with SN38 alone or in combination with BIO; data is presented as mean ± SD, n=3.

[0049] FIG. 8B shows the dose-response curves of A375 cells when treated with Mitomycin C alone or in combination with BIO; data is presented as mean ± SD, n=3.

[0050] FIG. 9A is a western blot of p-catenin, where total proteins were extracted from A375 cells treated with BIO, CuET, or various concentrations of the combination of BIO and CuET at a 1 :1 molar ratio.

[0051] FIG. 9B is a western blot of c-myc, where total proteins were extracted from A375 cells treated with BIO, CuET, or various concentrations of the combination of BIO and CuET at a 1:1 molar ratio.

[0052] FIG. 10A shows immunofluorescence (IF) images of cancer cells staining for p- catenin. Scale bar is 20 pm. IF images of fixed A375 cells treated with BIO, CuET, or the combination of BIO and CuET at a 1:1 molar ratio.

[0053] FIG. 10B shows immunofluorescence (IF) images of cancer cells staining for p- catenin. Scale bar is 20 pm. IF images of fixed A375 and B16F10 cells treated with BIO, DSF, or the combination of BIO and DSF + 1 pM CuSO4 at a 1:1 molar ratio.

[0054] FIGs. 11 A - 11 F show the formulation and characterization of polymer-stabilized lipid nanoparticles comprising BIO or BIO and CuET at a 1 :1 molar ratio. FIG. 11 A: Schematic outlining the components and the assembly of the lipid-polymer nanoparticle (LPN) formulation. FIG. 11B: Pictures of solutions formulated with various polymeric excipientsaccording to Table 7 showing the stability of the LPN formulation encapsulating BIO, and BIO with CuET. FIG. 11C: Transmission electron microscopy (TEM) image showing the morphology and size of a single LPN stabilized with polyvinylpyrrolidone (PVP) encapsulating BIO and CuET. Scale bar is 100 nm. FIG. 11D: Nanoparticle tracking analysis (NTA) graph showing the size distribution of the PVP-stabilized LPN encapsulating BIO and CuET in water. FIG. 11E: Aggregation kinetics of lipid nanoparticles formulated with or without PVP as a stabilizing excipient encapsulating BIO and CuET. FIG. 11 F: Stability of LPN stabilized with PVP and encapsulating BIO and CuET in human plasma over time.

[0055] FIGs. 12A -12F show absorption spectra. FIG. 12A shows the spectra of CuET, FIG. 12B shows the spectra of CuET BIO, and FIG. 12C shows the spectra of CuET the combination at a 1 :1 ratio dissolved in DMSO at various concentrations ranging from 100- 25 .g / mL. FIG. 12D is a standard curve of absorbance at 500 nm plotted against concentration in a solution of BIO and CuET at a 1 :1 ratio. FIGs. 12E -12F show the change in the absorbance spectra of various solutions of CuET and BIO at 1 : 1 , 1 :2, 1 :4, 1 :5 and 1 :10 ratios of CuET: BIO dissolved in DMSO (FIG. 12E) or in water as lipid nanoparticles (LPNs) (FIG. 12F).

[0056] FIG. 13 shows a nuclear magnetic resonance (NMR) spectrum of a solution of BIO in DMSO-de with or without the presence of PVP.

[0057] FIG. 14A and FIG. 14B shows high-performance liquid chromatography (HPLC) graphs of dissolved LPNs. FIG. 14A: HPLC graphs of LPNs encapsulating either BIO or CuET compared to control. FIG. 14B: HPLC graphs of dissolved lipid nanoparticles encapsulating BIO and CuET at various BIO:CuET molar ratios.

[0058] FIG. 15A - FIG. 15D show dynamic light scattering (DLS) size distribution graphs of nanoparticles formulated with BIO and poly p-amino ester (PBAE) polymers. FIG. 15A: DLS graph showing the size distribution of nanoparticles formulated with BIO and C32 polymer. FIG. 15B: DLS graph showing the size distribution of nanoparticles formulated with BIO, C32 polymer, and lipids. FIG. 15C: DLS graph showing the size distribution of nanoparticles formulated with BIO and C88 polymer. FIG. 15D: DLS graph showing the size distribution of nanoparticles formulated with BIO, C88 polymer, and lipids.

[0059] FIG. 16A shows DLS size distribution graphs of lipid nanoparticles formulated with polysorbate 20 as stabilizer.

[0060] FIG. 16B shows DLS size distribution graphs of lipid nanoparticles formulated with sorbitan monooleate 80 as stabilizer.

[0061] FIG. 17 shows fluorescent microscopy images of A375 cells treated with LPN stabilized with PVP containing BIO and CuET at a 1:1 molar ratio (LPBC) tagged with sulforhodamine 101. Scale bar is 100 pm.

[0062] FIG. 18A shows a IC50 curve of mouse melanoma cell lines treated with varying concentrations of LPBC; data is presented as mean ± SD, n=4.

[0063] FIG. 18B shows a IC50 curve of human melanoma cell lines treated with varying concentrations of LPBC; data is presented as mean ± SD, n=4.

[0064] FIG. 19 shows an invasion-migration assay across a collagen membrane of various human melanoma cell lines treated with vehicle control or 1 pM LPBC. Scale bar is 500 pm.

[0065] FIG. 20 shows a clonogenic assay of human melanoma cell lines treated with vehicle control, 1 pM or 2 pM LPBC.

[0066] FIGs. 21A - 21C show an XTT cell viability assay on YUMM1.7 and RPMI7951 spheroids. FIG. 21 A: Viability graph of spheroids treated with an increasing concentration of LPBC; data is presented as mean ± SD, n=3. FIG. 21 B: Representative light microscopy images of control and FIG. 21 C: LPBC treated YUMM1.7 spheroids. Scale bar is 200 pm.

[0067] FIGs. 22A - 22B show the acute toxicity of lipid nanoparticle formulations in healthy mice; data is shown as mean ± standard error from the mean (SEM), n=4. FIG. 22A: Change in mouse weight upon intravenous (IV) treatment of healthy mice with LPBC for two weeks every other day at concentrations of 2-8 mg / kg compared to mice treated with empty vector control. FIG. 22B: Change in mouse weight upon intraperitoneal (IP) treatment of healthy mice with lipid polymer nanoparticles comprising BIO and DSF at a 1:1 molar ratio (LPBD) for one week every other day at concentrations of 10 and 20 mg / kg

[0068] FIGs. 23A - 23B show the organ biodistribution profile of IV injected fluorescent LPBC. FIG. 23A: Ex vivo organ fluorescence imaging of major mouse organs explanted 24h after IV injection of fluorescent LPBC. FIG. 23B: Quantification of fluorescenceintensity of explanted organs; Welsch’s One-Way ANOVA with Dunnett’s 3T correction was used, circles represent individual mice, and data is shown as mean ± SD.

[0069] FIGs. 24A -24C show the in vivo efficacy of vehicle, lipid-polymer nanoparticles containing BIO (LPBIO), lipid polymer nanoparticles containing CuET (LPCuET), and LPBC in a subcutaneous B16F10 mouse model of melanoma. FIG. 23A: Schematic of the experimental setup and mouse treatment schedule. FIG. 23B: Tumor growth kinetics of treated mice bearing subcutaneous B16F10 tumors; Welsch’s One-Way ANOVA with Dunnett’s 3T correction was used at day 16; data shown as mean ± SEM. FIG. 23C: Difference in tumor weight between the treatment groups at endpoint; Welsch’s One-Way ANOVA with Dunnett’s 3T correction was used; empty circles represent individual mice, and data shown as mean ± SD.

[0070] FIGs. 25A - 25B show the in vivo efficacy of LPBC compared to vehicle and standard of care immune checkpoint inhibitors (ICI) in treating established B16F10 lung metastases. FIG. 25A: Representative mouse lung pictures showing the extent of tumor burden in vehicle and LPBC-treated mice; black nodules are individual tumors. FIG. 25B: Number of lung nodules counted on the lung surface at endpoint for vehicle (n=12), ICI (n=5), and LPBC (n=8); Welsch’s One-Way ANOVA with Dunnett’s 3T correction was used; empty circles represent individual mice, and data was pooled from two separate experiments and is shown as mean ± SD.

[0071] FIGs. 26A - FIG 26C show the in vivo efficacy of vehicle, LPBIO, LPCuET, and LPBC in a subcutaneous YUMM1 .7 mouse model of melanoma. FIG. 26A: Schematic of the experimental setup and mouse treatment schedule. FIG. 26B: Tumor growth kinetics of treated mice bearing subcutaneous YUMM1.7 tumors treated with LPBC compared to the other groups; Welsch’s One-Way ANOVA with Dunnett’s 3T correction was used at day 24; data pooled from two separate experiments shown as mean ± SEM. FIG. 26C: Kaplan-Meier survival analysis showing the increased survival of mice treated with vehicle compared to LPBC at a dose of 3 mg / kg; the log-rank test was used, n=10.

[0072] FIGs. 27A - FIG. 27L show immunohistochemistry (IHC) images of fixed YUMM1.7 tumors from mice treated with vehicle (FIGs. 27A, 27E and 27I), LPBIO (FIGs. 27B, 27F and 27J), LPCuET (FIGs. 27C, 27G and 27K), and LPBC (FIGs. 27D, 27H and27L) with staining for (FIGs. 27A-27D) p-catenin, cleaved caspase 3 (FIGs. 27E-27H), and Ki67 (FIGs. 27I-27L). Scale bar is 200 pm.

[0073] FIG. 28 shows the tumor volume of mice treated with vehicle control, lipid polymer nanoparticles comprising DSF (LPDSF), LPBIO, and LPBD in mice bearing subcutaneous MC38 tumors; Welsch’s One-Way ANOVA with Dunnett’s 3T correction was used; data is shown as mean ± SEM.

[0074] FIG. 29A - FIG. 290 show the serum concentration of (FIG. 29A) I FNy, (FIG. 29B) IL5, (FIG. 29C) IL6, (FIG. 29D) IL10, (FIG. 29E) CXCL2, (FIG. 29F) GM-CSF, (FIG. 29G) IL4, (FIG. 29H) IL7, (FIG. 29I) IL12p70, (FIG. 29J) IL13, (FIG. 29K) IL17A, (FIG. 29L) CXCL1 , (FIG. 29M) CXCL5, (FIG. 29N) CCL2, and (FIG. 290) TNFa cytokines and chem- okines in B16F10 mice after 24h of IV treatment with 3mg / kg of vehicle, LPBIO, LPCuET, or LPBC 24h; One-Way ANOVA with Dunnett’s 3T correction was used; empty circles represent individual mice and data is shown as mean.

[0075] FIG. 30 shows a heat map outlining the difference in the expression of select cytokine and chemokine mRNA extracted from subcutaneous B16F10 tumors of mice treated with LPBIO, LPCuET, and LPBC; data depicted as the geometric mean of the fold change in gene expression (2'AACt) from vehicle-treated mice.

[0076] FIGs. 31 A - 31 B show the effect of the drug-loaded LPNs on the metastatic spread and viability of treated B16F10 cells. FIG. 31A: Picture of mouse lungs harboring metastatic B16F10 lesions. FIG. 31B: the lesion count in mice injected IV with control B16F10 cells compared to B16F10 injected alongside 3 mg / kg LPBC, or B16F10 cells pre-treated with 1 pM and 5 pM of LPBC for 3h before IV injection; data is shown as mean ± SD, black nodules represent individual tumors, and empty circles represent individual mice. FIG. 31C: The viability of B16F10 cells pre-treated with 1 pM for 3 h. FIG. 31D: The viability of B16F10 cells pre-treated with 5 pM of LPBC for 3h.

[0077] FIGs. 32A - 32B show the differential fold change in the gene expression profile of surface receptors relevant for natural kill (NK) cell recognition in FIG. 32A: B16F10 cells and FIG. 32B: A375 cells; empty circles represent individual experiments and data is shown as geometric mean ± SD.

[0078] FIGs. 33A - 33B show the increase in the proportion of FIG. 31 A: NKp44L+ B16F10 cells and FIG. 31 B: LILBP1 + A375 cells when treated with LPBC; student’s t-test with Welch’s correction was used, empty circles represent individual experiments, and data is shown as mean.

[0079] FIG. 34A shows the proportion and FIG. 34B shows the mean fluorescence intensity (MFI) of CD69+ Jurkat cells when activated in the presence of BIO, CuET or the combination at a 1 : 1 molar ratio compared to vehicle control; Welsch’s One-Way ANOVA with Dunnett’s 3T correction was used; empty circles represent individual experiments and data is shown as mean.

[0080] FIG. 35A shows the proportion and FIG. 35B mean fluorescence intensity (MFI) of PD1+ Jurkat cells when activated in the presence of BIO, CuET or the combination at a 1 :1 molar ratio compared to vehicle control; Welsch’s One-Way ANOVA with Dunnett’s 3T correction was used; empty circles represent individual experiments and data is shown as mean.

[0081] FIG. 36 shows the IL2 secretion profile of Jurkat cells activated in the presence of vehicle, LPBIO, LPCuET, and LPBC; circles represent individual experiments and data is shown as mean.DETAILED DESCRIPTION

[0082] Many cancer types are associated with an overexpression of p97, also known as valosin-containing protein (VCP), and / or NPLOC4, such as lung cancer, breast cancer, colorectal cancer, gastric cancer, bladder cancer, pancreatic cancer, esophageal cancer, liver cancer, prostate cancer, bone cancer, head and neck cancer, thyroid cancer, hematological cancer, melanoma, glioblastoma, ovarian cancer, testicular cancer, and squamous cell carcinoma (Costantini S, Capone F, Polo A, Bagnara P, Budillon A. Valosin- Containing Protein (VCP) / p97: A Prognostic Biomarker and Therapeutic Target in Cancer. Int J Mol Sci. 2021 Sep 21 ;22(18):10177. doi: 10.3390 / ijms221810177. PMID: 34576340; PMCID: PMC8469696). There is provided herein a synergistic combination of molecules to achieve inhibition of the p97-UFD1-NPL4 protein complex to exacerbate endoplasmic reticulum (ER) stress and induce cytotoxicity in cancer cells. NPLOC4 codes for NPL4 which forms the p97-UFD1-NPL4 complex with p97 involved in shuttling misfolded proteins from the ER to the proteosome for degradation. In preferred embodiments, thecancer treated by the present methods and compositions overexpresses both p97 and NPLOC4 such that the 97-UFD1-NPL4 protein complex is formed.

[0083] To take advantage of the increased levels of ER stress that is generated inside the tumour microenvironment (TME) compared to homeostatic levels, as a therapeutic strategy, the metal chelate copper diethyldithiocarbamate (CuET), or its precursor disulfiram (DSF), are leveraged as potent inhibitors of the p97-UFD1-NPL4 protein complex. CuET or DSF are used as the first part of the combination to exacerbate ER stress and induce cytotoxicity in cancer cells. CuET and / or DSF are combined with an indirubin compound as an immunomodulatory and anti-inflammatory drug. The indirubin compound is generally of the following formula

[0084] Ri is H or CH3, each of R2, R3, and R4 are independently selected from H, Br, I, Cl and F, and Rs is selected from =0, =NOH, =NOCOCH3 or =NOCH3. The indirubin compound is for example 6-bromo-indirubin-3’-oxime (BIO). Some indirubins are potent glycogen synthase kinase-3 (GSK3) inhibitors and aryl hydrocarbon receptor (AHR) agonists, to prevent and / or modulate aberrant inflammation inside the TME as a result of CuET and / or DSF cytotoxicity or ER stress.

[0085] Since all of DSF, CuET and the indirubin compound are highly hydrophobic and exhibit poor bioavailability, an appropriate carrier is required to properly deliver this combination to the TME. It was presently found that a lipid nanoparticle coupled with a stabilizer formed an advantageous vehicle that is stable and delivers the combination to the TME.

[0086] There is therefore provided pharmaceutical compositions comprising a lipid nanoparticle that associates with the aforementioned drugs (i.e. disulfiram, the indirubin compound, and CuET). The association is stabilized by the stabilizer. The term associated means that there is a physical and / or chemical linkage between the lipid nanoparticle and the drugs and stabilizer. Although it is most likely that the lipid nanoparticle encapsulates at least a portion or a majority of the drugs and stabilizer, it is also possible they associate at least partially at the surface of the lipid nanoparticles. However, for simplicity, the present disclosure will make reference to encapsulation, however, the skilled person will appreciate that what is meant is association, including association with the surface of the nanoparticle.

[0087] Lipid nanoparticles are generally constructed with phospholipids or derivatives thereof which provide desirable properties for the treatment of cancers. The lipid nanoparticle is optionally modified with polyethylene glycol (PEG) to prolong circulation half-life. The term lipid nanoparticle can generally refer to a size of a size of from 10 nm to 1000 nm, from 10 nm to 500 nm, from 10 nm to 200 nm or from 10 nm to 100 nm. The lipid nanoparticle can be a liposome, a solid lipid nanoparticle, or a lipid-polymer hybrid. Liposomes have at least one phospholipid bilayer. Solid lipid nanoparticles have a solid lipid core made of, for example, glycerides, fatty acids, sterols, or wax type molecules that are surface stabilized by emulsifiers. The lipid-polymer hybrid particles have a polymeric component, typically as the core of the particle, that is surrounded by a lipid monolayer that can act as a surfactant. The lipid monolayer acts as the encapsulation that retains the drugs and prevents them from freely diffusing out.

[0088] The loading capacity is the amount of drug loaded per unit weight of the nanoparticle, indicating the percentage of mass of the nanoparticle that is due to the encapsulated drug. The present lipid nanoparticles have a loading capacity for the drugs (disulfiram and indirubin, CuET and indirubin, or for all three) of from 1 to 5 %, from 2 to 5%, from 3 to 5%, or from 4 to 5%. It was presently found that a 5% loading was the maximal loading capacity while still maintain proper stability (even with the stabilizer). Increasing the loading is desired to make delivery more efficient, and a loading of 4% or more was presently advantageously achieved.

[0089] The stabilizer is a species that creates a link at the interface between the medium inside the lipid nanoparticle and the encapsulated contents (or associated contents). Thestabilizer is preferably a surfactant or a polymer that may act similarly to a surfactant in this context. The surfactant properties of the stabilizer allow for the stabilization inside the lipid nanoparticle. The surfactant is preferably a lipid surfactant such as a phospholipid or other similar chemical species with a long hydrophobic chain coupled with a polar head. The polymer is preferably a pyrrolidone polymer (i.e. , a polymer comprising repeating pyrrolidone groups). However, other polymers are also contemplated other than pyrrolidone polymers such as C32. One example of a pyrrolidone polymer is polyvinylpyrrolidone (PVP), but other examples include C88 and Cp1. It should be understood that derivatives thereof are also suitable.

[0090] R represents a vinyl group, or an endcap amine group. N is an integer of from 2 to 20. The polymers may be branched or linear, capped or uncapped. Branching may be achieved using trimethylolpropane triacrylate, or other branching groups.

[0091] In some embodiments, repeating units comprise one of the following pyrrolidone groups:

[0092] In some embodiments, the pyrrolidone polymer is of one of the following formulas:

[0093] wherein R represents a vinyl group, or an endcap amine group, X is selected from substituted or unsubstituted C1-C10 alkyl, Ce-Cw aryl, C4-C10 heteroaryl or C3-C10 cycloalkyl, and Y is selected from H, substituted or unsubstituted C1-C10 alkyl, Ce-Cw aryl, C4-C heteroaryl or Cs-Cw cycloalkyl.

[0094] It should be noted that the pyrrolidone polymer could be a homopolymer, a copolymer or a crosslinked polymer as long as the pyrrolidone group remains sterically available. The stabilizer is preferably a pyrrolidone polymer, polysorbate (Tween™), sorbitan monooleate (SPAN™) and combinations thereof. Some particular examples but are not limited to PVP, Tween™ 20 and SPAN™ 80.

[0095] The susceptibility of cancer cells to proteotoxic stress is in part driven by their oncogenic transformation, which results in more strain being put on the growth and replication machinery of the cell, as well as the TME, which can be inhospitable to rapidly dividing cells due to the presence of hypoxia, inflammation, and metabolic constraints. The TME composition changes over time, resulting in the recruitment of stromal, endothelial and immune cells that are reprogrammed by cancer cells to support cancer development and growth. Importantly, the presence of aberrant inflammation inside the TME, often driven by pro-inflammatory cytokines like IL-6 as a result of cytotoxic drug treatment, represents an important driver of adaptive ER stress leading to acquired immunosuppression and resistance to cytotoxic drugs in cancer cells. Additionally, chromosomal instability (Cl), a hallmark of highly mutated and aggressive types of cancer like melanoma, can drive inflammation inside the TME in a more chronic manner via the presence of extra-nuclear DNA from poorly segregated chromosomes that activate the STING pathway. The chronic induction of STING-mediated inflammation can drastically alter the TME resulting in cancers that are notoriously metastatic and that can rapidly evolve resistance to immunotherapies.

[0096] The present disclosure shows that surprisingly, the induction of proteotoxic stress in cells can dampen the nuclear localization and signaling of p-catenin, which is considered an important limitation when using GSK3 inhibitors to treat cancer, p-catenin signaling may drive cancer cell survival, proliferation, and sternness, and is often associated with poor prognosis in many human malignancies, such as colorectal cancer. Cells treated with CuET or DSF, showed lower levels of p-catenin, and its nuclear localization was reduced, resulting in the reduction of p-catenin signaling during GSK3 inhibition which is a therapeutic modality in oncology.

[0097] Indirubin compounds such as 6-bromo-indirubin-3’-oxime (BIO), are GSK3 and tyrosine kinase 2 (TYK2) inhibitors, and can be leveraged as modulators of the inflammatory response in the TME. While TYK-2 is a tyrosine kinase that is clinically validated for the treatment of inflammatory diseases mainly driven by IL-10, IL-12, IL-23, and IFNa / p signaling, GSK3 is a more convoluted target due to its role in multiple cellular pathways. Nevertheless, GSK3, a serine / threonine kinase, has been shown to regulate NF-KB signaling via the NF-kappa-B essential modulator (NEMO), as well as through the interferon regulatory factor 3 (IRF3)-mediated transcription, which is involved in the STING signaling pathway. Moreover, GSK3 inhibition is considered a means for controlling inflammation,immune checkpoint receptors (e.g., PD1 and CTLA4) and ferroptosis, which have been shown to induce immunosuppression in the TME. Indirubin compounds are also AHR agonists. AHR is a transcription factor found in the cytoplasm of cells and is activated by binding to various ligands and endogenous compounds like tryptophan metabolites. Once activated, AHR translocates to the nucleus, dimerizes with the AHR nuclear translocator (ARNT), and regulates gene expression involved in inflammation and cell apoptosis.

[0098] A synergy has been demonstrated herein between the indirubin compound and CuET or DSF. The synergy with DSF instead of CuET was a surprising observation because DSF is less potent than CuET (individually). CuET is the active form of DSF in the body when the reduced form of DSF is coupled with available copper ions. Since the TME is generally rich in copper there may be no need to provide any copper supplement in the nanoparticle or as separate administration. It is optional to do so if there is enough copper in the TME. It may be beneficial to supplement copper in the nanoparticles or separately if the copper in the TME is not available to bind the reduced form of DSF. Preferred ratios between the active drugs have been determined.

[0099] In at least some embodiments, the ratio between the indirubin and CuET is from 2:1 to 1 :10, from 1.1 :1 to 1 :10, from 1.1 : 1 to 1 :5, from 1.1 :1 to 1 :2, from 1.1 :1 to 1 :1.1 or about 1 :1. Indeed, it was surprisingly found that a ratio close to 1 :1 or that has CuET in excess achieve synergy and low toxicity. Indeed, CuET or DSF should generally be administered in the range of 1 mg / kg to 10 mg / kg to avoid excess toxicity and providing an indirubin compound such as BIO at a dosage of more than 10 mg / kg may be too toxic.

[0100] In at least some embodiments, the ratio between the indirubin and DSF is from 1 :10 to 10:1 , from 1 :5 to 5:1 , from 1 :2 to 2:1 , from 2:3 to 3:2 or of about 1 :1. It was surprisingly found that disulfiram which is individual less potent than CuET can actually replace CuET and maintain synergy with the indirubin compound such as BIO.

[0101] The combination of the indirubin compound with both CuET and DSF has also been investigated, and the synergy is conserved. Accordingly, generally, the combination of the indirubin compound with either DSF or CuET provides increased levels of ER stress that is generated inside the TME. The synergistic combination is cytotoxic and reduces - catenin levels.

[0102] Many human cancers exhibiting aberrant p-catenin signaling through gain-of-func- tion mutations or the inactivation of the adenomatous polyposis coli (APC) protein, a tumor suppressor gene that regulates p-catenin signaling, exhibit an aggressive phenotype that is more resistant to various treatments, including immunotherapy. Given the role of GSK3 as part of the p-catenin destruction complex, consisting of AXIN-GSK3-APC, to mark p- catenin for proteasomal degradation, GSK3 inhibition by the indirubin compound results in a marked increase in p-catenin protein levels and singling in cells. Without wishing to be bound by theory, the mechanism of p-catenin modulation by the addition of CuET can be explained by its degradation which may be mediated by Siah1 / 2, as these ubiquitin ligases are upregulated during ER stress.

[0103] Indirubins, such as BIO, can become antagonistic even at low concentration of less than 100 nM. By blocking GSK3, BIO can induce p-catenin and c-myc signaling in cancer cells, increasing their resistance to apoptosis. The concentration of the indirubin compound is thus maintained close to the concentration of CuET and / or DSF in the body and TME to avoid amplification of survival pathways in cancer cells at sub-lethal doses of BIO (< 1 pM). This is done by providing enough nanoparticles which can have various loadings or concentrations inside.

[0104] Although the present disclosure has described the advantageous formulation of CuET and / or DSF with the indirubin compound as encapsulated in a lipid nanoparticle, there is still contemplated herein the separate administration of the drugs for effecting cancer treatment. For example, disulfiram and the indirubin compound such as BIO can be administered separately yet will still provide the synergistic effect at the TME as long as they are both provided with excipients that allow them to reach the TME.

[0105] In some embodiments, the treatment methods contemplated herein can be further combined with other therapies such as chemotherapy, radiation therapy, and immunotherapy, such as immune checkpoint blockade. The evolution of resistance in cancer populations is a major factor limiting patient remission and curing. One way to mitigate the development of resistance is to provide a combination therapy.

[0106] Administration is by any of the routes normally used for introducing an agent into ultimate contact with blood or tissue cells, such as oral or intravenous. A preferred administration route is intravenous which is enabled by the present formulation with the lipidnanoparticles and stabilizer. The agent described herein can be administered in any suitable manner, preferably with pharmaceutically acceptable carriers or excipients. The terms “pharmaceutically acceptable carrier”, “excipients” and “adjuvant” and “physiologically acceptable vehicle” and the like are to be understood as referring to an acceptable carrier or adjuvant that may be administered to a patient, together with the agent, and which does not destroy the pharmacological activity thereof. Further, as used herein "pharmaceutically acceptable carrier" or "pharmaceutical carrier" are known in the art. Additionally, such pharmaceutically acceptable carriers may be non-aqueous suspensions of the lipid nanoparticles.

[0107] As used herein, “pharmaceutical composition” means therapeutically effective amounts (dose) of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. A “therapeutically effective amount” as used herein in the context of the pharmaceutical composition refers to that amount which provides a therapeutic effect for a given condition and administration regimen.

[0108] Suitable methods of administering the agent are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. The preventive or therapeutic agents of the present invention may be administered, either orally or parenterally, systemically or locally. For example, intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppositories, intestinal lavage, oral enteric coated tablets, and the like can be selected, and the method of administration may be chosen, as appropriate, depending on the age and the conditions of the patient.Example 1Cellular Effects of BIO, CuET, and DSF

[0109] Cellular survival. Cellular survival was determined using the sulforhodamine B assay. Cancer cells were seeded in 96-well plates at a density of 3000-10000 cells per well and incubated overnight. The cells were treated at various concentrations with BIO, CuET, DSF, BOR, SN38, Mitomycin C, or the combination of BIO with CuET, DSF, BOR, SN38, Mitomycin C at various molar ratios for 48-72 h; all drugs were dissolved in DMSO andwere diluted from their stock concentrations prior to use. Cells were fixed with 50% trichloroacetic acid, stained with 0.4% suflorhodamine B, and resuspended in 10 mM TRIS buffer at a final volume of 200 pL per well. The optical density was measured at 492 nm using the SpectraMax™ i3 (Molecular Devices, San Jose, CA, USA). The data were fitted to a nonlinear regression model to obtain the IC50 values plotted as % control of DMSO-treated cells; all cells were treated with a total of 0.2% DMSO to control for the potential contribution of DMSO to cell toxicity. Drug synergy was calculated from dose-response matrices using the ZIP or HSA models of synergism using the application SynergyFinder+ (https: / / synergyfinder.org). Both the ZIP and HSA models are well established in the literature (Yadav B, Wennerberg K, Aittokallio T, Tang J. Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput Struct Bi- otechnol J. 2015 Sep 25;13:504-13. doi: 10.1016 / j.csbj.2015.09.001.).

[0110] Synergism of BIO and CuET. For this experiment, the B16F10 (RRID: CVCL_0159) cell line and a jak2 knockout (B16J2KO) were used since BIO was previously shown to cause cancer cell death via the inhibition of the JAK / STAT pathway. The YUMM1.7 (RRID: CVCL_JK16) cell line harboring driver mutations commonly found in humans, and its YUMMER1.7 counterpart with similar driver mutations, but with a higher mutagenic burden from being exposed to UV radiation were also used. For the human cell lines, well established A375 (RRID: CVCL_0132), Malme-3M (RRID: CVCL_1438), RPMI7951 (RRID: CVCL_1666), WM983B (RRID: CVCL_6809) with various driver mutations from both male and female patients were used. Before testing the combination of BIO and CuET on the cells, the IC50 values were obtained from individual dose-response curves of BIO and CuET, and the drug combination (BC) was added to cells at each of their respective IC50 molar concentration ratios according to Figs. 1A - 1 F.

[0111] There was a large difference between the sensitivity of B16F10 and YUMM1.7 cells to BIO (1.69 vs 6.62 pM) but not between B16F10 and B16J2KO (1.69 vs 1.28 pM) orYUMM1.7 and YUMMER1.7 (6.62 vs 7.19 pM). The sensitivity difference observed with BIO between the cell lines was not replicated in the CuET-treated group, wherein all mouse cell lines had narrower IC50 values ranging from 41.49 to 135.1 nM. The human lines exhibited broad sensitivity to BIO with IC50 values ranging from 0.84 to 3.59 pM, with narrower CuET sensitivity (41.49 to 92.93 nM). Since BIO inhibits multiple kinases (e.g., CDKs, JAKs) at micromolar concentrations, it is possible that the difference in kinase expression and activity can differ substantially between cell lines, which may explain thebroad toxicity range observed in the present experiments. Interestingly, such broad difference was not observed in the CuET group, likely due to a threshold effect whereby the accumulation of too many misfolded proteins and aggregates trigger cell death in the range of 40 to 140 nM.

[0112] To evaluate the combinatorial potential of CuET and BIO, the cells were treated with the two drugs at their respective IC50 values to test for toxicity in each cell line resulting in BIO:CuET ratios ranging from 10: 1 to 91 :1. The results show an additive effect in multiple cell lines marked by a significant sensitivity shift in the IC50 values for most cell lines as shown in Fig. 2. Because of the large difference in cell sensitivity between BIO and CuET (IC50 values in the nM vs pM range), the cytotoxicity synergism was not significant below a BIO:CuET ratio of 10:1 , calculated using the ZIP and HAS synergy models, but synergy may still be possible in other cancer cell lines, or melanoma lines that exhibit resistance to CuET. Moreover, there is a notable increase in cell proliferation for A375 and RPMI7893 cells when they are treated with a non-lethal CuET and BIO dose and when the ratio of BIO:CuET is above 1. To that end, the BIO concentration was kept as low as possible for in vivo studies, since large doses of BIO could be therapeutically restrictive (i.e. , outside the therapeutic index) at BIO:CuET concentration ratios larger than 10:1. The synergy results of BIO and CuET are succinctly summarized in Table 1.Table 1. IC50 values for melanoma cell lines treated with BIO, CuET and BC. For BC, the data is expressed in terms of the BIO concentration - the concentration of CuET can be calculated based on the combination ratio; data presented as mean ± standard deviation (SD).

[0113] Synergism of BIO and DSF. The following cell lines were used: B16F10, COLO 205 (RRID: CVCL_0218), PANC-1 (RRID: CVCL_0480), and A375. Cells were treated as part of a dose-response matrix as shown in Tables 2-5, which were used to calculate the ZIP and HSA synergy score at various drug combination ratios. In B16F10 cells, there was synergy between BIO and DSF at BIO concentrations of -1-10 pM and DSF concentrations of -0.1-100 pM, according to both the ZIP and HSA as shown in FIG. 3A and FIG. 3B. In COLO 205 cells, there was synergy between BIO and DSF at BIO concentrations of -0.01-10 pM, with the highest score at DSF concentrations of -0.1-0.001 pM, and lower scores at DSF concentrations below 10 pM, according to the ZIP model as shown in FIG. 4A. The HSA model shows synergy at BIO concentrations of -5-50 and pM -0.5-10 pM for both low (-0.1-0.01 pM) and high (-5-10 pM) DSF, respectively, as shown in FIG. 4B. In PANC-1 cells, there was synergy between BIO and DSF at BIO concentrations of -0.1- 10 pM and DSF concentrations of -0.5-20 pM as shown in FIG. 5A and FIG. 5B. In A375 cells, there was synergy between BIO and DSF at BIO concentrations of -0.01-50 pM and DSF concentrations of -0.5-50 pM, with the strongest synergy being at BIO concentrations of -1-50 pM (Figs. 6A-6B). The synergy experiments show that the combination of BIO and DSF is synergistic at various molar ratios depending on the tested cancer cell line, with most synergistic ratios falling within a BIO: DSF molar ratio of 10:1 to 1 :10. A person skilled in the art may understand that synergistic ratios outside of this range are also possible up to 100:1 to 1 : 100, depending on the cancer type.

[0114] Synergism of BIO and BOR. COLO 205 cells were treated as part of a dose-response matrix as shown in Table 6, which were used to calculate the ZIP and HSA synergy score at various drug combination ratios. There was synergy between BIO and BRO at BIO concentrations of -0.1-10 pM and BOR concentrations of -0.001-0.5 pM as shown in FIG. 7A and FIG. 7B. This outlines the possibility of combining BIO, or BIO and DSF / CuET, with BOR to treat various cancers that exhibit resistance to BOR, such as refractory multiple myeloma and solid tumors like colorectal adenocarcinoma.Table 2. Drug-response matrix for BIO and DSF in B16F10 cells. Data presented as % inhibition, n=3.BIO (pM)Table 3. Drug-response matrix for BIO and DSF in COLO 205 cells. Data presented as % inhibition, n=3.0 0.1 0.5 1 5 10BIO (pM)Table 4. Drug-response matrix for BIO and DSF in PANC-1 cells. Data presented as % inhibition, n=3.2015105DSF2(pM)1.510.50.200 1 5 10 50 100BIO (pM)Table 5. Drug-response matrix for BIO and DSF in A375 cells. Data presented as % inhibition, n=3.BIO (pM)0.50.10.050.0100 0.5 2.5 5 25 50DSF (pM)Table 6. Drug-response matrix for BIO and BOR in COLO 205 cells. Data presented as % inhibition, n=3.BIO (pM)

[0115] BIO sensitizes cancer cells to chemotherapy. A375 cells were treated with a range of concentrations of SN38 or Mitomycin C with or without 1 pM BIO. For bothchemotherapies, the viability of cancer cells with respect to untreated controls was lower in the presence of BIO compared to the chemotherapy alone, as shown in FIG. 8A and FIG. 8B, supporting the applicability of BIO, or BIO with DSF / CuET to improve the treatment of cancers that are resistant to chemotherapy, as melanoma is notoriously resistant to most forms of chemotherapy.

[0116] Western Blotting. A375 cells were seeded in 6-well plates at 3 x 105cells per well until confluent, and treated with 0.5 pM of BIO or CuET, or 0.5, 1 , 5, or 10 pM of BIO and CuET (1 :1 mol ratio) in DMSO for a duration of 5h. Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor and the lysates were centrifuged at 16,000 RCF for 10 min. Protein concentrations were measured using a Pierce™ BCA assay kit. Equal amounts of protein lysates (20 pg) were separated using a 4-20% Mini-PROTEAN® TGX™ Precast Protein Gel, 15-well, 15 pL sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blots were incubated with a primary antibody against GAPDH (clone 6C5, AM4300, Thermo Fisher, 3 pg / mL), a primary antibody against c-myc (clone 9E10, 14- 6784-82, eBioscience, 3 pg / mL), and a primary antibody against p-catenin (clone 15B8, 14-2567-82, Thermo Fisher, 5 pg / mL), both paired with a secondary goat anti-mouse HRP antibody (A16072, Invitrogen, 1 :5000). The blots were developed using Pierce™ ECL solution and imaged using the Bio-Rad ChemiDoc™ MP Imaging System. The BLLIeye™ pre-stained protein ladder was used as molecular weight reference.

[0117] The combination of BIO and CuET at a 1 :1 ratio was used to evaluate changes in P-catenin and c-myc levels using western blotting. Many human cancers exhibiting aberrant p-catenin signaling through gain-of- function mutations or the inactivation of the adenomatous polyposis coli (APC) protein, a tumor suppressor gene that regulates p-catenin signaling, may have an increase in c-myc transcription and demonstrate an aggressive phenotype that is more resistant to various treatments, including immunotherapy. Given the role of GSK3 as part of the p-catenin destruction complex, consisting of AXIN-GSK3- APC, to mark p-catenin for proteasomal degradation, GSK3 inhibition by BIO typically results in a marked increase in p-catenin and c-myc protein levels and singling in cells. As shown in FIG. 9A and FIG. 9B, the increase in p-catenin and c-myc levels may be reduced by the addition of CuET in a dose-dependent manner.

[0118] Immunocytochemistry imaging. A375 or B16F10 cells were seeded in a 35 mm glass-bottom petri dish at 3 x 105cells and treated with 0.5 pM of BIO, CuET, DSF or 1 :1 mol ratio of BIO and CuET or BIO and DSF for 24h. The cells were then washed with PBS, fixed with 4% paraformaldehyde (PFA), and permeabilized with 0.5% Triton X-100. The cells were incubated with a primary antibody against p-catenin (clone 15B8, 14-2567-82, Thermo Fisher, 3 pg / mL) and paired with a secondary goat anti-mouse Alexa Fluor™ 594 antibody (R37121 , Thermo Fisher, 1 :500). Cells were then stained with Hoechst 33342 to visualize the nucleus and imaged using the Zeiss LSM 800 confocal microscope. Laser intensity and gain were maintained constant during the imaging process for all samples. FIG. 10A and FIG. 10B show a noticeable increase in the content of p-catenin in both the cytoplasm and nucleus of BlO-treated cells, contributing to a phenotypical switch with more elongated cell morphology resembling fibroblasts. The addition of CuET or DSF (1 :1 mol ratio) abrogated the accumulation of p-catenin, as well as the associated change in cellular morphology.Example 2Formulations of BIO, CuET, and DSF

[0119] To safely co-administer BIO and CuET / DSF in vivo, the drugs need to be formulated in a drug delivery vehicle that is amenable for parenteral administration. Due to their strong hydrophobicity, the drugs were encapsulated in LPNs since lipid-based nanoparticles have a strong track record of safety and are considered industry standards for the delivery of multiple types of drugs, including chemotherapy. While CuET / DSF can be stably encapsulated into liposomes, as repeatedly demonstrated in the literature, preliminary attempts at loading BIO into liposomes resulted in their aggregation and precipitation out of solution. Herein are described lipid-based nanoparticle formulations that can stabilize both BIO and CuET / DSF.

[0120] The general protocol for the preparation of LPNs containing BIO, BIO with DSF, or BIO with CuET using the organic solvent injection method is shown in FIG. 11A. A mixture containing lipid:stabilizer:drug (mol ratio of approximately 6:2:1) may be added to anorganic solution in a closed container and heated until the complete dissolution of the mixture. The organic solution may be injected into an aqueous solution to form LPNs, which may be purified or concentrated using standard protocols. The organic solvent may be fully or partly water-miscible (e.g., ethanol). The polymer may be a poly p-amino ester (PBAE), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), poloxamer of various molecular weights, or combinations thereof. Preferably, the polymer is PVP, as it shows the greatest colloidal stability for the LPNs as shown in FIG. 11 B. A compilation of tested polymers can be found in Table 7.Table 7. Size and polydispersity of LPNs formulated with various polymeric excipients.idone

[0121] Preparation of LPNs containing BIO and CuET using 10 kDa PVP (PVP10) as a stabilizer. A mixture containing DSPC / DSPE-PEG2ooo / Cholesterol / PVP10 / BIO / CuET (mol ratio of 4 / 0.2 / 2 / 2 / 1 / 1) was added to 5 mL of pure ethanol in a closed container and was heated up to 50°C until the complete dissolution of the mixture. The hot ethanol solution was then injected into 45 mL of rapidly stirred ultrapure water at a constant rate. The resulting solution was transferred to a rotary evaporator to remove the ethanol and concentrate the nanoparticles. The solution was filtered using a centrifugal filter (100 kDa, Amicon®), and buffer exchange was performed using a Sephadex® G-50 column to suspend the LPNs in 1x PBS. The nanoparticles were then filter sterilized (0.22 pm) and stored at 4 °C. Fluorescent LPNs were fabricated in the same manner with the addition of 1 % (mol / mol DSPC) Texas Red™ DHPE. Vehicle LPNs (similar mol ratios with no drug added) were used as controls.

[0122] Preparation of LPNs containing BIO and DSF using PVP10 as a stabilizer. A mixture containing DSPC / DSPE-PEG2ooo / Cholesterol / PVP10 / BIO / DSF (mol ratio of4 / 0.2 / 2 / 2 / 1 / 1) was added to 5 mL of pure ethanol in a closed container and was heated up to 50°C until the complete dissolution of the mixture. The hot ethanol solution was then injected into 45 mL of rapidly stirred ultrapure water at a constant rate. The resulting solution was transferred to a rotary evaporator to remove the ethanol and concentrate the nanoparticles. The solution was filtered using a centrifugal filter (100 kDa, Amicon®), and buffer exchange was performed using a Sephadex® G-50 column to suspend the LPNs in 1x PBS. The nanoparticles were then filter sterilized (0.22 pm) and stored at 4 °C.

[0123] Preparation of LPNs containing BIO using PVP10 as a stabilizer. A stock solution of 1 mg / mL of BIO was prepared in ethanol. 0.625 mg / mL of PVP10 was added to the BIO solution under constant stirring. After the PVP10 was dissolved, 3.2 mg / mL of cholesterol, 3.2 mg / mL of DSPE-PEG2000, and 9.6 mg / mL of DSPC was added to the flask. The mixture was heated at 50°C for 15 minutes, after which it was injected into ultrapure water that was stirring at above 800 rpm at a constant rate. Once the ethanol solution was fully injected, the resulting solution was left to mix at low stirring speed for 10 min. The LPNs were concentrated and purified using tangential flow filtration using a filter with a molecular weight cutoff of 100 kDa.

[0124] Physicochemical characterization. The LPNs were diluted 1 :5000 and 1 : 10 for nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS), respectively. NTA measurements were recorded and analyzed using the NanoSight® NS300; DLS and Zeta potential measurements were performed using the Brookhaven Zeta-PALS light-scattering analyzer. Absorbance measurements were performed in 96-well plates at various dilutions using the SpectraMax™ i3 from Molecular Devices. Aggregation kinetics were analyzed in PBS or human serum at 25°C or 37°C, respectively, using the cuvette UV-Vis spectrophotometer on the NanoDrop® One C. For transmission electron microscopy (TEM), purified LPNs were drop-cast on a carbon-copper grid and stained with uranyl acetate for contrast. The imaging was performed using the FEI Tecnai G2 Spirit Twin TEM (Hillsboro, OR, USA) at a voltage of 120 kV. High performance liquid chromatography (HPLC) separation was performed on a reverse-phase column filled with C18 sorbent (Acclaim™ 120). Analysis was performed at room temperature and with a flow rate of 1 ,000 pL / min with isocratic chromatography. Mobile phase consisted of 70% HPLC grade acetonitrile and 30% HPLC grade methanol.

[0125] PVP is a well-known stabilizer in the field of pharmaceutics for oral drug formulations, and its safety profile is well documented. The ethanol injection method allowed the loading of both BIO and CuET at a 1 :1 mol ratio (LPBC) using PVP as a BIO stabilizer. The following experiments pertain to CuET and are exemplary in nature, but they are also replicable when DSF is use instead of CuET. Using transmission electron microscopy (TEM), the LPNs were observed to have a core-shell like structure and a mean diameter of approximately 112.9 ± 0.9 nm for vehicle and 135.6 ± 1.8 nm for drug-loaded LPNs (FIG. 11 C and FIG. 11 D). The addition of PVP resulted in a significantly more stable formulation with less aggregation over time in both PBS or human serum (FIG. 11 E and FIG. 11 F).

[0126] Determining the encapsulation efficiency and loading capacity of LPBC. The distinct absorbance spectra of CuET and BIO, along with their insolubility in water, were used to determine the amount of drug encapsulated in liposomes. First, the drugs were dissolved alone or in combination at a 1 : 1 ratio in DMSO at concentrations ranging from 100 to 0 pg / mL (FIG. 12A - FIG. 12C), and a standard curve was obtained by measuring the absorbance at 450 and 500 nm (FIG. 12D). After purification, the LPNs were diluted at various concentrations in PBS. The absorbance was then measured at 450 and 500 nm to fit the data with the standard curve of CuET and BIO dissolved in DMSO. At a 1 :1 drug ratio of BIO and CuET, the ratio of the absorbance at 450 and 500 is ~1 at a concentration < 100 pg / mL, shown in FIG. 12E, FIG. 12F, and Table 7, which allows for the quantification of both drugs in the system. Batches with deviations of more than 5% from a ratio of 1 were discarded. DMSO and empty LPNs were used as blanks.Table 8. Ratio of the absorbance at 450 / 500 nm of various ratios of CuET to BIO dissolved in DMSO or formulated in LPNs and solubilized in water.

[0127] The encapsulation efficiency (EE%) was determined using the following formula:100

[0128] Where Dtis the total amount of drug added during the liposome preparation and Dsis the amount of drug present in solution as determined from the standard curve extrapolation.

[0129] The loading capacity (LC%) was determined using the following formula: 100

[0130] Where DWtis the total amount of entrapped drug as determined from the standard curve extrapolation and NWtis the total weight of the nanoparticles. The encapsulation efficiency and loading capacity for both drugs was 86.96 ± 1.63% and 4.06 ± 0.08%, respectively.

[0131] The stability of BIO in the presence of PVP may be explained by the formation of hydrogen bonds with the oxime or amine groups, shown below. FIG. 13 shows the NMR spectra of BIO alone or BIO with PVP dissolved in DMSO-de with a distinct deshielding observed on the protons of the oxime and amine groups, indicative of the formation of hydrogen bonding between BIO and PVP.

[0132] The loading of both BIO and CuET into LPNs at a 1 :1 ratio was confirmed using analytical HPLC. After purification, LPBCs were freeze-dried, and the nanoparticles were resolubilized in methanol to detect and quantify the respective drug ratios that were retained in the LPNs. The LPNs were able to stably accommodate various ratios of BIO:CuET as shown in FIG. 14A and FIG. 14B.

[0133] Preparation of LPNs comprising BIO using PBAEs. Because LPNs cannot be stably formulated using amine-based polymers like PAA (FIG. 11 B), and since PVP is not biodegradable in humans post parenteral administration, formulating LPNs using biodegradable amine-based polymeric stabilizers is highly desirable. PBAEs have found multiple applications in the biotechnology and pharmaceutical industries ranging from their complexation with oligonucleotides to facilitate cellular transfection, to their ability to bind various metal ions serving as chelators. PBAEs offer a biocompatible solution for the controlled delivery and release of various types of molecular cargo. Three PBAE polymers were synthesized based on methods similar to those described in US8562966B2, the contents of which are incorporated herein in their entirety. Represented below are the linear structures of C32, C88, and Cp1 polymers.

[0134] C32 polymer was prepared by mixing 496 mg of 1 ,4-butanediol diacrylate (2.5 mmol) with 309 mg of 5-amino-1-pentanol (3 mmol). Polymerization was performed solvent free in screw cap vials under magnetic stirring at 90°C for 24 hours. Once the polymerization was complete, the viscous solution was washed with ether and desiccated.1H NMR (500 MHz, DMSO) 5 4.42 (s, 1 H), 4.06 - 3.94 (m, 2H), 3.39 (tt, J = 10.3, 6.0 Hz, 2H), 2.69 - 2.58 (m, 2H), 2.47 - 2.30 (m, 3H), 1 .59 (dq, J = 22.9, 6.4 Hz, 2H), 1 .50 - 1 .37 (m, 2H), 1.39 (s, 1 H), 1.35 (s, 1 H), 1.31 - 1.17 (m, 1 H).

[0135] C88 polymer was prepared by mixing 496 mg of 1 ,4-butanediol diacrylate (2.5 mmol) with 427 mg of 1-(3-aminopropyl)pyrrolidin-2-one (3 mmol) as described above.1H NMR (500 MHz, DMSO) 5 4.02 (h, J = 5.9 Hz, 3H), 3.31 (d, J = 14.0 Hz, 1 H), 3.15 (dt, J = 27.1 , 7.2 Hz, 2H), 2.74 - 2.66 (m, 1 H), 2.65 (d, J = 7.0 Hz, 2H), 2.39 (tt, J = 24.3, 7.0 Hz, 5H), 2.19 (td, J = 8.0, 2.9 Hz, 2H), 1.91 (p, J = 7.5 Hz, 2H), 1.62 (dq, J = 9.2, 4.7 Hz, 3H), 1.60 - 1.41 (m, 2H).

[0136] Cp1 polymer was prepared by mixing 496 mg of 1 ,4-butanediol diacrylate (3 mmol) with 482 mg of 4-(aminomethyl)-1-(2-methoxyethyl)pyrrolidin-2-one (2.8 mmol) as described above. The polymer was end capped with 1 ,3-pentanediamine (0.2 mmol) for 24h at room temperature with mixing. The solution was washed with ether and desiccated.1H NMR (400 MHz, DMSO) 54.02 (td, J = 7.8, 4.9 Hz, 3H), 3.39 (tt, J = 8.2, 4.2 Hz, 4H), 3.30 (t, J = 4.8 Hz, 2H), 3.08 (ddd, J = 24.9, 10.4, 5.3 Hz, 2H), 2.68 (pt, J = 12.3, 4.9 Hz, 3H), 2.40 (q, J = 7.4 Hz, 4H), 2.30 (dd, J = 17.7, 8.2 Hz, 2H), 2.16 (q, J = 7.3 Hz, 1 H), 1.90 (dt, J = 16.6, 5.5 Hz, 1 H), 1.67 - 1.55 (m, 3H), 1.52 - 1.40 (m, 2H), 1.21 (td, J = 14.1 , 7.1 Hz, 1 H), 0.85 (t, J = 7.4 Hz, 2H). FTIR 3300.96 cm’1(93.03%T), 2926.98 cm’1(80.66%T), 1729.30 cm’1(54.68%T), 1670.81 cm’1(50.56%T), 1548.19 cm-1(82.89%T), 1492.55 cm’1(83.77%T), 1448.11 cm’1(72.11%T), 1359.80 cm’1(75.95%T), 1263.95 cm-1(66.80%T), 1171.29 cm-1(51.38%T), 1115.59 cm’1(47.39%T), 1043.89 cm’1(65.05%T), 991.98 cm’1(71.69%T), 825.25 cm’1(81.66%T), 661.94 cm’1(82.47%T), 585.02 cm’1(79.25%T).

[0137] To test whether BIO and C32 can complex to form nanoparticles, both BIO and C32 were dissolved in ethanol at a C32:BIO weight ratio of 5: 1 and subsequently injected in ultrapure water forming particles with a mean diameter of 933.7 nm and a polydispersity index of 0.005, show in FIG. 15A. Preparation of LPNs containing BIO using C32 as a stabilizer. A mixture containing DSPC / DSPG / DSPE-PEG2000 / BIO (mol ratio of 4 / 0.8 / 0.2 / 1) was added to 5 mL of pure ethanol in a closed container and was heated up to 50°C untilthe complete dissolution of the mixture. C32 was added to the solution at a C32:BIO weight ratio of 5:1 and subsequently injected in ultrapure water forming particles with a mean diameter of 465.1 nm, a polydispersity index of 0.005, as shown in FIG. 15B and a zeta potential of -13.59 mV in dilute salt solution.

[0138] To test whether BIO and Cp1 can complex to form nanoparticles, both BIO and Cp1 were dissolved in ethanol at a Cp1 : BIO weight ratio of 5: 1 and subsequently injected in ultrapure water forming particles with a mean size of 302.1 nm, a polydispersity index of 0.317, as show in FIG. 15C, and a zeta potential of -5.18 mV in dilute salt solution. Preparation of LPNs containing BIO using Cp1 as a stabilizer. A mixture containing DSPC / DSPG / DSPE-PEG2000 / BIO (mol ratio of 4 / 0.8 / 0.2 / 1) was added to 5 mL of pure ethanol in a closed container and was heated up to 50°C until the complete dissolution of the mixture. Cp1 was added to the solution at a Cp1 :BIO weight ratio of 5:1 and subsequently injected in ultrapure water forming particles with a mean diameter of 243.7 nm, a polydispersity index of 0.005, as shown in FIG. 15D, and a zeta potential of -12.86 mV in dilute salt solution.

[0139] To test whether BIO and C88 can complex to form nanoparticles, both BIO and C88 were dissolved in ethanol at a C88:BIO weight ratio of 5: 1 and subsequently injected in ultrapure water forming particles with a mean size of 105.9 nm. LPNs may be formulated with C88:BIO as described above yielding a colloidally stable system capable of solubilizing BIO.

[0140] The results show the ability of PBAEs that contain pyrrolidone functional groups to stabilize BIO, and in conjunction with phospholipids to form LPNs. A person skilled in the art may envision the development of various types of PBAE polymers that contain pyrrolidone groups for various applications that involve the use of indirubin derivates like BIO.

[0141] Preparation of lipid nanoparticles containing BIO and DSF using emulsifiers as a stabilizer. A mixture containing DSPC / DSPE-PEG2ooo / Cholesterol / sorbitan monoole- ate / BIO / DSF (mol ratio of 4 / 0.2 / 2 / 2 / 1 / 1) was added to 5 mL of pure ethanol in a closed container and was heated up to 50°C until the complete dissolution of the mixture. The hot ethanol solution was then injected into 45 mL of rapidly stirred ultrapure water at a constant rate. The resulting solution was dialyzed against 0.9% salt solution to remove the ethanol. The solution was filtered using a centrifugal filter (100 kDa, Amicon®), and bufferexchange was performed using a Sephadex® G-50 column to suspend the lipid nanoparticles in 1x PBS. The protocol yields lipid nanoparticles with a mean diameter of 226.0 nm and polydispersity of 0.253. Other emulsifiers may be used instead of sorbitan monooleate, such as polysorbates or fatty acids (see Figs. 16A-16B).Example 3Biological Characterization of the Formulations

[0142] Cellular uptake of LPNs. Fluorescence imaging was performed using the Nikon Eclipse TE2000U epifluorescence microscope. A375, THP-1 and Jurkat cells were cultured in 6-well plates and treated with 1 pM of fluorescent LPNs for 6 hours. Adherent cells were then trypsinized, washed thrice with PBS and resuspended for 30 min in 4% (v / v) PFA for fixation. The cells were then stained with Hoechst 33342 for nuclear visualization and transferred to glass-bottom petri dishes for imaging. Laser intensity and gain were maintained constant during the imaging process for all samples. There was a clear preference in the uptake rate of melanoma cell lines compared to both THP1 and Jurkat, shown in FIG. 17, suggesting that the nanoparticle uptake may be cell-type dependent.

[0143] The cytotoxicity of LPBC was confirmed in both mouse and human melanoma cell lines using the SRB assay as described earlier. The IC50 value was shown to be below 1 pM, as shown in FIG. 18A and FIG. 18B, for mouse and human melanoma cell lines, respectively.

[0144] Migration-Invasion Assay. Serum-starved cells were detached with 0.25% trypsin- EDTA and seeded in FBS-free media at 3 x 105cells per well in 12-well 8.0 pm pore-size Transwell™ inserts (Corning™) coated with rat tail collagen I (Advanced BioMatrix™). In the bottom compartment, LPBC was introduced at 500 nM in media with 5% FBS. Cells were incubated at 37 °C with 5% CO2 for 24h. The inserts were fixed with 5% (v / v) glutaraldehyde, washed with PBS, and stained with 0.5% (w / v) crystal violet. The interior of the inserts was cleaned to remove non-migratory cells using cotton swabs. Inserts were photographed with a Zeiss Stemi 1000 microscope with at 20X objective. A375 cells displayed the largest invasion potential across a collagen membrane, which was significantly abrogated when LPBC nanoparticles were present, as shown in FIG. 19. A similar result wasnoticeable for all other melanoma cell lines. While the mechanism for the reduction in cell invasion-migration was not explored in this study, we speculate that the inhibition of GSK3, and potentially CDK5 / p25, which are involved in regulating cellular trafficking and cyto- skeletal dynamics, might dampen the ability of cancer cell to migrate across the extracellular matrix.

[0145] Clonogenic Assay. Cells were seeded at 300 cells / well in 6-well plates, treated with 1 or 2 pM LPBC every second day for seven days, and incubated at 37 °C with 5% CO2. On day 7, cells were washed and stained with 0.5% (w / v) crystal violet in 70% ethanol. The total number of colonies was then manually counted. Melanoma cells treated with 1 pM LPBC in a colony-forming assay showed that only Malme-3M cell were able to maintain their colony-forming ability, albeit with lower cell densities (FIG. 20). However, their colony-forming ability was completely abrogated at a concentration of 2 pM, showcasing the ability of LPBC to reduce not only the invasion of cancer cells, but also their ability to form persistent colonies in vitro.

[0146] The ability of LPBC to treat cancer cells was assessed in an environment that is more representative of the in vivo cell-cell architecture by assessing 3D spheroid viability. YUMM1.7 and RPM 17951 cells were added to a low adhesion 96-well U-bottom microplate at a density of 10000 cells per well, and cells were allowed to form spheroids for 24h. Spheroids were then treated with various concentrations of LPBC for 48h and the CyQUANT™ XTT assay kit was used to measure viability according to manufacturer instructions. A dose-dependent decrease in cell viability when treated with LPBC was observed (FIG. 21A), which shows a lower sensitivity to the drug combination compared to 2D cells. This is expected, as nanoparticles have a harder time penetrating 3D cell cultures to reach deeper into the core. Indeed, significant cell death towards the periphery of the spheroids was evident with significant cell detachment and aggregation, as these cells are exposed to a much higher initial concentration, as shown in Figs. 21 B-21C.

[0147] Mouse studies. C57BL / 6 mice were maintained under specific pathogen-free conditions. Subcutaneous tumor growth was monitored every second day through width (W) and length (L) measurements using a standard electronic caliper. Tumor volume (V) was calculated using the following formula: V= W2x L. Body weights were monitored every second day. Endpoint tumor volume was defined as 2 cm3. All mouse experiments contained at least three mice per treatment group.

[0148] For acute toxicity tests, healthy C57BL / 6 mice were injected intravenously with LPBC doses ranging from 2 mg / kg to 8 mg / kg every day for a week and their body weight was monitored every other day. As shown in FIG. 22A, there were no signs of any significant adverse effects in the mice given that they experienced no behavioral changes indicating discomfort, nor any loss in body weight compared to vehicle control (8 mg / kg equivalent) during the treatment period. A dosing range between 1-10 mg / kg of LPBC was evaluated, since the minimum effective dose of CuET was previously shown to be around 1 mg / kg in previous studies. In case of LPNs loaded with BIO and DSF at a 1 :1 ratio (LPBD), the mice were able to tolerate a dose of up to 20 mg / kg when treated intraperitoneally before the development of any noticeable side effects or changes in their body weight (FIG. 22B).

[0149] Hematoxylin and eosin (H&E) staining of major organs in mice treated with 3 mg / kg of vehicle, LPBIO, LPCuET, LPBC or LPBD for 1-2 weeks indicated no clear signs of morphological defects in the microstructure of any major organs highlighting the safety profile of the formulation at the tested doses. Given that PVP is not a biodegradable polymer in the liver, it is expected that some higher molecular weight PVP would accumulate in scavenger cells, which could result in the formation of granulomas under continuous exposure, but lower molecular weights of the polymer, typically <20 kDa such as the one used in this study, can be more readily filtered via the kidneys. Importantly, there was minimal accumulation of LPBC in the kidneys, and the lack of observable the toxicity on H&E images is encouraging given that the drugs may be cytotoxic to podocytes.

[0150] The biodistribution profile of LPBC was also evaluated using subcutaneous YUMM1.7 mouse models to see whether the LPNs could localize to the tumors. The YUMM1.7 model was used since the cells are amelanotic, and they would not impede with ex vivo fluorescence imaging (performed with the Bruker Xtreme™). Mice were injected subcutaneously (dorsally) with 1 x 105YUMM1.7 cells in 100 pL PBS to establish the subcutaneous mouse model. Tumor-bearing mice were injected IV with 3 mg / kg of fluorescent LPBC, and ex vivo fluorescent imaging of major organs showed a significant accumulation of LPBC in the tumors of mice compared to any of their major organs after 24 hours, shown in FIG. 23A and quantified in FIG. 23B. While nanoparticles have the propensity to accumulate in the liver sinusoids and be sequestered by tissue-resident macrophages like Kupffer cells, there is only minimal accumulation of LPBC in the liver at 24 hours. The lower fluorescence intensity in the liver may be due to the fast sequestrationand metabolic turnover in the liver tissue compared to YLIMM1 .7 tumors, suggesting that LPBC may remain in the tumors for a longer period. Alternatively, the addition of PVP to the system could have influenced the LPNs tumor tropism, showing preferential accumulation of the LPNs into the tumors.

[0151] The therapeutic effect of LPBC was first tested in a subcutaneous B16F10 melanoma mouse model. C57BL / 6 mice that were subcutaneously (dorsally) injected with 3 x 105B16F10 cells in 100 pL of PBS to establish the model. The mice were treated IV with 1 mg / kg of vehicle, LPBIO, LPCuET and LPBC starting on day 7 as shown in FIG. 24A. At endpoint (day 17), there was a large and significant decrease in the tumor volume and tumor weight (-47%) in mice treated with LPBC compared to the other control groups (FIG. 24B and FIG. 24C).

[0152] The ability of LPBC to treat established B16F10 lung metastases was compared to standard of care immune checkpoint inhibitors (ICI) (aPD1 and aCTLA4 antibodies). B16F10 cells were used in this experiment since they have strong affinity to the lung if injected IV, due to multiple passages selecting for lung tropism. C57BL / 6 mice were injected IV with 3 x 105B16F10 cells to establish metastases, and on day 5, the mice were treated with 3 mg / kg of LPBC, standard ICI therapy (100 pg of each antibody), or vehicle control every other day. The explanted lungs (on day 15) showed a remarkable reduction of the tumor burden present on the lung surface in the LPBC group compared to vehicle control (Figs. 25A). Quantification of the number of tumor nodules on the surface of the lungs shows a significant reduction (-46%) in the LPBC-treated group compared to both vehicle control and ICI-treated mice, which is consistent with the reduction seen in the subcutaneous model (Figs. 25B). This remarkable difference in tumor burden between the treated and control mice is noteworthy because the B16F10 model is known for its insensitivity to ICI checkpoint inhibition, mimicking resistance seen in humans.

[0153] To further test the ability of LPBC to treat melanoma, YUMM1.7 mouse model was used since these cells are genetically engineered to harbor driver mutations that are more frequently encountered in human melanoma, making them a more representative model. C57BL / 6 mice were subcutaneously injected with 1 x 105YUMM1.7 cells in 100 pL PBS to establish the model. The mice were treated IV with 3 mg / kg of vehicle control, LPBIO, LPCuET, and LPBC starting on day 7 as shown in FIG. 26A. By day 25 (endpoint), the tumor volume in the LPBC group (221 ± 256 mm3) was significantly lower than vehicle(940 ± 699 mm3), LPBIO (1140 ± 216 mm3), and LPCuET (1331 ± 370 mm3) shown in FIG. 26B. This represents a reduction of -76% in the tumor volume compared to the vehicle control during the treatment period. LPBC-treated mice also showed an increase in the overall survival (median of 31.5 days) compared to vehicle control (median of 26.5 days) as shown in FIG. 26C.

[0154] To further assess how treatment with LPBC can result in the observed decrease in tumor growth, explanted YUMM1.7 tumors were stained for the presence of p-catenin, cleaved caspase 3, and Ki67, which are important markers for sternness (and BIO activity), apoptosis, and proliferation, respectively. The internal organs and tumors from treated mice were removed 24 hours after the last injection, and fixed in 10% buffered formalin for 48h, embedded in paraffin, and sectioned at 4 pm. H&E staining was performed on all tissues. Immunostaining was performed using the Discovery XT automated system (Ventana Medical Systems). Tumor tissue sections were stained with anti- -catenin, anticleaved caspase 3, and anti-Ki67. Scanning was performed using a Leica Aperio AT Turbo digital pathology scanner at 40X magnification and 25 microns / pixel. FIGs. 27A - 27L show that the treatment with LPBC results in a decrease in p-catenin and Ki67 staining, and an increase in cleaved caspase 3 in YUMM1.7 tumors.

[0155] To test whether this therapy works in other types of cancers, C57BL / 6 mice were injected subcutaneously (dorsally) with 3 x 105MC38 cells to establish a colorectal model. On day 7, the mice were treated intraperitoneally with 5 mg / kg of vehicle control, LPN with DSF (LPDSF), LPBIO, and LPBD every day. At endpoint, there was a reduction in the tumor volume of -50% in the mice treated with LPBD compared to the vehicle control group (FIG. 28), suggesting that the combination works in more than one cancer type. Interestingly, in MC38, it seems that BIO alone also acts to reduce tumor growth.

[0156] To evaluate cytokine production in mice after treatment, mouse plasma was isolated 24 hours after treatment and used to perform a multiplexed ELISA on a variety of relevant cytokines using a high-sensitivity T cell Milliplex® kit (MHSTCMAG-70K) on the Luminex MAGPIX® system according to manufacturer instructions. Treatment with LPCuET alone elicited a strong inflammatory response as evidenced by a large increase in the plasma cytokine levels, being significantly large for IL-6, and trending toward significance for the rest (FIGs. 29A - 290). The addition of BIO abrogated the increase in the inflammatory cytokines observed in the plasma of mice treated with LPCuET. Theseresults show that BIO has a strong immunomodulatory effect when co-administered with a cytotoxic compound like CuET.

[0157] To evaluate whether this effect is replicated at the tumor level, RNA was extracted from snap frozen subcutaneous B16F10 tumors and RT-qPCR analysis was performed to probe relevant cytokines. T umor tissues were harvested at the end of the treatment period, snap-frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted using Au- rum™ Total RNA kit (Bio-Rad, #7326820), following the manufacturer’s protocol. cDNA was synthesized using iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad, #1708841). RNA / cDNA purity and concentrations were assessed using a NanoDrop™ spectrophotometer. Real-time PCR (RT-PCR) was performed with SsoFast EvaGreen™ Supermix (Bio-Rad, #172-5201). Expression levels of target genes (pro-inflammatory cytokines) were normalized to the housekeeping gene 18s. Primer sequences for mouse IL- 1 , IL4, IL5, IL6, IL10, CXCL2, CCL2, and 18S mRNAs are listed in Table 9. The foldchange was calculated using the formula 2"AACt. The results show a consistent upregulation in the mRNA levels of inflammatory cytokines when mice are treated with LPCuET, and a significant downregulation once BIO was added as shown by the heat map in FIG. 30. These results strongly suggest that BIO plays an important immunomodulatory role in vivo that may be responsible, at least in part, for the observed response to treatment at a combination mol ratio of 1 :1 . This synergistic effect is not replicated in vitro, as the synergy ratios for melanoma cells was over 10:1 of BIO:CuET, which strongly indicates an immune component is responsible for the observed synergistic effect in vivo.Table 9. Primer sequences that were used to measure inflammatory markers in B16F10 tumors.

[0158] To further explore the ability of LPBC to reduce the metastatic spread of melanoma by targeting circulating cancer cells, 3 mg / kg of LPBC was injected concurrently with the B16F10 cells IV only once (FIGs. 31A-31 B). There was a noticeable decrease of the tumor burden in the lungs of LPBC-treated mice on day 15 (endpoint) compared to the vehicle controls, suggesting that LPBC may directly affect the circulating cancer cells since there was no difference in the tumor burden when mice were pre-treated with 3 mg / kg of LPBC 1 hour before B16F10 cells were injected (data not shown). To further probe this observation, B16F10 cells were pre-treated in vitro with 1 or 5 pM of LPBC for 3h, which was not enough exposure time to result in cell death as melanoma cells remained viable and proliferated if kept in culture post-PBS wash (FIGs. 31C-31 D). The pre-treated cells were checked for viability before being injected into mice IV, and on day 15 (endpoint) the explanted lungs showed a complete abrogation of metastatic spread from cells pre-treated with 5 pM.

[0159] This finding suggests that 1) melanoma cells post treatment fail to adhere to the lung tissue to form colonies; 2) they are more readily recognized by circulating immune cells because tumor cells might display increased: a) number of MHC (class I or / and class II) molecules following the treatment compared to untreated cells, or b) expression of stress ligands (e.g., ULBP1 and NKp44L) on cancer cells triggered by the treatment to improve the immune recognition of cancer cells by NK cells and CD8+ T cells; or a combination of the above mentioned possibilities.

[0160] Since there is a lack of trained immunity against B16F10 cells, we hypothesized that the cells could be more recognizable by NK cells given their innate ability to identify cellular stress. We therefore performed RT-qPCR analysis on A375 and B16F10 cells to look for the expression of receptors that may be upregulated under LPBC treatment, which show a strong upregulation in the mRNA of NKp44L and ULBP1 genes in B16F10 and A375 cells, respectively (FIG. 32A and FIG. 32B). The treatment of B16F10 cells with CuET also decreased the expression of PD-L1 mRNA.

[0161] Flow cytometry was performed on A375 and B16F10 to confirm the upregulation of the NK receptors. Cells were seeded in 6-well plates and treated with 100 nM of vehicle control or LPBC for 24 hours. Single-cell suspensions were washed with PBS and labeled with primary rabbit anti-mouse NKp44L (A6142, ABclonal) and primary rabbit anti-human LILBP1 (A10483, ABclonal) with secondary goat anti-rabbit (A11008, invitrogen). Data were acquired using the Attune™ CytPix™ flow cytometer. A strong upregulation of the NK receptors NKp44L and LILBP1 on B16F10 and A375 cells, respectively, was identified on the cell surface (FIG. 33A and FIG. 33B). Both receptors are strong NK cell activators in the context of cellular stress and play an important role in the clearance of infected or mutated cells. These results support the possibility that LPBC, or LPBD, may help activate immune recognition modules to help clear the cells, which could be further amplified if administered with other immunotherapies, such as ICI or oncolytic viruses.

[0162] To verify the effect of LPBC on immune cells, Jurkat T cells were used as a model. The cells were seeded in low-adherence 6-well plates at a density of 1 x 106cells per well and were activated with 50 ng / mL of phorbol 12-myristate-13-acetate (PMA), 1 pg / mL of ionomycin and 1 pg / mL of phytohemagglutinin-L (00-4977-03, eBioscience) in the presence of 100 nM of vehicle, LPBIO, LPCuET or LPBC for 24 hours. Single-cell suspensions were washed with PBS and labeled with anti-human CD69 (clone H1.2F3, 45-0691-82, eBioscience) and anti-human PD-1 (clone J43, 67-9985-82, eBioscience) as activation markers. The treatment of Jurkat cells with BIO significantly reduced the expression of the activation marker CD69 (FIG. 34A and FIG. 34B) but has little impact on the baseline PD- 1 expression (FIG. 35A and FIG. 35B). Treatment with CuET significantly increased the number of cells expressing PD-1 , which were decreased back to baseline by the addition of BIO. This is consistent with literature findings showing the ability of GSK3 inhibitors to downregulate PD-1 expression through the transcription factor T-bet.

[0163] The secretion of IL2 by Jurkat cells was assessed by ELISA. Cells were seeded in low-adherence 6-well plates at a density of 1 x 106cells per well and were activated with 50 ng / mL of phorbol 12-myristate-13-acetate (PMA), 1 pg / mL of ionomycin and 1 pg / mL of phytohemagglutinin-L (00-4977-03, eBioscience) in the presence of 100 nM of vehicle, LPBIO, LPCuET or LPBC for 24 hours. ELISA against human IL-2 was performed on the supernatant according to manufacturer instructions. Interestingly, IL-2 secretion by activated Jurkat cells was also found to be significantly reduced in the presence of CuET (FIG. 36) suggesting that CuET may also act directly on immune cells to modulate theiractivation. Taken together, these results show that the combination of BIO and CuET has powerful immunomodulatory abilities, making it well positioned for its translation into the clinic.

[0164] The person skill in the art understands that CuET can be interchanged with DSF in the presence of copper. In a biological context, DSF is unstable and gets reduced to diethyldithiocarbamate, which in the presence of copper rapidly chelates and forms CuET. For example, in cell culture conditions, DSF is almost always present in the form of CuET since the media contains 10% serum with enough copper levels to allow the formation of CuET. Therefore, CuET and DSF supplemented with Cu (DSF / Cu) may be understood as interchangeable in the present context. Further, in a biological context where copper ions are abundant in solution, for example at a Cu:DSF ratio of 1 :1 or more, DSF / Cu may be interchangeable with DSF. The biological context is important and may explain why DSF is synergistic with BIO at lower BIO:DSF mol ratios compared to BIO:CuET given the ability of cancer cells to sequester copper from their environment, which is highly dependent on the number of seeded cancer cells. Copper supplementation during treatment with DSF may prove beneficial to recapitulate the effects observed with CuET when there is a shortage of copper ions that are free to bind to the reduced form of DSF.

[0165] Statistical analysis. Experiments were carried out independently in at least technical and biological triplicates (n > 3). One-way ANOVA with Dunnett’s correction or Welsch’s One-Way ANOVA with Dunnett’s 3T correction were employed for multiple comparisons to assess the statistical significance between groups at 95% confidence. When comparing only two conditions for a single variable, a t-test was used with Welch’s correction. Statistical analyses were performed using the GraphPad Prism™ 10 software. Data are presented as the mean unless otherwise indicated (*p<0.05, **p<0.01 , ***p<0.005, and ****p<0.001).

[0166] The above description is meant to be exemplary only, and one skilled in the art will recognize that changes may be made to the embodiments described without departing from the scope of the invention disclosed. Still other modifications which fall within the scope of the present invention will be apparent to those skilled in the art, in light of a review of this disclosure, and such modifications are intended to fall within the appended claims.

Claims

WHAT IS CLAIMED IS1. A pharmaceutical composition comprising disulfiram, an indirubin compound, and a stabilizer, wherein the disulfiram, the indirubin compound, and the stabilizer are associated with a lipid nanoparticle.

2. The pharmaceutical composition of claim 1 , wherein the indirubin compound is of formulawherein Ri is H or CH3, each of R2, R3, and R4 are independently selected from H, Br, I, Cl and F, and Rs is selected from =0, =NOH, =N0C0CH3 or =N0CH3.

3. The pharmaceutical composition of claim 1 or 2, wherein the indirubin compound 6- bromo-indirubin-3’-oxime (BIO).

4. The pharmaceutical composition of claim 3, wherein a molar ratio of disulfiram to BIO is from 10:1 to 1 :10.

5. The pharmaceutical composition of any one of claims 1 to 4, further comprising a metal ion associated with the lipid nanoparticle.

6. The pharmaceutical composition of claim 5, wherein the metal ion is copper.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the stabilizer is of formulawherein R is vinyl or an amine endcap and n is an integer of from 2 to 20.

8. The pharmaceutical composition of any one of claims 1 to 6, wherein the stabilizer is of formulawherein R is vinyl or an amine endcap and n is an integer of from 2 to 20.

9. The pharmaceutical composition of any one of claims 1 to 6, wherein the stabilizer is selected from a pyrrolidone polymer, polysorbate (Tween™), sorbitan monooleate (SPAN™) and combinations thereof.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the lipid nanoparticle is a liposome having a size of from 10 nm to 1000 nm.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the lipid nanoparticle has a loading capacity of from 1 to 5 %.

12. A pharmaceutical composition comprising metal chelate copper diethyldithiocarbamate (CuET) and an indirubin compound in a molar ratio of from about 1 :1 to about 1 :10, and a stabilizer, and wherein the CuET, the indirubin compound and the stabilizer are associated with a lipid nanoparticle.

13. The pharmaceutical composition of claim 12, wherein the lipid nanoparticle has a size of from about 10 to about 1000 nm.

14. The pharmaceutical composition of claim 12 or 13, wherein the CuET and the indirubin compound are encapsulated in the lipid nanoparticle at a loading capacity of from about 1 to about 5 %.

15. The pharmaceutical composition of any one of claims 12 to 14, wherein the indirubin compound is 6-bromo-indirubin-3’-oxime (BIO).

16. Use of the pharmaceutical composition as defined in any one of claims 1 to 15, for treating a cancer characterized by an overexpression of p97 and / or NPLOC4.

17. A method of treating a cancer characterized by an overexpression of p97 and / or NPLOC4, the method comprising administering to a subject in need thereof the pharmaceutical composition as defined in any one of claims 1 to 15.

18. Use of disulfiram and an indirubin compound for treating a cancer characterized by an overexpression of p97 and / or NPLOC4.

19. The use of claim 18, wherein the indirubin compound is 6-bromo-indirubin-3’-oxime (BIO).

20. A method of treating a cancer characterized by an overexpression of p97 and / or NPLOC4, the method comprising administering to a subject in need thereof disulfiram and an indirubin compound.

21. The method of claim 20, wherein the indirubin compound is 6-bromo-indirubin-3’- oxime (BIO).

22. A polymer of formulawherein R is vinyl or an amine endcap and n is an integer of from 2 to 20.

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