Compositions and methods for enhancing WNT signaling to treat cancer - Patents.com

Abstract

Methods and compositions for enhancing Wnt signaling pathway activity in tissues of a subject have been developed for the treatment of cancer, particularly cancers with one or more mutations in the APC (adenomatous polyposis coli) gene. Preferably, the amount of the composition for enhancing Wnt signaling does not reduce or inhibit the proliferation or viability of normal healthy cells in the subject. In some embodiments, a pharmaceutical composition comprising an effective amount of one or more GSK-3 inhibitors is administered to reduce the proliferation or viability of cancer cells in a subject. A preferred GSK-3 inhibitor is LY2090314 encapsulated in or associated with nanoparticles. A dosage form of LY2090314 encapsulated in or associated with nanoparticles for administration is also described.

Description

[Technical field]

[0001] STATEMENT REGARDING GOVERNMENT-FUNDED RESEARCH This invention was made with Government support under Grant Nos. R01 CA211184 and R01 CA254314 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] CITED RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 63 / 253,380, filed October 7, 2021, which is incorporated by reference in its entirety herein.

[0003] Reference to sequence listing The sequence listing submitted as an xml file named "MIT_23378.xml" (created on October 6, 2022, size: 6,332 bytes) is incorporated by reference into this specification pursuant to 37 CFR § 1.834(c)(1).

[0004] FIELD OF THEINVENTION The present invention is generally in the field of anti-tumor compositions and methods, most particularly the use of small molecule GSK inhibitors to modulate the Wnt signaling pathway to treat cancer, particularly colon cancer. [Background technology]

[0005] 2. Background of the Invention Colorectal cancer (CRC) is the second leading cause of cancer morbidity and mortality worldwide. It is estimated that nearly half of the population will develop at least one benign colon adenomatous polyp during their lifetime, with less than 3% of cases going on to develop colorectal cancer. Most cases go undetected, as symptoms are rare until very late stages. Colon cancer presents itself as a polypoid growth that progresses into a malignant tumor; it then metastasizes to the lymph nodes, liver, and lungs. They are the leading cause of death in patients with advanced disease.

[0006] The Wnt signaling pathway is essential for cell proliferation, cell polarity, developmental cell fate determination, and tissue homeostasis (Logan, CY & Nusse, R. Annu. Rev. Cell Dev. Biol. 20, 781-810 (2004)). As a result, deregulation of Wnt signaling is often associated with cancer and other diseases (Clevers, H. Cell 127, 469-480 (2006); MacDonald, BT et al., Dev. Cell 17, 9-26 (2009)). Notably, more than 90% of colorectal cancers (CRCs) harbor mutations that activate the Wnt pathway, and more than 80% contain mutations in the Wnt antagonist Adenomatous Polyposis Coli (APC) (Nature 487, 330-337 (2012)). Human APC mutations generally occur in the central region of the open reading frame, termed the "mutation cluster region" (MCR), resulting in a truncated protein product. APC truncation results in the loss of multiple β-catenin binding sites (20R), an Axin interaction site (SAMP), a nuclear localization sequence, and a C-terminal basic region that mediates cytoskeletal interactions. Germline or sporadic APC mutations in colonic stem cells lead to polyp formation and are considered the initiating event in colorectal tumorigenesis. It is well established that in the context of Wnt signaling, APC acts as a scaffold for the β-catenin destruction complex.

[0007] Colon cancer strictly follows a single "gatekeeper gene" paradigm. Mutations that inactivate the APC (adenomatous polyposis coli) gene are found in approximately 80% of all human colon tumors, and heterozygosity for such mutations results in autosomal dominant colon cancer predisposition in humans and mouse models. APC mutations and hypermethylation have also been found in a variety of other cancer types, including pancreatic and gastric cancer. Wnt / β-catenin signaling is essential for intestinal homeostasis and is aberrantly activated in the majority of CRCs through mutations in the tumor suppressor APC. APC is an essential component of a cytoplasmic protein complex that targets β-catenin for destruction.

[0008] The central lesion in both hereditary and sporadic colon tumors results in activation of the Wnt signaling pathway (Kennell JA and Cadigan, KM Adv. Exp. Med. Biol. 656: 1-12 (2009)). Inactivating APC or GSK3β mutations or stabilizing CTNNB1 (encoding β-catenin) mutations are present in nearly all tumors (Fearnhead NS et al., Br Med Bull. 64:27-43 (2002)). More specifically, the canonical tumor suppressor function of APC is to form a "destruction complex" with Axin / Axin2 and GSK-3β that promotes ubiquitination and subsequent proteasomal degradation of the proto-oncogene β-catenin in the absence of Wnt signaling. Loss of APC function results in the accumulation of β-catenin, which translocates to the nucleus and binds to Tcf / Lef transcription factor complexes to activate the transcription of a number of target genes, including cyclin D1, c-myc, and CRD-BP (Noubissi FK et al., Nature. 441:898-901 (2006)). The tumorigenic consequences of unregulated β-catenin activity may involve both direct stimulation of cell growth and proliferation, as well as disruption of differentiation programs.

[0009] The complexity of molecular pathways in colon cancer presents a challenge for clinical treatment.Even though the survival of patients diagnosed with colon cancer early is often excellent, the incidence of colon cancer continues to increase, especially among young people.Therefore, new and effective treatments are needed for colon cancer and other cancers associated with APC dysregulation and hypermethylation.

[0010] It is an object of the present invention to provide compositions and methods of use thereof for the treatment of colon cancer.

[0011] Another object of the present invention is to provide compositions and methods for treating cancers associated with mutations that inactivate the APC gene.

[0012] It is yet another object of the present invention to provide compositions and methods for treating cancer with little or no systemic toxicity. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Logan, CY & Nusse, R. Annu. Rev. Cell Dev. Biol. 20, 781-810 (2004) [Non-Patent Document 2] Clevers, H. Cell 127, 469-480 (2006) [Non-Patent Document 3] MacDonald, BT et al., Dev. Cell 17, 9-26 (2009) [Non-Patent Document 4] Nature 487, 330-337 (2012) [Non-Patent Document 5] Kennell JA and Cadigan, KM Adv. Exp. Med. Biol. 656: 1-12 (2009) [Non-Patent Document 6] Fearnhead NS et al., Br Med Bull. 64:27-43 (2002) [Non-Patent Document 7] Noubissi FK et al., Nature. 441:898-901(2006) Summary of the Invention [Means for solving the problem]

[0014] Summary of the Invention A pharmaceutical composition and its method of use have been developed for treating cancer, particularly colon cancer associated with high Wnt signaling activity and mutation that inactivates APC gene.The composition delivers one or more active agents that increase Wnt signaling in cancer associated with dysregulated Wnt activity.Excessive Wnt activity in cancer cells that exceeds a threshold level leads to reduced cancer cell proliferation and reduced tumor burden.

[0015] A method for treating cancer in a subject in need of such treatment includes administering to the subject an effective amount of a composition for increasing Wnt signaling activity in cancer cells of the subject to reduce cancer cell proliferation and / or reduce cancer cell viability in the subject. Typically, the amount of the composition does not reduce the proliferation and / or viability of healthy cells in the subject. In a preferred embodiment, the composition for increasing Wnt signaling activity comprises a glycogen synthase kinase 3 (GSK-3) inhibitor. An exemplary GSK-3 inhibitor has the following structure: [ka] [ka] Includes.

[0016] In a preferred embodiment, the GSK-3 inhibitor is LY2090314, SAR502250, AZD2858, or an analog, derivative, or prodrug thereof. Typically, the GSK-3 inhibitor is encapsulated in and / or associated with a delivery vehicle that increases the serum half-life of the GSK-3 inhibitor compared to the serum half-life of the same amount of the GSK-3 inhibitor alone. A preferred delivery vehicle is a polymeric nanoparticle or microparticle. In a particular embodiment, the polymeric nanoparticle comprises poly(lactic-co-glycolic acid) (PLGA) and / or polyhydroxyalkanoate. In another embodiment, the composition for increasing Wnt signaling activity comprises isolated Wnt-3a protein.

[0017] Typically, the cancer cells are characterized by increased Wnt signaling activity compared to normal control cells. In an exemplary embodiment, the cancer cells have one or more mutations in the adenomatous polyposis coli (APC) gene. The cancer may be colon cancer, rectal cancer, peritoneal carcinomatosis, pancreatic cancer, adenocarcinoma, ovarian cancer, multiple myeloma, and sarcoma of the pancreas, bone, bladder, brain, breast, cervix, esophagus, kidney, liver, lung, nasopharynx, prostate, skin, stomach, and uterus. In a preferred embodiment, the cancer is colon cancer.

[0018] In some embodiments, the method includes administering to the subject one or more additional active agents (e.g., chemotherapeutic agents, anti-infective agents, anti-inflammatory agents, and diagnostic agents). In some embodiments, the method also includes administering adoptive T cell therapy, surgery, or radiation therapy, and / or a cancer vaccine or an immune checkpoint modulator (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists). In an exemplary embodiment, the subject has had surgery to remove cancer, and the composition is administered to reduce or prevent the proliferation of cancer cells in the subject and / or to enhance the growth of normal tissue in the subject.

[0019] The method for enhancing the activity of the Wnt signaling pathway in cancer cells comprises contacting cancer cells with an effective amount of GSK-3 inhibitor to increase the Wnt signaling in the cells.Typically, the method reduces the proliferation and / or survival rate of the cancer cells that have one or more mutations on APC gene.An exemplary GSK-3 inhibitor is LY2090314.

[0020] The dosage form for injection comprises a GSK-3 inhibitor encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in cancer cells of a subject and to reduce cancer cell proliferation and / or reduce cancer cell viability in the subject. Typically, the amount effective to increase Wnt signaling activity in cancer cells does not reduce healthy cell proliferation and / or viability in the subject. A preferred dosage form comprises LY2090314 encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in cancer cells of a subject and to reduce cancer cell proliferation and / or reduce viability of cancer cells selected from colon cancer, rectal cancer, carcinomatosis, pancreatic cancer, and adenocarcinoma. Typically, the effective amount is effective to reduce tumor size.

[0021] The kit comprises a GSK-3 inhibitor encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in cancer cells of a subject, as well as to reduce cancer cell proliferation and / or reduce cancer cell viability in the subject, and instructions for use according to the described methods.

[0022] Methods of making nanoparticle compositions and pharmaceutical formulations comprising an effective amount of one or more active agents (e.g., one or more GSK-3 inhibitors) for administration to a subject in need thereof to reduce tumor growth or size are also provided. Methods of more efficiently purifying PEG proteins have also been developed.

[0023] The pharmaceutical composition comprising the combination of effective amounts of GSK-3 inhibitor and Wnt agonist can be administered together or separately. Methods of selecting and treating subjects with cancer are also provided. Typically, administering the combination of the two active agents (i.e., GSK-3 inhibitor and Wnt agonist) is effective to reduce cancer cell proliferation or viability, or one or more associated symptoms to a greater extent or for a longer duration than administering the same amount of GSK-3 inhibitor alone or the same amount of Wnt agonist alone to the subject. In the most preferred embodiment, the reduction in cancer cell proliferation or viability in the subject with cancer is greater than the additive reduction achieved by administering the GSK-3 inhibitor and Wnt agonist alone. In some subjects with tumors, the combination is effective for reducing tumor burden, reducing tumor progression, reducing the rate of tumor cell proliferation, or a combination thereof. Exemplary GSK-3 inhibitors include LY2090314, SAR502250, AZD2858, or analogs, derivatives, or prodrugs thereof. Exemplary Wnt agonists include isolated R-spondin1 or R-spondin3 proteins, or derivatives thereof. A method for treating cancer in a subject in need of cancer treatment comprises administering to the subject an effective amount of a pharmaceutical composition to increase Wnt signaling of cancer cells in the subject to reduce cancer cell proliferation and / or reduce cancer cell viability in the subject. Typically, the amount of the composition does not significantly reduce healthy cell proliferation and / or viability in the subject. In a preferred embodiment, an effective amount of a GSK-3 inhibitor and a Wnt agonist are administered. Preferably, the composition does not reduce or minimizes healthy cell proliferation and / or viability in the subject. Typically, the composition is administered to the subject parenterally or enterally. In some embodiments, the GSK-3 inhibitor and the Wnt agonist are administered either orally or by injection.In some embodiments, the GSK-3 inhibitor and the Wnt agonist are administered by different routes and / or times within a treatment cycle. In a preferred embodiment, the GSK-3 inhibitor and the Wnt agonist are encapsulated in and / or associated with a delivery vehicle that increases the serum half-life of the GSK-3 inhibitor and the Wnt agonist compared to the serum half-life of the same amount of the GSK-3 inhibitor or the Wnt agonist in the absence of the delivery vehicle. Exemplary delivery vehicles include nanoparticles or microparticles (e.g., liposomes, polymeric particles, virus-like particles, and protein nanostructures). In a preferred embodiment, the delivery vehicle is a polymeric nanoparticle comprising poly(lactic-co-glycolic acid) (PLGA) and / or polyhydroxyalkanoates. Typically, the cancer cells are characterized by increased Wnt signaling activity compared to normal control cells (e.g., those with one or more mutations in the adenomatous polyposis coli (APC) gene). In some embodiments, the cancers suitable for treatment are colon cancer, rectal cancer, peritoneal carcinomatosis, pancreatic cancer, adenocarcinoma, ovarian cancer, multiple myeloma, and sarcoma (cancer of the pancreas, bone, bladder, brain, breast, cervix, esophagus, kidney, liver, lung, nasopharynx, prostate, skin, stomach, and uterus). In one embodiment, the cancer is colon cancer. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a line graph showing the relative growth of cells (0-650) as a function of concentration (Log10) for each of the following, respectively: [ka]

[0025] [Diagram 2]Figure 2 is a bar graph showing cell growth rate versus concentration of LY2090314, where growth (0-1500) and off-target inhibition (0-0.001) are indicated above and below thresholds for 0 growth rate, respectively. The points at which growth rate decreases and off-target inhibition begins are indicated by arrows.

[0026] [Diagram 3] Figure 3 shows, respectively, [ka] FIG. 1 is a line graph showing organoid growth normalized to non-treated (0-1.4) versus concentrations of LY2090314 (0-1,000 nM) in each of the cells.

[0027] [Figure 4] FIG. 4 is a graph of LY nanoparticle size distribution showing counts (0-30) across particle diameters (0-1000,000 nm).

[0028] [Figure 5A] 5A-5C show quantification of tumor burden following LY nanoparticle treatment. FIG. 5A is a graph of TdT+ tumors / mouse showing the number of tumors (0-150) in each of the untreated and LY nanoparticle-treated samples, respectively. FIG. 5B is a graph of mean fluorescence / tumor showing the mean fluorescence (0-80) in each of the untreated and LY nanoparticle-treated samples, respectively. FIG. 5C is a graph of tumor burden / mouse showing the area x fluorescence intensity (0-1.5 x 108) in each of the untreated and LY nanoparticle-treated samples, respectively. [Figure 5B-C]5A-5C show quantification of tumor burden following LY nanoparticle treatment. FIG. 5A is a graph of TdT+ tumors / mouse showing the number of tumors (0-150) in each of the untreated and LY nanoparticle-treated samples, respectively. FIG. 5B is a graph of mean fluorescence / tumor showing the mean fluorescence (0-80) in each of the untreated and LY nanoparticle-treated samples, respectively. FIG. 5C is a graph of tumor burden / mouse showing the area x fluorescence intensity (0-1.5 x 108) in each of the untreated and LY nanoparticle-treated samples, respectively.

[0029] [Figure 6] 6 is a graph of percent survival of APC min mice in the control group versus that of the treatment group in which mice were treated with 2.5 mg / kg / day LY nanoparticles in their diet for two weeks. The treatment window is indicated by the two dotted lines.

[0030] [Figure 7] FIG. 7 shows the results of the [ka] The LY concentration (M) for each of the following (0 to 1.5 × 10 -5 ) human adenocarcinoma treatment, survival rate (0-1.1).

[0031] [Figure 8A-B] 8A and 8B are graphs showing the survival percentage of colon cancer organoids in the presence of increasing concentrations of LY2090314 and 5-fluorouracil (5-FU).

[0032] [Figure 9] Figure 9 is a graph of WRN (Wnt3a R-spondin 3, Noggin) concentration showing the relative growth AKPVT (0-2.0) against the WRN enrichment factor (0.125-32).

[0033] [Figure 10A-C]10A-10C are graphs showing the number of tumors (FIG. 10A), mean fluorescence / tumor (FIG. 10B), and tumor burden (FIG. 10C) in mice treated with doxycycline (Dox) or a control without doxycycline (no Dox).

[0034] [Figure 11] FIG. 11 is a Kaplan-Meier plot showing percent survival versus days after surgery in mice treated with doxycycline (Dox) or a no doxycycline control (no Dox).

[0035] [Figure 12A] 12A-12E are graphs showing the change in body weight relative to day 0 one week after administration of 5-fluorouracil in mice treated with PBS or 5-fluorouracil at doses of 6.25 mg / kg / day, 12.5 mg / kg / day, 25 mg / kg / day, and 50 mg / kg / day (FIG. 12A); tumor number (FIG. 12B), mean fluorescence / tumor (FIG. 12C), and tumor burden / mouse (FIG. 12D) in control mice and mice treated with 5FU; and the change in body weight over an 8-day period after administration of LY2090314 nanoparticles used to treat disseminated colon cancer (FIG. 12E). [Figure 12B-D] 12A-12E are graphs showing the change in body weight relative to day 0 one week after administration of 5-fluorouracil in mice treated with PBS or 5-fluorouracil at doses of 6.25 mg / kg / day, 12.5 mg / kg / day, 25 mg / kg / day, and 50 mg / kg / day (FIG. 12A); tumor number (FIG. 12B), mean fluorescence / tumor (FIG. 12C), and tumor burden / mouse (FIG. 12D) in control mice and mice treated with 5FU; and the change in body weight over an 8-day period after administration of LY2090314 nanoparticles used to treat disseminated colon cancer (FIG. 12E). [Figure 12E]12A-12E are graphs showing the change in body weight relative to day 0 one week after administration of 5-fluorouracil in mice treated with PBS or 5-fluorouracil at doses of 6.25 mg / kg / day, 12.5 mg / kg / day, 25 mg / kg / day, and 50 mg / kg / day (FIG. 12A); tumor number (FIG. 12B), mean fluorescence / tumor (FIG. 12C), and tumor burden / mouse (FIG. 12D) in control mice and mice treated with 5FU; and the change in body weight over an 8-day period after administration of LY2090314 nanoparticles used to treat disseminated colon cancer (FIG. 12E).

[0036] [Figure 13A-B] Figures 13A-13C are graphs showing dose response of SAR502250 x LY2090314 on wild type colon organoids, clearly showing additive response and independent ability to grow wild type organoids for each drug on the x and y axes, respectively (Figure 13A); dose response of SAR502250 on cancer organoids showing relative growth of AKPVT (0-1.5) over a concentration range of SAR502250 from 6 x 10-8 to 2 x 10-5 (Figure 13B); and dose response of AZD2858 on wild type organoid growth showing fluorescence units over a concentration range of AZD2858 from 1 x 10-7 to 1 x 10-4 (Figure 13C). [Figure 13C] Figures 13A-13C are graphs showing dose response of SAR502250 x LY2090314 on wild type colon organoids, clearly showing additive response and independent ability to grow wild type organoids for each drug on the x and y axes, respectively (Figure 13A); dose response of SAR502250 on cancer organoids showing relative growth of AKPVT (0-1.5) over a concentration range of SAR502250 from 6 x 10-8 to 2 x 10-5 (Figure 13B); and dose response of AZD2858 on wild type organoid growth showing fluorescence units over a concentration range of AZD2858 from 1 x 10-7 to 1 x 10-4 (Figure 13C).

[0037] [Figure 14]FIG. 14 is a two-dimensional dose response of RSPO1 by LY2090314 on inhibition of colon cancer organoid growth (AKPVT) as assessed by a resazurin-based assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description of the Invention I. Definition The term "dosage regime" refers to drug administration in terms of formulation, route of administration, drug dose, dosing interval and duration of treatment.

[0039] The terms "individual," "host," "subject," and "patient" are used interchangeably and refer to mammals, including, but not limited to, primates (e.g., humans), as well as rodents (e.g., mice and rats) and other laboratory animals.

[0040] The term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disease state being treated, or otherwise provide the desired pharmacological and / or physiological effect. The exact dosage will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune health, etc.), the disease, and the treatment being administered. The effect of the effective amount can be relative to a control. Such controls are known in the art and discussed herein, and can be, for example, the condition of the subject before or in the absence of administration of the drug or drug combination, or in the case of a drug combination, the effect of the combination can be compared to the effect of administration of only one of the drugs.

[0041] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic responses, or other problems or complications, commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying substances, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0042] The term "pharmaceutical acceptable salt" as used herein refers to the derivative of the compound defined herein, where the parent compound is modified by making its acid salt or base salt.The examples of pharmaceutical acceptable salt include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids.The above pharmaceutical acceptable salts include, for example, the conventional non-toxic salts or quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids. Such conventional non-toxic salts include those derived from inorganic acids (e.g., hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids); and salts prepared from organic acids (e.g., acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, naphthalenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, and isethionate salts). Pharmaceutically acceptable salts of the compounds can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or free base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704; and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use", P. Heinrich Stahl and Camille G. Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

[0043] The term "prodrug," as used herein, refers to a pharmacological agent (drug) that is administered in an inactive (or significantly less active) form. Once administered, the prodrug is metabolized within the body (in vivo) to the active compound.

[0044] The term "inhibit" or "reduce" in the context of inhibition means to reduce or decrease in activity and amount. This can be a complete inhibition or reduction or a partial inhibition or reduction in activity or amount. The inhibition or reduction can be compared to a control or standard level. The inhibition can be 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. For example, a composition comprising one or more inhibitors of cancer cells can inhibit or reduce the activity and / or amount of cancer cells by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% from the activity and / or amount of the same cells in an equivalent tumor tissue of a subject that has not received or been treated with the inhibitor composition. In some embodiments, the inhibition and reduction are compared at the mRNA, protein, cell, tissue, and organ levels. For example, inhibition and reduction in tumor growth or tumor size / volume.

[0045] The terms "treating" or "preventing" a disease, disorder, or condition include preventing the disease, disorder, and / or condition from occurring in an animal that may be predisposed to, but has not yet been diagnosed as having, the disease, disorder, and / or condition; inhibiting the disease, disorder, or condition, e.g., preventing its progression; and alleviating the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating the disease or condition includes improving at least one symptom of the disease or condition, even if the underlying pathophysiology is not affected (e.g., treating pain in a subject by administering an analgesic, even if such an agent does not treat the cause of the pain). Desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease condition, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated (including, but not limited to, a reduction in the proliferation of cancerous cells, a reduction in symptoms resulting from the disease, an increase in the quality of life of an individual suffering from the disease, a reduction in the dosage of other medications required to treat the disease, a delay in the progression of the disease, and / or a prolongation of the individual's survival).

[0046] The term "biodegradable" generally refers to a material that breaks down or decays under physiological conditions into smaller units or chemical species that can be metabolized, eliminated, or excreted by the subject. Degradation time is a function of composition and morphology.

[0047] The term "targeting moiety" refers to a moiety that is localized or separated from a specific location. The moiety can be, for example, a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule. The location can be a tissue, a specific cell type, or a subcellular component. In one embodiment, the targeting moiety directs the localization of an active agent.

[0048] The term "prolonged residence time" refers to an increase in the time required for a drug to be cleared from a patient's body or from an organ or tissue of the patient. In certain embodiments, "prolonged residence time" refers to an agent that is cleared with a half-life that is 10%, 20%, 50%, or 75% longer than a comparative standard (e.g., a comparable agent without association with or encapsulation into a delivery vehicle (e.g., nanoparticles)). In certain embodiments, "prolonged residence time" refers to an agent that is cleared with a half-life that is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, or 10000-fold longer than a comparative standard (e.g., a comparable agent without nanoparticles that specifically target a particular cell type associated with a tumor).

[0049] The terms "incorporated" and "encapsulated" refer to incorporating, formulating, or otherwise including an active agent into and / or on a composition that allows for controlled release (e.g., sustained release) of such agent in a desired application. The active agent or other material may be incorporated into the particle by encapsulation, physical mixing, etc., of the agent within, within, or to one or more surface functional groups of such particles (by covalent, ionic, or other binding interactions).

[0050] The term "Wnt signaling" or "Wnt signaling activity" or "Wnt signaling pathway activity" is used interchangeably and refers to a group of signaling pathways that start with proteins that pass signals to cells through cell surface receptors. The term "Wnt pathway" refers to any one of three Wnt signaling pathways, including the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt / calcium pathway, which are activated by binding of Wnt-protein ligands to Frizzled family receptors. Exemplary assays used to evaluate the activity of Wnt signaling pathway include the TCF / LEF Reporter kit, which is a functional readout of beta-catenin binding to TCF LEF transcription factor binding domain with reporter activity; or by stem cell growth through wild-type organoids. In some embodiments, the activity of Wnt signaling pathway can be evaluated using stable reporter-expressing cell lines using luminescence or fluorescence.

[0051] II. Composition A. Compounds that Increase Wnt Activity or Levels Wnt Pathway Wnt signaling is initiated when Wnt proteins bind to the N-terminal extracellular cysteine-rich domain of Frizzled (Fz) family receptors. These receptors span the plasma membrane seven times and constitute a distinct family of G protein-coupled receptors (GPCRs). However, to promote Wnt signaling, co-receptors may be required along with the interaction between Wnt proteins and Fz receptors. Examples include lipoprotein receptor-related protein (LRP)-5 / 6, receptor tyrosine kinase (RTK), and ROR2. Upon receptor activation, a signal is sent to the phosphoprotein Dishevelled (Dsh), which is located in the cytoplasm. This signal is transmitted through a direct interaction between Fz and Dsh. Dsh proteins are present in all organisms and all share the following highly conserved protein domains: an amino-terminal DIX domain, a central PDZ domain, and a carboxy-terminal DEP domain. These different domains are important because following Dsh, Wnt signaling can branch into multiple pathways, each pathway interacting with a different combination of the three domains.

[0052] The three best-characterized Wnt signaling pathways are the canonical Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt / calcium pathway. As their names suggest, these pathways belong to one of two categories: canonical or non-canonical. The difference between the categories is that the canonical pathway involves the protein β-catenin, while the non-canonical pathway operates independently of it. The canonical Wnt pathway (or Wnt / β-catenin pathway) is the Wnt pathway that causes the accumulation of β-catenin in the cytoplasm and its eventual translocation to the nucleus to act as a transcriptional coactivator for transcription factors belonging to the TCF / LEF family. Without Wnt, β-catenin does not accumulate in the cytoplasm because a destruction complex would normally degrade it. This destruction complex contains the following proteins: axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK-3) and casein kinase 1 alpha (CK1 alpha). Thus, in some embodiments, the composition of active agents for enhancing the Wnt signaling pathway in cancer cells contains one or more inhibitors of axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK-3) and casein kinase 1 alpha (CK1 alpha). In a preferred embodiment, the active agent for enhancing the Wnt signaling pathway in cancer cells contains one or more inhibitors of glycogen synthase kinase 3 (GSK-3).

[0053] It has been established that the increase of Wnt signaling activity in cancer cells characterized by abnormally high levels of Wnt activity and / or reduced APC function leads to the inhibition of cancer cell proliferation.Excessive Wnt activity in cancer cells that exceeds a threshold level leads to reduced cancer cell proliferation and reduced tumor burden.In a preferred embodiment, the active agent that increases Wnt activity is the agent that reduces the level or activity of one or more inhibitors of Wnt signaling pathway.

[0054] One agent that enhances Wnt activity is an inhibitor of APC-mediated activity (e.g., an inhibitor of GSK-3). Thus, in some embodiments, compositions and methods of one or more GSK-3 inhibitors increase Wnt activity above a threshold in cancer cells characterized by increased Wnt activity to treat cancer in a subject.

[0055] The composition of active agent for enhancing Wnt signaling pathway in cancer cells comprises one or more active agents that enhance Wnt signaling.In some embodiments, the active agent is a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule suitable for enhancing Wnt signaling pathway.In a preferred embodiment, the active agent is a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule suitable for enhancing Wnt signaling pathway by inhibiting glycogen synthase kinase 3 (GSK-3).

[0056] 1. Glycogen synthase kinase 3 inhibitors In some embodiments, the active agent is an inhibitor of glycogen synthase kinase 3 (GSK-3). In a preferred embodiment, the GSK-3 inhibitor is the small molecule inhibitor, LY2090314.

[0057] Typically, the GSK-3 inhibitor, for example, LY2090314, is administered to the cancer cells of the subject in an amount effective to reduce or inhibit GSK-3 activity in the cancer cells of the subject. For example, in some embodiments, the LY2090314 is administered in an amount effective to treat or prevent one or more symptoms of cancer in the subject. Due to the short serum half-life of LY2090314, the effectiveness of LY2090314 in treating and preventing cancer in the subject is increased by one or more shielding agents or delivery systems that protect the LY2090314 molecule in the body of the subject. A preferred delivery system is a nanoparticle that encapsulates, complexes with, or otherwise associates with one or more GSK-3 inhibitors, for example, LY2090314. GSK-3 is a ubiquitously expressed serine / threonine kinase. It plays a fundamental role in many metabolic processes, especially as the final enzyme involved in glycogen synthesis. There are two major known isoforms of GSK-3, known as GSK-3α and GSK-3β. Unless otherwise specified, an inhibitor of GSK-3 as described herein inhibits GSK-3α and GSK-3β. Thus, in a preferred embodiment, an inhibitor of GSK-3 inhibits GSK-3α and GSK-3β. In other embodiments, the inhibitor is an inhibitor of GSK-3α, which inhibits the GSK-3α isoform but has little or no activity against the GSK-3β isoform. In other embodiments, the inhibitor is an inhibitor of GSK-3β, which inhibits the GSK-3β isoform but has little or no activity against the GSK-3α isoform.

[0058] The expression of GSK-3 is ubiquitous, and different concentrations of its isoforms exist in human tissues, and it plays an important role in many cellular pathways, including PI3K / PTEN / Akt / mTORC1, and Ras / Raf / MEK / ERK. GSK-3 is constitutively active in human cells under normal conditions, and phosphorylation at S9 and Y216 regulates GSK-3 activity. Given its broad function, when dysregulated, GSK-3 is associated with the development of several human diseases, including diabetes, bipolar disorder, neurodegenerative diseases (Alzheimer's disease), and cardiovascular disease. Thus, in a preferred embodiment, the inhibitor of GSK is in an amount effective to treat or prevent cancer in a subject without inducing toxicity in said subject.

[0059] GSK-3 also plays an important role in the Wnt and Hedgehog (HH) pathways, which are involved in cell survival and morphology. GSK-3, in part through the Wnt and HH pathways, affects the tumorigenesis and progression of various human cancers and is associated with the development of melanoma, hepatocellular carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, and colorectal cancer (Takahashi-Yanaga F et al., Biochem. Pharmacol., 86(2013), pp. 191-199; Cervello, M. et al., Adv. Biol. Regul., 65(2017), pp. 59-76).

[0060] APC In some embodiments, the GSK-3 inhibitor mediates the activity of the APC gene product. Up to 70% of colon cancers contain mutations in the APC gene. APC is a key regulator of Wnt signaling. Activation of this pathway leads to nuclear accumulation of β-catenin, a process mediated by GSK-3 inhibition. Nuclear β-catenin interacts with T cell factor 4 to induce the transcription of VEGF and c-myc. Phosphorylation of β-catenin by GSK-3 targets it for degradation (Vidri, et al., Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, V 1867, (4), 118626, (2020), ISSN 0167-4889; Kwong, et al., Advances in experimental medicine and biology, V656 85-106. (2009) doi:10.1007 / 978-1-4419-1145-2_8; and Mancinelli, et al., Oxidative Medicine and Cellular Longevity, V. 2017, Article ID 4629495, doi.org / 10.1155 / 2017 / 4629495).

[0061] In some embodiments, the active agent is a small molecule capable of inhibiting GSK-3. The term "small molecule" refers to a small organic compound having a molecular weight greater than about 100 Daltons (Da) and less than about 2,500 Da, preferably between about 100 Da and about 2,000 Da, inclusive, more preferably between about 100 Da and about 1,250 Da, inclusive, more preferably between about 100 Da and about 1,000 Da, inclusive, more preferably between about 100 Da and about 750 Da, inclusive, more preferably between about 200 Da and about 600 Da, inclusive.

[0062] i. LY2090314 In some embodiments, the GSK3αβ inhibitor is LY2090314, which has a molecular structure as shown in Formula I below: [ka]

[0063] LY2090314 (CAS# 603288-22-8) has a molecular weight of 512.53 Da and the formula C 28 H 25It is a small molecule with FN6O3 and a solubility of 31 mg / mL (60.48 mM) in DMSO. LY2090314 is a potent inhibitor of glycogen synthase kinase-3 (GSK-3), with IC50 values ​​of 1.5 nM and 0.9 nM for GSK-3α and GSK-3β, respectively. LY2090314 (20 nM) promotes time-dependent stabilization of β-catenin total protein and induction of axin 2. LY2090314 is highly selective for GSK3, as shown by its fold selectivity against a large panel of kinases. LY2090314 highly induces apoptotic cell death in a panel of melanoma cell lines, independent of BRAF mutation status. LY2090314-induced cell death is dependent on β-catenin, and GSK3β knockdown increases the sensitivity of cells to LY2090314. LY2090314 remains active in cell lines resistant to PLX4032 and has an independent mechanism of action. LY2090314 exhibits high clearance (close to hepatic blood flow) and a moderate volume of distribution (approximately 1-2 L / kg), resulting in rapid elimination (half-lives approximately 0.4 h, 0.7 h, and 1.8-3.4 h in rats, dogs, and humans, respectively). LY2090314 is rapidly eliminated by extensive metabolism, with no apparent intestinal reabsorption and negligible circulating metabolite exposure due to biliary excretion of metabolites into feces (Zamek-Gliszczynski, et al., Drug Metab Dispos, 2013 Apr;41(4):714-26. doi: 10.1124 / dmd.112.048488. Epub 2013 Jan 10). LY2090314 (25 mg / kg Q3D, iv) increases Axin2 gene expression in vivo, demonstrates single agent activity in the A375 xenograft model of melanoma, and enhances the efficacy of DTIC.

[0064] LY2090314 is available from multiple commercial sources, including MedChem Express, Catalogue No. HY-16294.

[0065] ii. Other small molecule GSK-3 inhibitors In some embodiments, the inhibitor of GSK-3 is another small molecule drug that inhibits the activity of GSK-3.Exemplary small molecule GSK-3 inhibitors include CHIR-99021, SB 216763, tideglusib, TWS119, AR-A014418, TDZD-8, GSK 3 inhibitor IX, Kenpaullone, cromolyn sodium, CHIR-98014, AZD1080, R547, RGB-286638, 9-ING-41, SB 415286, BRD0705, IM-12, AZD2858, indirubin-3'-monoxime, 1-azakempaullone, CP21R7, bikinin, BIO-acetoxime, VP3.15 dihydrobromide, GNF4877, GSK-3β inhibitor 1, and hSMG-1 inhibitor 11j.

[0066] In some embodiments, the GSK-3 inhibitor is a small molecule inhibitor, SAR502250, as shown in Formula II. [ka]

[0067] In other embodiments, the GSK-3 inhibitor is a small molecule inhibitor, AZD2858, as shown in formula III. [ka]

[0068] 2.Wnt3a Protein In some embodiments, the one or more active agents are isolated Wnt3a protein (Wingless-type MMTV integration site family member 3A). Wnt3a is a member of the Wnt family of gene products, which activates the canonical Wnt signaling pathway and exhibits multiple biological functions. Wnt3a is associated with embryonic development, neurogenesis, cell differentiation, proliferation, and tumorigenesis. Wnt3a acts as a ligand for members of the frizzled family of seven-transmembrane receptors. Wnt-3 and Wnt-3a play distinct roles in intercellular signaling during the morphogenesis of the developing neural tube. It has been established that exposure of cells to Wnt3a can enhance signaling through, for example, the Wnt signaling pathway, enhancing Wnt activity.

[0069] An exemplary human Wnt3a polypeptide comprises approximately 385 amino acids and has a molecular weight of approximately 60-70 kDa. In some embodiments, the isolated Wnt3a protein has the amino acid sequence shown below: [ka]

[0070] In some embodiments, the isolated Wnt3a protein is recombinantly expressed, optionally with one or more purification tags, such as a histidine tag.

[0071] In some embodiments, the isolated Wnt3a protein is co-expressed or fused with one or more additional components for increased yield, enhanced stability, ease of purification, etc., compared to isolated Wnt3a protein expression alone. In one embodiment, the Wnt3a protein has the amino acid sequence shown below and is fused to afamin (AFM) to allow purification of functional unmodified Wnt3a via purification of the complexed protein AFM modified with a TEV (tobacco echovirus) moiety having a histidine tag (e.g., 6×HIS tag) for pulldown purification: [ka] [ka]

[0072] In another embodiment, the Wnt3a protein is directly tagged with a TEV (tobacco echovirus) moiety having a histidine tag (e.g., a 6xHIS tag) having the amino acid sequence shown below: [ka]

[0073] In some embodiments, the Wnt3a protein is isolated or purified using precipitation with polyethylene glycol. In a preferred embodiment, the Wnt3a protein is precipitated with high concentrations of polyethylene glycol, for example, a final PEG concentration of more than 40%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, or more than 5%, or between about 40% and about 10%, inclusive; or between about 30% and about 20%, inclusive. In a specific embodiment, the Wnt3a protein conjugated to a TEV (tobacco echovirus) moiety with a histidine tag is precipitated with high concentrations of polyethylene glycol, for example, 30%, 25%, 20%, 15%, 10%, 5%. In some embodiments, the PEG for protein precipitation is PEG400, PEG1500, PEG6000, PEG8000, PEG10,000, and / or PEG20,000.

[0074] Thus, in some embodiments, one or more active agents is an isolated Wnt3a protein (e.g., a soluble polypeptide having the amino acid sequence of SEQ ID NO:1, or a variant thereof having 50% or greater sequence identity to SEQ ID NO:1 (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 99.5% sequence identity to SEQ ID NO:1). Recombinant human Wnt3a protein is available from several commercial sources, including Abcam Catalogue No. ab153563.

[0075] In some embodiments, the one or more active agents are one or more of an isolated Wnt3a protein and R-spondin3 and noggin. An exemplary R-spondin3 protein has the amino acid sequence shown below: [ka]

[0076] B. Nanoparticles and Microparticles In some embodiments, the composition of an active agent for enhancing the Wnt signaling pathway in cancer cells comprises one or more particles for delivery of the active agent that enhances Wnt signaling to the body.

[0077] Suitable delivery vehicles for the compounds are known in the art and can be selected to suit the particular active agent. For example, in some embodiments, the compositions are incorporated into, encapsulated by, or attached to nanoparticles, microparticles, microspheres, micelles, synthetic lipoprotein particles, or carbon nanotubes.

[0078] For example, the compositions may be incorporated into vehicles (e.g., polymeric microparticles or nanoparticles) that provide controlled release of the active agent(s). In some embodiments, release of drug(s) is controlled by diffusion of the active agent(s) from the particle and / or degradation of the polymer particle by hydrolysis and / or enzymatic degradation.

[0079] In a preferred embodiment, the composition is incorporated into polymeric microparticles or nanoparticles that provide controlled release of the active agent(s), reduce rapid clearance from the system, and / or reduce rapid hepatic metabolism of the active agent(s).

[0080] Generally, the particles are formed from one or more polymers, lipids, or other suitable materials that encapsulate, complex, or otherwise associate with the one or more GSK-3 inhibitors. The particles shield the GSK-3 inhibitor from degradation or destruction in the body, thereby enhancing the serum half-life and residence time of the GSK-3 inhibitor. In some embodiments, the particles include one or more targeting agents, for example, to deliver the GSK-3 inhibitor to any targeted site (e.g., a specific cell type, a specific organelle).

[0081] The particles permit and / or enhance the biological activity of the encapsulated or associated active agent. Typically, the particles protect the active agent, effectively extending the residence time of the active agent in vivo. Thus, the particles effectively increase the serum half-life of the active agent(s) in vivo compared to the half-life of the active agent in the absence of the particle. In some embodiments, the particles enhance the serum half-life of the active agent by 10%, 20%, 50%, 75%, 100%, 200%, 300%, 400% or more than the serum half-life of the active agent without association with or encapsulation within a delivery vehicle such as a particle. In certain embodiments, the agent encapsulated to enhance Wnt signaling activity is cleared with a half-life that is 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or 10000 times longer than a standard for comparison (e.g., a comparable agent without particles that specifically targets a particular cell type associated with a tumor). The particles typically release the GSK-3 inhibitor into the body over a defined time course that is typically at least 2, 3, 4, 5, 10, or 20 times the half-life of the same amount of the same GSK-3 inhibitor alone in serum. In some embodiments, two active agents (with or without a targeting moiety) are incorporated into the same particle and formulated for release at different times and / or over different periods of time. For example, in some embodiments, one of the agents is completely released from the particle before release of the second agent begins. In other embodiments, release of the first agent begins, followed by release of the second agent before all of the first agent is released, hi yet other embodiments, both agents are released simultaneously, over the same or different periods of time.

[0082] 1. Polymer nanoparticles In some embodiments, the active agent is encapsulated within or complexed with a polymeric nanoparticle.

[0083] The particle can be a polymer particle, a lipid particle, a solid lipid particle, an inorganic particle, or a combination thereof. For example, the particle can be a lipid-stabilized polymer particle. In a preferred embodiment, the particle is a polymer particle, a solid lipid particle, or a lipid-stabilized polymer particle. The particle can include a polymer particle formed from a biodegradable polymer, a non-biodegradable polymer, or a combination thereof. The polymer particle core can be a fully or partially biodegradable polymer core.

[0084] FDA approved biodegradable polymers In some embodiments, the active agent is encapsulated and / or complexed with a polyhydroxy acid ester, such as poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or poly(glycolic acid) (PGA), to form particles with nanometer dimensions. Poly(lactic-co-glycolic acid) (PLGA), or (PLG), is a copolymer used in many Food and Drug Administration (FDA) approved therapeutic devices due to its biodegradability and biocompatibility. During polymerization, successive (glycolic or lactic) monomer units are linked together in PLGA by ester linkages, thus resulting in a linear aliphatic polyester as the product.

[0085] Other FDA approved polymers include polyanhydrides, polyorthoesters, polyhydroxyalkanoates, and some non-biodegradable polymers (e.g., polymethacrylates and cyanomethacrylates). Particles containing polyhydroxyalkanoates are particularly suitable for delivery to areas along the digestive tract and / or for systemic delivery via absorption through the digestive tract following enteral administration. In a preferred embodiment, the particles include polyhydroxyalkanoates.

[0086] In some embodiments, the particles include a biodegradable polymer that is blended with or covalently bonded to one or more additional polymers, which may be present within the inner core and / or on the outer surface of the particle upon formation, e.g., blended with PLGA or exclusively bonded to the exterior of the particle.

[0087] Polyalkylene oxide (PEO) polymers (also referred to as polyalkylenes, polyalkylene glycols, or polyalkylene oxides) are frequently attached to the surface of the polymer or covalently attached to hydrophobic biodegradable polymers, which self-assemble to form particles with the PEO polymers on the surface and the hydrophobic polymer in the core. A preferred PEO is polyethylene glycol (PEG).

[0088] Other biodegradable polymers In some embodiments, the active agent is encapsulated and / or complexed with a biodegradable polymer. Exemplary biodegradable polymers include water-insoluble or slightly soluble polymers that are converted chemically or enzymatically into water-soluble materials in the body. Biodegradable polymers may include soluble polymers crosslinked by hydrolyzable crosslinking groups to make the crosslinked polymers water-insoluble or slightly soluble. Representative biodegradable polymers include polyamides, polycarbonates, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidones, polyglycolides, polysiloxanes, polyurethanes and their copolymers, modified celluloses, and acrylate polymers.

[0089] Excipients can also be added to the core polymer to alter its porosity, permeability, and or degradation profile.

[0090] Hydrophilic Polymers In some embodiments, the active agent is encapsulated and / or complexed with one or more hydrophilic polymers.Representative hydrophilic polymers include cellulose polymers, such as starch and polysaccharides; hydrophilic polypeptides and poly(amino acids), such as poly-L-glutamic acid (PGS), γ-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides, such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyols); poly(olefin alcohols); polyvinylpyrrolidone; poly(hydroxyalkyl methacrylamide); poly(hydroxyalkyl methacrylate); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohols), and copolymers thereof.

[0091] Hydrophobic Polymers Representative hydrophobic polymers include polyhydroxy acids, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid); polyhydroxyalkanoates, such as poly-3-hydroxybutyrate or poly-4-hydroxybutyrate; polycaprolactone; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactone); polycarbonates, such as tyrosine polycarbonate; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids). poly(oxyethylene) / poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acid), as well as copolymers thereof.

[0092] Amphiphilic Polymers Representative amphiphilic polymers include block copolymers of any of the hydrophobic and hydrophilic polymers listed above. Amphiphilic compounds also include phospholipids (e.g., 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphosphatidylcholine (DLPC)) incorporated in a ratio (lipid weight / polymer weight) between 0.01 and 60, most preferably between 0.1 and 30.

[0093] PEGylation As noted above, modification of the surface of the particle, typically by incorporation of surface PEO polymers to increase available hydroxyl groups, can be used to decrease the rate of clearance from the blood and to enhance cellular or tissue uptake. Methods for surface modification of the polymer particles or incorporation of PEO polymers are known to those skilled in the art.

[0094] 2. Lipid particles In some embodiments, the particle is a lipid particle, a liposome or a micelle, or comprises a lipid core. Lipid particles and lipid nanoparticles are known in the art. Lipid particles are formed from one or more lipids, which can be neutral, anionic or cationic at physiological pH. The lipid particles are preferably made from one or more biocompatible lipids. The lipid particles can be formed from a combination of more than one lipid. For example, a charged lipid can be combined with a non-ionic or uncharged lipid at physiological pH.

[0095] Representative neutral and anionic lipids include, but are not limited to, sterols and lipids, such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids.Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (e.g., egg PC, soybean PC) (including 1,2-diacyl-glycero-3-phosphocholine); phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids, such as sphingomyelin and sphingoglycolipids (also known as 1-ceramidylglucoside), such as ceramide galactopyranoside, ganglioside and cerebroside; fatty acid, sterols containing carboxylic acid group (e.g., cholesterol).

[0096] Representative cationic lipids include, but are not limited to, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (also referred to as TAP lipids (e.g., methyl sulfate salts)). Representative TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Representative cationic lipids in liposomes include, but are not limited to, dimethyldioctadecylammonium bromide (DDAB), 1,2-diacyloxy-3-trimethylammonium propane, N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethylamine (N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethylamine). amine) (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dialkyloxy-3-dimethylammonium propane, dioctadecylamidoglycylspermine (DOGS), 3-[N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), β-alanylcholesterol, cetyltrimethylammonium bromide (CTAB), diC 14-amidine, N-tert-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine, N-(α-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonioacetyl)diethanolamine chloride, 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N,N,N',N'-tetramethyl-,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide. In one embodiment, the cationic lipid may be a 1-[2-(acyloxy)ethyl]-2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivative, such as 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM).In one embodiment, the cationic lipid is a 2,3-dialkyloxypropyl quaternary ammonium compound derivative containing a hydroxyalkyl moiety on the quaternary amine, such as 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP ... It can be methyl-hydroxybutylammonium bromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethylammonium bromide (DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE).

[0097] Micellar In some embodiments, the particle or particle core is a lipid micelle. Lipid micelles can be formed, for example, as a water-in-oil emulsion with a lipid surfactant. An emulsion is a blend of two immiscible phases, where a surfactant is added to stabilize the dispersed droplets. In some embodiments, the lipid micelle is a microemulsion. A microemulsion is a thermodynamically stable system composed of at least water, oil, and lipid surfactant that produces a clear and thermodynamically stable system. The droplet size is less than 1 micron, about 10 nm to about 500 nm, or about 10 nm to about 250 nm. Lipid micelles are generally useful for encapsulating hydrophobic active agents, including hydrophobic therapeutic agents, hydrophobic prophylactic agents, or hydrophobic diagnostic agents.

[0098] Liposomes In some embodiments, the particle or particle core is a liposome. Liposomes are small vesicles that are composed of an aqueous medium surrounded by lipids arranged in a spherical bilayer. Liposomes can be classified as small unilamellar vesicles, large unilamellar vesicles, or multilamellar vesicles. Multilamellar liposomes contain multiple concentric lipid bilayers. Liposomes can be used to encapsulate targeted agents by entrapping hydrophilic agents in the aqueous interior or between the bilayers, or hydrophobic agents within the bilayers.

[0099] The lipid micelles and liposomes typically have an aqueous core. The aqueous core may contain water or a mixture of water and alcohol. Representative alcohols include, but are not limited to, methanol, ethanol, propanol (e.g., isopropanol), butanol (e.g., n-butanol, isobutanol, sec-butanol, tert-butanol), pentanol (e.g., amyl alcohol, isobutyl carbinol), hexanol (e.g., 1-hexanol, 2-hexanol, 3-hexanol), heptanol (e.g., 1-heptanol, 2-heptanol, 3-heptanol, and 4-heptanol), or octanol (e.g., 1-octanol), or combinations thereof.

[0100] In one embodiment, the liposomes are prepared from long chain fatty acids and a phytosterol formulation.

[0101] solid lipid particles In some embodiments, the particles are solid lipid particles or contain a solid lipid core. Solid lipid particles provide an alternative to colloidal micelles and liposomes. Solid lipid particles are typically submicron in size, i.e., about 10 nm to about 1 micron, 10 nm to about 500 nm, or 10 nm to about 250 nm. Solid lipid particles are formed from lipids that are solid at room temperature. They are obtained from oil-in-water emulsions by replacing liquid oil with solid lipids.

[0102] Representative solid lipids include, but are not limited to, higher saturated alcohols, higher fatty acids, sphingolipids, synthetic esters, and mono-, di-, and triglycerides of higher saturated fatty acids. Solid lipids may include fatty alcohols having 10-40, preferably 12-30 carbon atoms (e.g., cetostearyl alcohol). Solid lipids may include higher fatty acids having 10-40, preferably 12-30 carbon atoms (e.g., stearic acid, palmitic acid, decanoic acid, behenic acid). The solid lipids may include glycerides (including monoglycerides, diglycerides, and triglycerides) of higher saturated fatty acids having 10 to 40, preferably 12 to 30, carbon atoms (e.g., glyceryl monostearate, glycerol behenate, glycerol palmitostearate, glycerol trilaurate, tricaprin, trilaurin, trimyristin, tripalmitin, tristearin, and hydrogenated castor oil). Exemplary solid lipids may include cetyl palmitate or beeswax. Cyclodextrins may also be used.

[0103] 3.Inorganic particles In some embodiments, the particles include a core formed from or formed from an inorganic particle, such as a metal, metal oxide, or semiconductor particle. The particles can be metal nanoparticles, semiconductor nanoparticles, or core-shell nanoparticles. Inorganic particles and inorganic nanoparticles can be assembled into a variety of shapes, such as rods, shells, spheres, and cones. The inorganic particles can have any dimensions. The inorganic particles can have a maximum dimension of less than 1 micron, from about 10 nm to about 1 micron, from about 10 nm to about 500 nm, or from 10 nm to about 250 nm.

[0104] The inorganic particles or particle cores may include metal oxides. Any metal oxide of the above metals is contemplated. Suitable metal oxides may include metal oxides that include one or more of the following metals: titanium, scandium, iron, tantalum, cobalt, chromium, manganese, platinum, iridium, niobium, vanadium, zirconium, tungsten, rhodium, ruthenium, copper, zinc, yttrium, molybdenum, technetium, palladium, cadmium, hafnium, rhenium, and combinations thereof. Suitable metal oxides may include cerium oxide, platinum oxide, yttrium oxide, tantalum oxide, titanium oxide, zinc oxide, iron oxide, magnesium oxide, aluminum oxide, iridium oxide, niobium oxide, zirconium oxide, tungsten oxide, rhodium oxide, ruthenium oxide, alumina, zirconia, silicon oxide (e.g., silica-based glass and silicon dioxide), or combinations thereof. The metal oxides may be non-biodegradable. The metal oxide may be a biodegradable metal oxide, which may include silicon dioxide, aluminum oxide, and zinc oxide.

[0105] Hybrid Particles In some embodiments, the particle or particle core is a hybrid particle. Hybrid particle, as used herein, refers to a particle that combines features of two or more of polymer particles, lipid particles, and inorganic particles. Examples of hybrid particles can include polymer-stabilized liposomes, polymer-coated inorganic particles, or lipid-coated polymer particles. The hybrid particles can include a polymeric internal region, a lipidic internal region, or an inorganic internal region. The hybrid particles can include a polymeric, lipidic, or inorganic external layer.

[0106] 4. Dendrimer particles In some embodiments, the particle or particle core is a dendrimer. Dendrimers are three-dimensional, hyperbranched, monodisperse, spherical, and polyvalent macromolecules that contain a high density of surface end groups. The term "dendrimer" includes, but is not limited to, a molecular structure having an inner core and layers (or "generations") of repeating units attached to and extending from the inner core, each layer having one or more branch points, and an outer surface of end groups attached to the outermost generation. In some embodiments, dendrimers have a regular dendrimer or "starburst" molecular structure.

[0107] Suitable dendrimer scaffolds that may be used include poly(amidoamine) (also known as PAMAM), or STARBURST TM Dendrimers include polypropylamine (POPAM), polyethyleneimine, polylysine, polyester, iptycene, aliphatic poly(ether), and / or aromatic polyether dendrimers. The dendrimers may have carboxylic acid, amine, and / or hydroxyl termini. In a preferred embodiment, the dendrimers are hydroxyl terminated. Each dendrimer in the dendrimer complex may have the same, similar, or different chemical nature from the other dendrimers (e.g., a first dendrimer may comprise a PAMAM dendrimer, while a second dendrimer may be a POPAM dendrimer).

[0108] Typically, dendrimers have a diameter between about 1 nm and about 50 nm, more preferably between about 1 nm and about 20 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 5 nm. In some embodiments, the diameter is between about 1 nm and about 2 nm. Conjugates are generally in the same size range, although larger proteins such as antibodies may increase in size by as much as 5-15 nm. Typically, drugs are conjugated at a drug to dendrimer weight ratio of between 0.1:1 and 4:1 inclusive.

[0109] In some embodiments, the dendrimer has a molecular weight between about 500 daltons and about 100,000 daltons, preferably between about 500 daltons and about 50,000 daltons, and most preferably between about 1,000 daltons and about 20,000 daltons.

[0110] Methods for making dendrimers are known to those skilled in the art and generally involve a two-step repeating reaction sequence that produces concentric shells (generations) of dendritic β-alanine units around a central initiator core (e.g., ethylenediamine-core). Each subsequent growth step represents a new "generation" of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately twice the molecular weight of the preceding generation. Dendrimer scaffolds suitable for use are commercially available in various generations. Preferred dendrimer compositions are based on generation 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 dendrimer scaffolds. Such scaffolds have 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 reactive sites, respectively. Thus, the dendrimer compounds based on these scaffolds can have up to a corresponding number of targeting moieties (if any) and drugs combined.

[0111] 5. Targeting or Binding Moieties In some embodiments, the particles contain one or more tissue targeting or tissue binding moieties to target nanoparticles to specific locations in vivo and / or enhance in vivo residence time at desired locations in the body. For example, in some embodiments, the particles are sequestered or bound to one or more distinct tissues or organs after local or systemic administration to the body. Thus, the presence of a targeting or binding moiety can enhance the delivery of an active agent to a target site compared to the nanoparticle and active agent in the absence of a targeting or binding moiety. The conjugation of the nanoparticles to one or more targeting or binding moieties can be via a spacer and a link between the spacer and the nanoparticle, and / or the spacer and targeting agent can be designed to provide a releasable or non-releasable form of the nanoparticle-targeting agent complex.

[0112] In some embodiments, the particles are retained along the digestive tract for an extended period of time following enteral administration.

[0113] 6.Particle properties The particles for delivery of active agents encoding Wnt signaling have properties optimized for cell penetration, delivery of agents to tissues, and certain routes of administration. The particles may have a diameter between about 10 nm and about 100,000 nm, inclusive. For example, the nanoparticles may have a diameter of 10 nm to 900 nm, 10 nm to 800 nm, 10 nm to 700 nm, 10 nm to 600 nm, 10 nm to 500 nm, 20 nm to 500 nm, 30 nm to 500 nm, 40 nm to 500 nm, 50 nm to 500 nm, 60 nm to 400 nm, 50 nm to 350 nm, 50 nm to 300 nm, or 50 nm to 200 nm. In preferred embodiments, the nanoparticles may have a diameter of less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm. In some embodiments, the microparticles may have a diameter of between about 1 micron and about 100 microns (inclusive), between about 1 micron and 20 microns (inclusive), and between about 2 microns and 10 microns (inclusive).

[0114] In some embodiments, the nanoparticles encapsulating one or more active agents for enhancing Wnt signaling have any diameter between about 10 nm and about 500 nm inclusive, between about 20 nm and about 500 nm inclusive, or between about 25 nm and about 250 nm inclusive. In preferred embodiments, the particle core is a nanoparticle core having a diameter between about 25 nm and about 250 nm inclusive. In most preferred embodiments, the particle has a diameter between 10 nm and 150 nm inclusive.

[0115] One embodiment provides nanoparticles engineered to maximize half-life and targeting of the nanoparticle to the tumor microenvironment and / or tumor vasculature, for example, by adjusting the amount of one or more compounds present on or otherwise associated with the surface of the particle. In one embodiment, nanoparticles are engineered to maximize half-life and targeting of the nanoparticle to the tumor microenvironment and / or tumor vasculature, for example, by adjusting the amount of PEG on the nanoparticle.

[0116] C. Additional Active Agents to be Delivered The nanoparticle or microparticle compositions described above can be used to deliver an active agent that enhances Wnt signaling and one or more additional active agents, particularly one or more active agents that prevent or treat one or more symptoms of cancer. Suitable therapeutic, diagnostic, and / or prophylactic agents can be biomolecules, such as enzymes, proteins, polypeptides, or nucleic acids, or small molecule agents (e.g., molecular weight less than 2000 amu, preferably less than 1500 amu), including organic, inorganic, and organometallic agents.

[0117] 1. Therapeutic Agents In some embodiments, the one or more additional therapeutic, prophylactic or diagnostic agents include, but are not limited to, chemotherapeutic agents, anti-infective agents, and combinations thereof.

[0118] For example, in some embodiments, the nanoparticles comprise LY2090314 and / or one or more GSK-3 inhibitors (CHIR-99021, SB 216763, tideglusib, TWS119, AR-A014418, TDZD-8, GSK-3 inhibitor IX, kenpaullone, cromolyn sodium, CHIR-98014, AZD1080, R547, RGB-286638, 9-ING-41, SB 415286, BRD0705, IM-12, AZD2858, indirubin-3'-monoxime, 1-azakempaullone, CP21R7, bikinin, BIO-acetoxime, VP3.15 dihydrobromide, GNF4877, GSK-3β inhibitor 1, and hSMG-1 inhibitor 11j), and / or isolated Wnt3a protein, and optionally one or more additional therapeutic agents.

[0119] In some embodiments, the additional therapeutic agent is any inhibitor that targets one or more of APC, GSK-3α, GSK-3β, or one or more components of the Wnt signaling pathway. In other embodiments, the additional therapeutic agent is an inhibitor such as crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, vemurafenib, dabrafenib, ibrutinib, palbociclib, sorafenib, ribociclib, cabozantinib, gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, trametinib, axitinib, lenvatinib, nintedanib, pazopanib, regorafenib, sunitinib, vandetanib, dacomitinib, and ponatinib.

[0120] In some embodiments, the additional therapeutic agent is a tyrosine kinase inhibitor (e.g., a HER2 inhibitor, an EGFR tyrosine kinase inhibitor). Exemplary EGFR tyrosine kinase inhibitors include gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib.

[0121] In some embodiments, the additional therapeutic agent is an anti-angiogenic agent. Exemplary anti-angiogenic agents include, but are not limited to, antibodies against vascular endothelial growth factor (VEGF) (e.g., bevacizumab (Avastin®) and rhuFAb V2 (ranibizumab, Lucentis®) and other anti-VEGF compounds (including aflibercept (Eylea®); Macugen® (pegaptanib sodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals; pigment epithelium-derived factor(s) (PEDF); COX-2 inhibitors (e.g., celecoxib (Celebrex®) and rofecoxib (Vioxx®)); interferon alpha; interleukin-12 (IL-12); thalidomide (Thalomid®) and its derivatives (e.g., lenalidomide (Revlimid®); squalamine; endostatin; angiostatin; ribozyme inhibitors (e.g., Angiozyme® (Sirna Therapeutics)); multifunctional antiangiogenic agents (e.g., NEOVASTAT® (AE-941) (Aeterna Laboratories, Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors (e.g., sunitinib (Sutent®)); tyrosine kinase inhibitors (e.g., sorafenib (Nexavar®) and erlotinib (Tarceva®)); antibodies against the epidermal growth factor receptor (e.g., panitumumab (Vectibix®) and cetuximab (Erbitux®)), as well as other anti-angiogenic agents known in the art.

[0122] In some embodiments, one or more additional therapeutic agents include conventional cancer therapeutic agents, such as chemotherapy agents, cytokines, chemokines, and radiation therapy. Most chemotherapy agents can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs affect cell division or DNA synthesis and function in some way. Additional therapeutic agents include monoclonal antibodies and tyrosine kinase inhibitors (e.g., imatinib mesylate (GLEEVEC®) or GLIVEC®), which directly target molecular abnormalities in certain types of cancer (chronic myeloid leukemia, gastrointestinal stromal tumors).

[0123] Representative chemotherapeutic agents that may be encapsulated or mixed with one or more active agents that enhance Wnt signaling include, but are not limited to, amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, innotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, daunorubici), lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentastatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and its derivatives, trastuzumab (Herceptin®), cetuximab, and rituximab (Rituxan® or MabThera®), bevacizumab (Avastin®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5, and combinations thereof.

[0124] In some embodiments, the compositions and methods are used prior to or in conjunction with immunotherapy (e.g., inhibition of checkpoint proteins such as components of the PD-1 / PD-L1 / 2 axis or the CD28-CTLA-4 axis using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists), adoptive T cell therapy, and / or cancer vaccines. Exemplary immune checkpoint modulators used in immunotherapy include pembrolizumab (anti-PD1 mAb), durvalumab (anti-PDL1 mAb), PDR001 (anti-PD1 mAb), atezolizumab (anti-PDL1 mAb), nivolumab (anti-PD1 mAb), tremelimumab (anti-CTLA4 mAb), avelumab (anti-PDL1 mAb), and RG7876 (CD40 agonist mAb). In some embodiments, the compositions and methods are used prior to or in conjunction with an anti-PDL2 mAb.

[0125] In some embodiments, the additional therapeutic agent is an anti-infective agent. Exemplary anti-infective agents include antiviral agents, antibacterial agents, antiparasitic agents, and antifungal agents. Exemplary antibiotics include moxifloxacin, ciprofloxacin, erythromycin, levofloxacin, cefazolin, vancomycin, tigecycline, gentamicin, tobramycin, ceftazidime, ofloxacin, gatifloxacin; antifungal agents: amphotericin, voriconazole, natamycin.

[0126] III. Methods for making particles Provided is a method for making anti-cancer compositions that increase Wnt signaling activity.The method generally comprises preparing a composition for delivering one or more active agents to increase Wnt signaling activity in cancers associated with dysregulated Wnt activity.Typically, the method comprises nanoparticle preparation, including polymer synthesis and / or encapsulation of the composition.

[0127] A. Emulsion Method In some embodiments, the nanoparticles are prepared using an emulsion solvent evaporation method. For example, a polymeric material is dissolved in a water-immiscible organic solvent and mixed with a drug solution or a combination of drug solutions. In some embodiments, a solution of a therapeutic, prophylactic, or diagnostic agent to be encapsulated is mixed with the polymer solution. The polymer can be one or more of the following, but is not limited to: various forms of PLA, PGA, PCL, copolymers thereof, polyacrylates, the aforementioned PEGylated polymers, the aforementioned polymer-drug conjugates, the aforementioned polymer-peptide conjugates, or the aforementioned fluorescently labeled polymers, or combinations thereof. The drug molecule can be one or more of the following, but is not limited to: PPARγ activators (e.g., rosiglitazone, (RS)-5-[4-(2-[methyl(pyridin-2-yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione, pioglitazone, (RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,4-dione, troglitazone, (RS)-5-(4-[(6-hydroxy-2, 5,7,8-tetramethylchroman-2-yl)methoxy]benzyl)thiazolidine-2,4-dione, etc.), prostaglandin E2 analogs (PGE2, (5Z,11α,13E,15S)-7-[3-hydroxy-2-(3-hydroxyoct-1-enyl)-5-oxo-cyclopentyl]hept-5-enoic acid, etc.), beta3 adrenergic receptor agonists (CL 316243, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate hydrate disodium, etc.), fibroblast growth factor 21 (FGF-21), irisin, RNA, DNA, chemotherapy compounds, nuclear magnetic resonance (NMR) imaging agents, or combinations thereof. The water immiscible organic solvent can be one or more of the following, but is not limited to: chloroform, dichloromethane, and acyl acetate.The drug may be dissolved in one or more of the following, but is not limited to: acetone, ethanol, methanol, isopropyl alcohol, acetonitrile, and dimethylsulfoxide (DMSO).

[0128] In some embodiments, the polymer solution comprises one or more polymer conjugates as described above. The polymer solution may comprise a first amphiphilic polymer conjugate having a hydrophobic polymer block, a hydrophilic polymer block, and a targeting moiety conjugated to the hydrophilic end. In a preferred embodiment, the polymer solution comprises one or more additional polymers or amphiphilic polymer conjugates. For example, the polymer solution may comprise, in addition to the first amphiphilic polymer conjugate, one or more hydrophobic polymers, hydrophilic polymers, lipids, amphiphilic polymers, polymer-drug conjugates, or conjugates comprising other targeting moieties. By controlling the ratio of the first amphiphilic polymer to the additional polymer or amphiphilic polymer conjugate, the density of the targeting moiety may be controlled. The first amphiphilic polymer may be present at 1% to 100% by weight of the polymer in the polymer solution. For example, the first amphiphilic polymer may be present at 10%, 20%, 30%, 40%, 50%, or 60% by weight of the polymer in the polymer solution.

[0129] Then, an aqueous solution is added to the resulting mixture solution to obtain an emulsion solution by emulsification.The emulsification technique can be, but is not limited to, probe sonication or homogenization via homogenizer.The plaque targeting peptide or fluorophore or drug may be associated with the surface of the polymer matrix of the particle, encapsulated within the matrix, surrounded by the matrix, and / or distributed throughout the matrix.

[0130] Solvent Evaporation In solvent evaporation, the polymer is dissolved in a volatile organic solvent (e.g., methylene chloride). The drug (either dissolved or dispersed as fine particles) is added to the solution, and the mixture is suspended in an aqueous solution containing a surfactant (e.g., poly(vinyl alcohol)). The resulting emulsion is stirred until most of the organic solvent is evaporated, leaving solid microparticles. The resulting microparticles are washed with water and dried overnight in a freeze dryer. Microparticles with different sizes (0.5-1000 microns) and morphologies can be obtained by this method. This method is useful for relatively stable polymers such as polyester and polystyrene.

[0131] B. Nanoprecipitation method In another embodiment, the nanoparticles are prepared using nanoprecipitation or microfluidic devices. The polymeric material is mixed with the drug or drug combination in a water-miscible organic solvent. The polymer can be one or more of the following, but is not limited to: PLA, PGA, PCL, copolymers thereof, polyacrylate, the aforementioned PEGylated polymers, the aforementioned polymer-drug conjugates, the aforementioned polymer-peptide conjugates, or the aforementioned fluorescently labeled polymers, or various forms of these combinations. The drug molecule can be one or more of the following, but is not limited to: PPARγ activators (e.g., rosiglitazone, (RS)-5-[4-(2-[methyl(pyridin-2-yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione, pioglitazone, (RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,4-dione, troglitazone, (RS)-5-(4-[(6-hydroxy-2, 5,7,8-tetramethylchroman-2-yl)methoxy]benzyl)thiazolidine-2,4-dione, etc.), prostaglandin E2 analogs (PGE2, (5Z,11α,13E,15S)-7-[3-hydroxy-2-(3-hydroxyoct-1-enyl)-5-oxo-cyclopentyl]hept-5-enoic acid, etc.), beta3 adrenergic receptor agonists (CL 316243, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate hydrate disodium, etc.), RNA, DNA, chemotherapy compounds, nuclear magnetic resonance (NMR) imaging agents, or combinations thereof. The water-miscible organic solvent can be one or more of the following, but is not limited to: acetone, ethanol, methanol, isopropyl alcohol, acetonitrile, and dimethylsulfoxide (DMSO). The resulting mixture solution is then added to a polymer non-solvent (e.g., an aqueous solution) to obtain a nanoparticle solution.The plaque targeting peptide or fluorophore or drug may be associated with the surface of, encapsulated within, surrounded by, and / or distributed throughout the polymer matrix of the particle.

[0132] C. Microfluidics Methods for making nanoparticles using microfluidics are known in the art. Suitable methods include those described in US Patent Application Publication No. 2010 / 0022680 A1 (Karnik et al.). Generally, a microfluidic device includes at least two channels that converge into a mixing device. The channels are typically formed by lithography, etching, embossing, or molding of a polymer surface. A fluid source is attached to each channel, and application of pressure to the sources induces fluid flow in the channels. The pressure can be applied by a syringe, pump, and / or gravity. Inlet streams of solutions with polymers, targeting moieties, lipids, drugs, payloads, etc. converge and mix, and the resulting mixture is combined with a polymer non-solvent solution to form nanoparticles with the desired size and density of moieties on its surface. By varying the pressure and flow rate in the inlet channel, as well as the nature and composition of the fluid sources, nanoparticles with reproducible size and structure can be generated.

[0133] D. Spray drying In this method, the polymer is dissolved in an organic solvent. A known amount of active drug is suspended (insoluble drug) or co-dissolved (soluble drug) in the polymer solution. The solution or dispersion is then spray-dried. Typical process parameters for a miniature spray dryer (Buchi) are: polymer concentration = 0.04 g / mL, inlet temperature = -24°C, outlet temperature = 13-15°C, aspirator setting = 15, pump setting = 10 mL / min, spray flow = 600 Nl / hr, and nozzle diameter = 0.5 mm. Microparticles ranging between 1 and 10 microns are obtained with a morphology depending on the type of polymer used.

[0134] E. Hydrogel Microparticles Microparticles made from gel-type polymers (e.g., alginate) are produced through traditional ionic gelation techniques. The polymer is first dissolved in an aqueous solution, mixed with barium sulfate or certain bioactive agents, and then extruded through a microdroplet forming device, which in some cases uses a nitrogen gas stream to break off the droplets. A slowly stirred (approximately 100-170 RPM) ionic hardening bath is placed below the extrusion device to catch the forming microdroplets. The microparticles are allowed to incubate in the bath for 20-30 minutes to allow sufficient time for gelation to occur. The particle size of the microparticles is controlled by using different size extruders or varying either the flow rate of the nitrogen gas or the polymer solution. Chitosan microparticles can be prepared by dissolving the polymer in an acid solution and crosslinking it with tripolyphosphate. Carboxymethylcellulose (CMC) microparticles can be prepared by dissolving the polymer in an acid solution and precipitating the microparticles with lead ions. In the case of negatively charged polymers (eg, alginate, CMC), positively charged ligands of different molecular weights (eg, polylysine, polyethyleneimine) can be ionically bound.

[0135] IV. Formulations Formulations and pharmaceutical compositions are provided that include an effective amount of the above-described compositions for increasing Wnt signaling activity in a pharmaceutical carrier suitable for administration to an individual in need thereof to treat one or more symptoms of cancer.

[0136] In a preferred embodiment, the active agent that increases Wnt activity is an agent that reduces one or more inhibitors of the Wnt signaling pathway. One such agent that enhances Wnt activity is an inhibitor of APC-mediated activity (e.g., an inhibitor of GSK-3). Thus, in some embodiments, a pharmaceutical preparation comprising one or more GSK-3 inhibitors is described for increasing Wnt activity above a threshold in cancer cells characterized by increased Wnt activity to treat cancer in a subject. In a preferred embodiment, the pharmaceutical preparation comprises a small molecule inhibitor, LY2090314, having the molecular structure shown below: [ka] or a derivative, or analog, or prodrug thereof.

[0137] Due to the short serum half-life of LY2090314, the effectiveness of LY2090314 in treating and preventing cancer in a subject is increased by one or more shielding agents or delivery systems that protect the LY2090314 molecule in the body of the subject.Thus, in a preferred embodiment, a pharmaceutical formulation comprises LY2090314 protected in one or more shielding agents or delivery systems.A preferred delivery system is a nanoparticle that encapsulates, complexes with, or otherwise associates with LY2090314.Thus, a pharmaceutical formulation is described that comprises one or more GSK-3 inhibitors associated with nanoparticles.

[0138] The appropriate formulation depends on the route of administration selected. In a preferred embodiment, the composition for increasing Wnt signaling activity is formulated for parenteral delivery (e.g., by intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection or infusion). In some embodiments, the composition is formulated for intratumoral injection. It may also be possible to administer locally (e.g., to mucosal surfaces such as the mouth, lung, intranasal, intravaginal, etc.). Thus, the composition for increasing Wnt signaling activity is designed to be administered locally or systemically. The composition may be lyophilized and stored in a single-use vial for rehydration immediately before use. Thus, in some embodiments, the composition for increasing Wnt signaling activity is lyophilized. For example, in some embodiments, the composition for increasing Wnt signaling activity is lyophilized in a single-use vial for rehydration immediately before use. Other means for rehydration and administration are known to those skilled in the art.

[0139] The pharmaceutical preparation of active agent for increasing Wnt signaling activity typically comprises one or more active agents as described above in combination with one or more pharma- ceutically acceptable excipients.Representative excipients include solvents, diluents, pH adjusters, preservatives, antioxidants, suspending agents, wetting agents, viscosity adjusters, isotonicity agents, stabilizers, and combinations thereof.Suitable pharma-ceutically acceptable excipients are preferably selected from generally regarded as safe (GRAS) materials and can be administered to individuals without causing undesirable biological side effects or unwanted interactions.

[0140] Generally, pharmaceutically acceptable salts can be prepared by reacting the free acid or base form of active agent with stoichiometric amount of suitable base or acid in water or organic solvent, or in the mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include the salts of active agents obtained from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as the salts formed by reacting drugs with suitable organic ligands (e.g., quaternary ammonium salts). A list of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 20th edition, Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704.

[0141] In some embodiments, the composition for increasing Wnt signaling activity is formulated in dosage unit form for ease of administration and uniformity of dosage. The phrase "dosage unit form" refers to a physically discrete unit of active agent appropriate for the patient to be treated. However, it is understood that every single administration of the composition will be determined by the attending physician within the scope of sound medical judgment. In some embodiments, the therapeutically effective dose of the agent for increasing Wnt signaling activity is first predicted in cell culture assays or animal models, usually mice, rabbits, dogs, or pigs. The animal models are also used to achieve the desired concentration range and route of administration. Such information should then be useful for determining useful doses and routes of administration in humans. The therapeutic efficacy and toxicity of the conjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal to 50% of the population)). The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio, LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for human use.

[0142] Pharmaceutical compositions are described that are formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), enteral routes of administration.

[0143] A. Parenteral Administration The phrases "parenteral administration" and "administered parenterally" are art-recognized terms and include modes of administration other than enteral and topical administration (e.g., injection), including, but not limited to, intravenous, intramuscular, intravascular, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, and intraspinal injection and infusion. Compositions for increasing Wnt signaling activity can be administered parenterally, for example, by subdural, intravenous, intrathecal, intravenous, intravenous, intraarticular, intraarticular, intraarticular, intrasynovial, intraamniotic, intraperitoneal, or subcutaneous routes.

[0144] For liquid preparations, pharma- ceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, and fixed oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions, or suspensions, including saline and buffered media. The composition for increasing Wnt signaling activity can also be administered in emulsion, for example, water-in-oil form. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral.Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0145] Formulations suitable for parenteral administration may contain aqueous and non-aqueous sterile suspensions, which may include antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose). In general, water, saline, aqueous dextrose and related sugar solutions, and glycols (e.g., propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions.

[0146] Injectable pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Eds. Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th ed., pp. 622-630 (2009)).

[0147] B. Enteral Administration In some embodiments, the composition for increasing Wnt signaling activity is administered enterally. The carrier or diluent may be a solid carrier or diluent for a solid formulation, a liquid carrier or diluent for a liquid formulation, or a mixture thereof.

[0148] For liquid preparations, pharma- ceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils.Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters (e.g., ethyl oleate).Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions or suspensions (including saline and buffered media).

[0149] Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral.Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0150] Vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, and fixed oils. Formulations include, for example, aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Vehicles may include, for example, fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose). In general, water, saline, aqueous dextrose, and related sugar solutions are preferred liquid carriers. They may also be formulated with proteins, fats, saccharides, and other components of infant formula.

[0151] In certain embodiments, the composition is formulated for oral administration.Oral formulations can be in the form of chewing gum, gel strips, tablets, capsules, or lozenges.Encapsulating materials for preparing enteric coated oral formulations include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and methacrylic acid ester copolymers.Solid oral formulations (e.g., capsules or tablets) are preferred.Elixirs and syrups are also well-known oral formulations.

[0152] V. How to use Methods of using the compositions to increase Wnt signaling activity in cancer cells are provided.

[0153] A. Cancer Treatment It has been established that increasing Wnt activity above a threshold in cancers that show high Wnt activity leads to reduced cancer cell proliferation and reduced tumor burden.Therefore, a method for treating cancer is provided, particularly cancers associated with high Wnt signaling activity and the mutation that inactivates APC gene.Said method typically comprises administering to a subject in need of cancer treatment an effective amount of a composition for increasing Wnt signaling activity, so as to treat or prevent the cancer characterized by increased Wnt activity in said subject.

[0154] In some embodiments, the method comprises administering to a subject identified as having or at risk of having a cancer associated with dysregulated Wnt activity one or more active agents that increase Wnt signaling, encapsulated in or associated with nanoparticles. In a preferred embodiment, the method comprises administering an inhibitor of GSK-3. Thus, in a preferred embodiment, the method comprises administering to a subject identified as having or at risk of having a cancer associated with dysregulated Wnt activity one or more GSK-3 inhibitors, encapsulated in or associated with nanoparticles. A preferred GSK-3 inhibitor is the small molecule inhibitor, LY2090314. Thus, in a preferred embodiment, the method comprises administering to a subject identified as having or at risk of having a cancer associated with dysregulated Wnt activity LY2090314, encapsulated in or associated with nanoparticles.

[0155] In a preferred embodiment, one or more active agents that increase Wnt signaling are administered to the cancer cells in the subject in an amount effective to increase Wnt signaling activity in the cancer cells of the subject.For example, in some embodiments, one or more active agents that reduce or inhibit GSK-3 activity are administered to the cancer cells in the subject in an amount effective to reduce or inhibit GSK-3 activity in the cancer cells of the subject.For example, in some embodiments, LY2090314 is administered to the cancer cells in the subject in an amount effective to reduce or inhibit GSK-3 activity in the cancer cells of the subject.Since inactivating GSK-3 enhances Wnt activity above the threshold required for cancer cell survival and proliferation, administering an agent that reduces or inhibits GSK-3 activity to the subject in an amount effective to inactivate GSK-3 is effective for treating or preventing one or more symptoms of cancer in the subject. Thus, in a preferred embodiment, a method of treating cancer in a subject includes administering to the subject one or more GSK-3 inhibitors (e.g., LY2090314) encapsulated in or associated with nanoparticles in an amount effective to prevent or reduce one or more symptoms of cancer in the subject.

[0156] In some embodiments, the method of GSK3 inhibition is used in combination with one or more Wnt agonists to enhance the efficacy of treatment. In some embodiments, the method involves using a combination of one or more Wnt agonists and one or more GSK3 inhibitors. For example, in a preferred embodiment, R-spondin1 and / or R-spondin3 is used in combination with one or more GSK3 inhibitors (e.g., LY2090314) to treat colon cancer. Based on the tests to date, the effective dose of each class of compound is less than when the compound is administered alone. Because the combination shows a "synergistic effect", i.e., the results of treating cancer cells with both classes of compounds are greater than would be expected from treatment with either class alone, or their additive effective doses. In some embodiments, the effective amount of one or both agents used in combination is lower than the effective amount of each agent when administered separately. This may provide benefits to efficacy. Because a larger dose of the compound can be used in combination, which is safer than using the same amount of only one class of compound. The term "combination" or "combined" can be used to refer to either the concomitant, simultaneous, or sequential administration of the GSK3 inhibitor and the Wnt agonist.The combination can be administered separately but simultaneously (e.g., to the same subject via separate intravenous lines; one agent is given orally while the other is given by infusion or injection, etc.), or sequentially (e.g., one agent is given first, followed by the second).There can be different periods between the administration of the two compounds, and different periods between treatment cycles.

[0157] Preferably, one or more components of the combination treatment are encapsulated within nanoparticles, with or without a controlled release formulation.

[0158] B. Treatment Regimens The method of using the composition for increasing Wnt signaling activity in cancer cells can include one or more treatment regimens. The treatment regimen can include one or more administrations of the composition that increases Wnt signaling to achieve a desired physiological change. For example, in some embodiments, the treatment method includes administering to a subject (e.g., a mammal, particularly a human) an effective amount of the composition that increases Wnt signaling to treat cancer characterized by increased Wnt signaling activity or its symptoms and / or produce a physiological change in the subject.

[0159] 1. Dosage and Effective Amount In some embodiments, the method of treating cancer in a subject comprises administering a composition for increasing Wnt signaling activity in cancer cells in an amount effective for treating the cancer.The method typically comprises administering to the subject an effective amount of a composition for increasing Wnt signaling activity in the cancer cells of the subject to reduce cancer cell proliferation and / or reduce cancer cell viability in the subject.Exemplary methods of increasing Wnt signaling activity include GSK3 inhibition (e.g., using a GSK3 inhibitor) and / or increasing Wnt signaling (e.g., using a Wnt agonist).Preferably, when one or more classes of active agents are used in combination to increase Wnt signaling, the reduction in cancer cell proliferation or viability in the subject with cancer is greater than the additive reduction achieved by administering each of the agents alone.

[0160] It has been established that the proliferation and survival rate of cancer cells in a subject with cancer can be reduced and prevented by increasing the Wnt activity of the cancer cells, and the same increase in Wnt activity enhances the growth and survival rate of healthy cells in the same subject. This makes compositions for increasing Wnt signaling activity for treating cancer particularly safe and effective, reducing or minimizing the side effects associated with conventional chemotherapeutic agents for a given type of cancer. Increasing the proliferation or survival rate of healthy cells in the subject can be beneficial to the subject, for example, to increase healing or tissue regeneration in the subject. Thus, in some embodiments, the amount of the composition increases or stimulates the proliferation and / or survival rate of healthy cells in the subject. In an exemplary embodiment, the amount of the composition for increasing Wnt signaling activity administered to a subject is effective to reduce tumor cell proliferation and survival rate and enhance the proliferation and survival rate of normal healthy cells in the subject. Thus, in some embodiments, the amount of the composition is effective to enhance healing or tissue repair in the subject (e.g., a subject that has undergone surgery, chemotherapy, or another harmful treatment or procedure). In an exemplary embodiment, the amount of the composition enhances regeneration of colon tissue in a subject (e.g., a subject that has undergone surgery to remove colon cancer) while reducing colon cancer cell viability, proliferation, or metastasis.

[0161] Dosage and administration regimen depends on the severity and location of the disorder or injury and / or the method of administration and is known to those skilled in the art. A therapeutically effective amount of the composition used in the treatment of cancer is typically sufficient to reduce or alleviate one or more symptoms of cancer. The symptoms of cancer can be physical (e.g., tumor burden) or biological (e.g., cancer cell proliferation). Thus, the amount of the composition can be effective, for example, to kill tumor cells or inhibit tumor cell proliferation or metastasis. Preferably, the composition comprising one or more active agents (e.g., GSK3αβ inhibitors) is preferentially delivered into or around tumor tissue. Preferably, the active agent does not target or otherwise modulate the activity or amount of healthy cells that are not in or associated with tumor tissue, or modulates at a reduced level compared to cancer or cancer-associated cells. In this way, by-products and other side effects associated with the composition are reduced, preferably resulting directly or indirectly in cancer cell death. In some embodiments, the composition for increasing Wnt signaling activity directly or indirectly reduces cancer cell migration, angiogenesis, immune evasion, or a combination thereof.In some embodiments, the composition for increasing Wnt signaling activity directly or indirectly induces changes in cancer cell itself or its microenvironment, which suppresses the proliferation of the cancer cell, induces the apoptosis of the cancer cell, or induces the activation of immune response against the cancer cell, or a combination thereof.

[0162] In some in vivo approaches, the composition for increasing Wnt signaling activity is administered to a subject in a therapeutically effective amount to reduce tumor size.For example, in some embodiments, the effective amount of the composition for increasing Wnt signaling activity is used to put cancer into remission and / or maintain cancer in remission.Also provided is an effective amount of the composition for increasing Wnt signaling activity that reduces or stops cancer stem cell proliferation.

[0163] The actual effective amount of the composition may vary depending on factors including the specific active agent administered, the specific composition formulated, the mode of administration, and the age, weight, condition, and route of administration and disease or disorder of the subject being treated.In some embodiments, the composition for increasing Wnt signaling activity is administered by a route selected from intravenous, intramuscular, intravascular, intrapericardial, intrathecal, intracapsular, intraorbital, intracardiac, intraperitoneal, subcutaneous, intraarticular, subarachnoid, intraspinal, and oral.In a preferred embodiment, the composition for increasing Wnt signaling activity is administered parenterally, for example, by subdural, intravenous, intrathecal, intravenous, intravenous, intraarterial, intraperitoneal, or subcutaneous route.In other embodiments, the composition for increasing Wnt signaling activity is administered enterally.Generally, for intravenous injection or infusion, the dosage may be lower.

[0164] In general, the timing and frequency of administration is adjusted to balance the effectiveness of a given treatment or diagnostic schedule and the side effects of a given delivery system. Exemplary administration frequencies include continuous infusion, one or more administrations (e.g., hourly, daily, weekly, monthly or yearly administrations).

[0165] In some embodiments, a dosage is administered to a human every day, every other day, or once, twice, or three times every 2, 3, 4, 5, or 6 days. In some embodiments, a dosage is administered about once or twice every week, every 2, 3, or 4 weeks. In some embodiments, a dosage is administered about once or twice every month, every 2, 3, 4, 5, or 6 months.

[0166] It is understood by those skilled in the art that the administration regimen can be any length of time sufficient to treat the condition in the subject.In some embodiments, the regimen includes a drug holiday (e.g., drug-free) following one or more treatment cycles.A drug holiday can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days; or 1 week, 2 weeks, 3 weeks, 4 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0167] Also described is a dosage form of the composition for increasing Wnt signaling activity in cancer cells.In some embodiments, the dosage form of the GSK-3 inhibitor encapsulated in nanoparticles is in a form suitable for parenteral or enteral administration, and is in an amount effective for increasing Wnt signaling activity in cancer cells of a subject, and reducing cancer cell proliferation and / or reducing cancer cell viability in the subject.In an exemplary embodiment, the dosage form is in an amount effective for increasing Wnt signaling activity in cancer cells, but does not reduce the proliferation and / or viability of healthy cells in the subject.

[0168] In some embodiments, the effective concentration of the GSK-3 inhibitor suitable for administration is between about 0.1 micromolar and about 10 micromolar.

[0169] 2. Control The therapeutic results of the composition for increasing Wnt signaling activity can be compared to a control. Suitable controls are known in the art and include, for example, untreated cells or untreated subjects. A representative control is a comparison of a condition or symptom of a subject before and after administration of a targeted agent. The condition or symptom can be a biochemical, molecular, physiological, or pathological readout. For example, the effect of the composition on a particular symptom, pharmacological, or physiological indicator can be compared to an untreated subject, or to the condition of the subject before treatment. In some embodiments, the symptom, pharmacological, or physiological indicator is measured in the subject before treatment and measured again one or more times after treatment has begun. In some embodiments, the control is a reference level or an average determined based on the measurement of the symptom, pharmacological, or physiological indicator in one or more subjects (e.g., healthy subjects) that do not have the disease or condition to be treated. In some embodiments, the effect of the treatment is compared to a conventional treatment known in the art.

[0170] C. Combination Treatments and Procedures The compositions for increasing Wnt signaling activity may further be administered alone or in combination with one or more conventional treatments or procedures (eg, conventional cancer treatments or surgery).

[0171] In some embodiments, conventional cancer treatment is in the form of one or more additional active agents. Thus, in some embodiments, the method administers a composition for increasing Wnt signaling activity in combination with one or more additional active agents. The combination treatment may include administering the composition for increasing Wnt signaling activity and additional active agents together in the same mixture or in separate mixtures. Thus, in some embodiments, the method administers a pharmaceutical preparation that includes a composition for increasing Wnt signaling activity and one, two, three, or more additional active agents. Such preparations typically include an effective amount of a composition for increasing Wnt signaling activity and an effective amount of an additional therapeutic, preventive, or diagnostic agent. The additional active agents may have the same or different mechanisms of action. In some embodiments, the combination produces an additive effect on the treatment of cancer. In some embodiments, the combination produces a greater than additive effect on the treatment of the disease or disorder.

[0172] Said additional treatment or procedure can be simultaneous or sequential with the administration of said composition for increasing Wnt signaling activity.In some embodiments, said additional treatment is performed during drug cycle or during drug holiday that is part of said composition dosing regimen.For example, in some embodiments, said additional treatment or procedure is surgery, radiation therapy, or chemotherapy.

[0173] Additional therapeutic agents include conventional cancer treatments, such as chemotherapy agents, cytokines, chemokines, and radiation therapy, as discussed above. Most chemotherapy drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor drugs. These drugs affect cell division or DNA synthesis and function in some way. Additional therapeutic agents include monoclonal antibodies and tyrosine kinase inhibitors (e.g., imatinib mesylate (GLEEVEC®) or GLIVEC®), which directly target molecular abnormalities in certain types of cancer (chronic myeloid leukemia, gastrointestinal stromal tumors).

[0174] In some embodiments, the additional treatment is a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate ... Rexartan, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentastatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and their derivatives, trastuzumab (Herceptin®), cetuximab, and rituximab (Rituxan® or MabThera®), bevacizumab (Avastin®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine staurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5, and combinations thereof.

[0175] When treating colorectal cancer, the additional chemotherapy treatments and regimens include FOLFOX (leucovorin calcium, fluorouracil, and oxaliplatin), CAPEOX (capecitabine and oxaliplatin), FOLFIRI (leucovorin calcium, fluorouracil, and irinotecan), FOLFOXIRI (leucovorin calcium, fluorouracil, oxaliplatin, and irinotecan), and 5-FU / LV (5-fluorouracil and leucovorin calcium), preferably as directed by the NCCN guidelines. An exemplary regimen for FOLFOX includes: Day 1: Oxaliplatin 85 mg / m over 2 hours. 2 IV, Day 1: Leucovorin 400 mg / m over 2 hours 2 IV, followed by days 1–2: fluorouracil 400 mg / m on day 1; 2 IV injection, followed by 1,200 mg / m 2 / day × 2 days (total 2,400 mg / m over 46-48 hours) 2 ) IV continuous infusion; cycle repeated every 2 weeks. Further exemplary regimens for FOLFOX include: Day 1: Oxaliplatin 85 mg / m over 2 hours 2 IV, Day 1: Leucovorin 400 mg / m over 2 hours 2 IV, followed by days 1-2: fluorouracil 1,200 mg / m 2 / day (total of 2,400 mg / m over 46-48 hours) 2 ) IV continuous infusion; repeated every 2 weeks. In some embodiments, the additional chemotherapy treatment is FOLFOX + bevacizumab; FOLFOX + cetuximab; or FOLFOX + panitumumab, preferably as prescribed by the NCCN guidelines.

[0176] In some embodiments, the compositions and methods are used prior to or in conjunction with immunotherapy (e.g., inhibition of checkpoint proteins such as components of the PD-1 / PD-L1 axis or the CD28-CTLA-4 axis using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists), adoptive T cell therapy, and / or cancer vaccines. Exemplary immune checkpoint modulators used in immunotherapy include pembrolizumab (anti-PD1 mAb), durvalumab (anti-PDL1 mAb), PDR001 (anti-PD1 mAb), atezolizumab (anti-PDL1 mAb), nivolumab (anti-PD1 mAb), tremelimumab (anti-CTLA4 mAb), avelumab (anti-PDL1 mAb), and RG7876 (CD40 agonist mAb).

[0177] In some embodiments, the additional treatment is adoptive T cell therapy. Methods of adoptive T cell therapy are known in the art and used in clinical practice. In general, adoptive T cell therapy involves the isolation and ex vivo expansion of tumor-specific T cells to achieve a higher number of T cells than can be obtained by vaccination alone. The tumor-specific T cells are then infused into a patient with cancer in an attempt to give the patient's immune system the ability to overwhelm the residual tumor through T cells, which can attack and kill the cancer. Several forms of adoptive T cell therapy can be used for cancer treatment, including but not limited to culturing tumor-infiltrating lymphocytes or TILs; isolating and expanding one specific T cell or clone; and using T cells that are engineered to recognize and attack tumors. In some embodiments, the T cells are taken directly from the patient's blood. Methods of priming and activating T cells in vitro for adaptive T cell cancer therapy are known in the art. See, e.g., Wang et al., Blood, 109(11):4865-4872(2007) and Hervas-Stubbs et al., J. Immunol., 189(7):3299-310(2012).

[0178] Historically, adoptive T cell therapy strategies have focused primarily on the infusion of tumor antigen-specific cytotoxic T cells (CTLs) that can directly kill tumor cells. However, CD4+ T helper (Th) cells (e.g., Th1, Th2, Tfh, Treg, and Th17) can also be used. Th can activate antigen-specific effector cells and recruit cells of the innate immune system (e.g., macrophages and dendritic cells) to assist antigen-presenting cells (APCs), and antigen-primed Th cells can directly activate tumor antigen-specific CTLs. As a result of activating APCs, antigen-specific Th1 has been implicated as an initiator of epitope or determinant spreading, the expansion of immunity to other antigens in tumors. The ability to induce epitope spreading may lead to more efficient tumor cell killing due to the ability to expand the immune response to many potential antigens in tumors and initiate heterologous responses. In this way, adoptive T cell therapy can be used to stimulate endogenous immunity. In some embodiments, the T cells express a chimeric antigen receptor (CAR, CAR T cell, or CART). Artificial T cell receptors are engineered receptors that transfer specific specificity to immune effector cells. Typically, these receptors are used to transfer the specificity of monoclonal antibodies into T cells and can be engineered to target virtually any tumor-associated antigen. First generation CARs typically had an intracellular domain derived from the CD3 ζ chain, which is the main transmitter of signals from endogenous TCR. Second generation CARs add intracellular signaling domains from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cells, and third generation CARs combine multiple signaling domains (e.g., CD3z-CD28-41BB or CD3z-CD28-OX40) to enhance efficacy.

[0179] In some embodiments, the compositions and methods are used prior to or with a cancer vaccine, such as a dendritic cell cancer vaccine. Vaccination typically involves administering to a subject an antigen (e.g., a cancer antigen) together with an adjuvant to induce therapeutic T cells in vivo. In some embodiments, the cancer vaccine is a dendritic cell cancer vaccine that has been primed ex vivo so that the antigen delivered by the dendritic cell presents the cancer antigen. Examples include PROVENGE® (sipuleucel-T), which is a dendritic cell-based vaccine for the treatment of prostate cancer (Ledford et al., Nature, 519, 17-18 (05 March 2015). Such vaccines and other compositions and methods for immunotherapy are reviewed in Palucka et al., Nature Reviews Cancer, 12, 265-277 (April 2012).

[0180] In some embodiments, the compositions and methods are used prior to or in conjunction with surgical removal of a tumor, for example, in preventing metastasis of a primary tumor. In some embodiments, the compositions and methods are used to enhance the body's own anti-tumor immune function.

[0181] D. Subjects to be Treated Generally, the method of administering the composition for increasing Wnt signaling activity is useful in the context of treating cancer, including tumor therapy.All of the described methods can include the step of identifying and selecting the subject in need of treatment or the subject who will benefit from administration with the composition.

[0182] Typically, the subject to be treated has a proliferative disease (e.g., a benign or malignant tumor). In some embodiments, the subject to be treated has been diagnosed with stage I, stage II, stage III, or stage IV cancer.

[0183] The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancerous cells migrate from the tumor, invade blood or lymphatic vessels, and are transported to other tissues, where they continue to grow. In this way, a primary tumor at one site can give rise to a secondary tumor at another site.

[0184] The compositions and methods are useful for treating a subject having a benign or malignant tumor by slowing or inhibiting tumor growth in the subject, reducing tumor growth or size, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor initiation or growth.

[0185] Malignant tumors that can be treated are classified according to the developmental origin of the tissue from which the tumor originates. Cancers are tumors that originate from endodermal or ectodermal tissues (e.g., skin or epithelial lining of internal organs and glands). The above compositions are particularly effective in treating cancer. Sarcomas, which occur less frequently, originate from mesodermal connective tissues (e.g., bone, fat and cartilage). Leukemias and lymphomas are malignant tumors of hematopoietic cells in the bone marrow. Leukemias grow as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors can appear in many organs or tissues of the body to establish cancer.

[0186] The types of cancer that can be treated with the provided compositions and methods include, but are not limited to, colorectal cancer, peritoneal carcinomatosis, pancreatic cancer, (the term adenocarcinoma is not tissue specific), multiple myeloma, sarcoma, brain, breast, esophageal, liver, lung, stomach, and uterine cancer. In some embodiments, the composition is used to treat multiple cancer types together. The composition can also be used to treat metastases or tumors in multiple locations. Some cancer types are directly driven by WNT (e.g., hepatocellular carcinoma, cholangiocarcinoma, and medulloblastoma). Thus, in a preferred embodiment, the cancer to be treated is hepatocellular carcinoma, cholangiocarcinoma, and medulloblastoma.

[0187] Exemplary cancers that may be treated include: brain tumors (including but not limited to glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma); breast cancer (adenocarcinoma, lobular carcinoma (small cell carcinoma), intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, cancer, Paget's disease, and inflammatory breast cancer); adrenal cancer (including, but not limited to, pheochromocytoma and adrenocortical carcinoma); thyroid cancer (e.g., papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer); pancreatic cancer (including, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors); pituitary cancer (including, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus) insipius); eye cancer (including, but not limited to, ocular melanomas such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma); vaginal cancer (including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma); vulvar cancer (including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease); cervical cancer (including, but not limited to, squamous cell carcinoma, and adenocarcinoma); uterine cancer (including, but not limited to, endometrial carcinoma and uterine sarcoma); ovarian cancer (including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor); esophageal cancer (including, but not limited to, squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, and ovarian cancer); carcinoma), mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma);Gastric cancer (including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma); colon cancer; rectal cancer; liver cancer (including, but not limited to, hepatocellular carcinoma and hepatoblastoma), gallbladder cancer (including, but not limited to, adenocarcinoma); cholangiocarcinoma (including, but not limited to, papillary, nodular, and diffuse types); lung cancer (including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer); testicular cancer (including, but not limited to, embryonal, seminoma, undifferentiated, classical (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, carcinoma), choriocarcinoma (yolk sac tumor), prostate cancer (including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma); penal cancer; oral cancer (including but not limited to squamous cell carcinoma); basal carcinoma; salivary gland cancer (including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma); pharyngeal cancer (including but not limited to squamous cell carcinoma, and verrucous melanoma); skin cancer (including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentigo melanoma); kidney cancer (including but not limited to renal cell carcinoma, adenocarcinoma, adenocarcinoma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); Wilms' tumor; bladder cancer (including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma).

[0188] In some embodiments, the cancer is characterized as having one or more mutations in the Adenomatous polyposis coli (APC) gene. The methods and compositions as described are useful for both prophylactic and therapeutic treatments.

[0189] Therapeutic treatment involves administering to a subject after cancer has been diagnosed a therapeutically effective amount of the composition as described, or a pharma- ceutically acceptable salt thereof.

[0190] In further embodiments, the compositions are used for prophylactic use, i.e., to prevent, delay onset, reduce, eradicate, or delay progression of signs or symptoms after onset, and prevent recurrence. In some embodiments, the subject has adenomatous polyps (adenomas) of the colon and / or rectum, which are benign (non-cancerous) growths but may be precursor lesions to colorectal cancer. For prophylactic use, a therapeutically effective amount of the compounds and compositions or pharmacologic acceptable salts thereof as described are administered to the subject before onset (e.g., before obvious signs of cancer), during early onset (e.g., at the time of early signs and symptoms of cancer), or after onset of cancer has been established. Prophylactic administration may occur from days to years before onset of symptoms. Prophylactic administration may be used, for example, in chemo-preventative treatment of subjects exhibiting precancerous lesions, subjects diagnosed with early stage malignancies, and subpopulations (e.g., familial, racial, and / or occupational) susceptible to particular cancers.

[0191] 1. Adenomatous Polyposis Coli (APC) Mutations and Related Cancers Adenomatous polyposis coli (APC) is widely accepted as a tumor suppressor gene that is highly mutated in colorectal cancer (CRC). Mutation and inactivation of this gene are critical and early events observed almost exclusively in colorectal tumorigenesis. Alterations in the APC gene generate a truncated gene product, leading to activation of the Wnt signaling pathway and deregulation of multiple other cellular processes.

[0192] Thus, in some embodiments, the compositions and pharmaceutical formulations thereof are suitable for use in treating one or more symptoms of cancer associated with one or more mutations or inactivation in the APC gene. In a preferred embodiment, the compositions and pharmaceutical formulations thereof are suitable for use in treating one or more symptoms of colon cancer associated with one or more mutations or inactivation in the APC gene.

[0193] VI. Kits Medical kits are also disclosed. The medical kits may include, for example, a dosage supply of GSK-3 inhibitors encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in cancer cells in vivo. The active agent may be provided alone (e.g., lyophilized) or in a pharmaceutical composition. The active agent may be present in a unit dose or in a stock that should be diluted before administration. In some embodiments, the kit includes a supply of a pharma- ceutically acceptable carrier. The kit may also include a device (e.g., a syringe) for administration of the active agent or composition. The kit may include printed instructions for administering the compound in the use as described above.

[0194] The invention will be further understood by reference to the following non-limiting examples. EXAMPLES

[0195] Example 1: Screening of GSK3αβ inhibitors for their ability to support colonic organoid growth material and method Mouse and human organoids were grown in standard organoid culture system using Advanced DMEM / F12 medium with MATRIGEL® and 5% fetal bovine serum, and GLUTAMAX® supplement.The dose response of labeled drugs was applied to organoid cultures, and resazurin viability assay was performed after 4 days of culture.Relative growth was assessed by the conversion of resazurin to resorufin and is shown on the y-axis.

[0196] result Screening of various drugs highlights that LY2090314 potently enables the growth of wild-type colon organoids over a wide drug concentration range, compared to the more restricted range and absolute growth of some of the other GSK3 inhibitors (Figure 1).

[0197] Based on on-target vs. off-target function in organoids, the best performing drugs, in order of decreasing efficacy, are: LY2090314 (Figure 2), SAR502250, A-1070722, and CHIR 99021. Most of the other drugs exhibit limited on-target vs. off-target properties and may function by other mechanisms, especially in colonic epithelial cells.

[0198] Example 2: GSK3 inhibition reduces APC at all concentrations - / - Potently inhibits complex colon cancer organoid genotypes material and method Same as Figure 1, except that the genotype of the organoids tested was varied rather than wild type and contained mutations as indicated in the figure legend.

[0199] result At all concentrations, LY2090314 inhibited the growth of organoids harboring APC mutations, even when additional cancer-causing mutations were added on top of the APC mutation (Figure 3). This compares to the general improvement in growth that can be seen in wild-type organoid growth, highlighting the differential sensitivity of tumor organoid growth to GSK3 inhibition.

[0200] Example 3: Nanoparticle formulations of GSK3αβ inhibitors material and method Nanoparticles were formulated using PLGA 5-10k mw, 50:50 lactic acid to glycolic acid ratio. Synthesized using double emulsion technique with 2% soy lecithin as emulsifier. LY2090314 was encapsulated at 0.1mg / ml and 1:100mg / mg w / w to PLGA in double emulsion using dichloromethane as solvent. Emulsion was induced using 75W sonication power for 2 minutes through a tip probe. The particles were agitated and dried overnight at room temperature.

[0201] result DLS results show that the particles have a polydisperse size range with a bimodal distribution at 500 nm and 10,000 nm (Figure 4).

[0202] Example 4: Treatment of peritoneal carcinomatosis in a mouse model using GSK3αβ inhibitors formulated in nanoparticles material and method Mouse colon tumor organoids were injected into the peritoneum of B6 mice. These tumor organoids were of APC- / - KRAS(G12D), P53- / - genotype (the most common genotype of human colon cancer). One arm (treatment group) received daily injections of the nanoparticles described above containing LY2090314, while the other non-treatment group received nothing. The tumors were imaged via dissection microscopy after one week of treatment or no treatment, since the tumors also expressed TdTomato fluorescent protein.

[0203] result The number of tumors, mean fluorescence, and overall tumor burden (mean fluorescence x area) in treated vs. untreated groups showed a statistically significant reduction in tumor burden in all metrics evaluated (Figures 5A-5C). A 93% reduction in the mean tumor burden per mouse was observed.

[0204] Example 5: Enteral formulation of LY nanoparticles increased survival in a mouse model material and method APC, Min / + mice were treated with LY2090314-containing nanoparticles in their good for 2 weeks as indicated by the dotted line in Figure 6. The dose was given at approximately 2.5 mg / kg / day.

[0205] result Min (multiple intestinal neoplasia) is a mutant allele of the mouse Apc (adenomatous polyposis coli) locus that encodes a nonsense mutation at codon 850. Like humans who carry germline mutations in APC, Min / + mice are predisposed to intestinal adenoma formation. Min mice provide a good animal model to test the role of Apc and interacting genes in the initiation and progression of intestinal and mammary tumorigenesis.

[0206] The data indicate that drug-loaded nanoparticles may increase intestinal regeneration in disease settings, directly stimulate underlying stem cells, and possibly treat adenomas. When the nanoparticles were incorporated into the diet of tumor-prone mice (APC min model), survival was significantly improved by enteral feed-based delivery to the intestines of tumor-prone mice (Figure 6). However, the formulation needs to increase luminal residence time and limit systemic absorption, since we wanted to treat the entire length of the intestine. Therefore, a combination with PHA nanoparticles or other carriers that limit early small intestinal absorption was ideal. Enema may also work and may be the best delivery method for colon or rectal cancer.

[0207] Example 6: Human colon cancer cells are sensitive to the GSK3αβ inhibitor, LY2090314 material and method Human colon cancers were collected from the operating room at MGH. Colon tumor organoid lines were generated from resections and maintained under standard colon organoid culture conditions. LY2090314 dose response was applied to various human colon cancers and resazurin cell counts were performed after 4 days of culture.

[0208] result All human colon cancers showed sensitivity to LY2090314, confirming that this phenomenon is applicable from mouse studies to colon cancers of human origin (Figure 7).

[0209] Furthermore, the use of enriched WRN or Wnt3a, R-spondin3, and Noggin demonstrated the inhibitory ability of the enriched recombinant proteins on mouse colon cancer organoids in a dose-dependent manner (Figure 9).

[0210] Example 7: Combination treatment using a GSK3αβ inhibitor, LY2090314, and other conventional chemotherapeutic agents 5-Fluorouracil (5-FU) is an essential component of systemic chemotherapy for colorectal cancer (CRC) in the palliative and adjuvant setting. Despite encouraging advances in CRC treatment to date, patient response rates to therapy remain low and patient benefits from 5-FU-based regimens are frequently compromised by the development of chemotherapy resistance.

[0211] LY2090314 was used with 5-FU on colon cancer organoids. The data show that this mechanism of cell killing or tumor inhibition by LY2090314 is independent of 5-FU (Figures 8A and 8B). The cell killing or tumor inhibition effect of LY2090314 is additive with 5-FU. Thus, LY2090314 can be added to many of the existing chemotherapy regimens for colon cancer to improve outcomes.

[0212] Example 8: Isolated Wnt proteins can inhibit AKPVT tumor organoid growth Figure 9 shows the relative growth of AKPVT (0-2.0) against WNT enrichment factor (0.125-32). The data clearly showed that isolated Wnt proteins inhibited AKPVT tumor organoid growth at higher concentrations.

[0213] Example 9: In vivo results using GSK3α − / −, GSK3β − / −, KRAS G12D + / −, P53 − / −, TdTomato shAPC (3ABKPT shAPC) organoids material and method Ten RAG2 immunodeficient mice were injected with 300,000 cells of 3ABKPT shAPC organoid cells and divided into two groups (treated with and without doxycycline). Doxycycline 625mg / kg was incorporated into the diet. Over a week, the amount of TdTomato fluorescence was imaged and quantified.

[0214] result These organoids are Wnt-independent by double knockout of both the alpha and beta genes of GSK3. They also have mutations in both copies of KRAS, and P53, so that they mimic their oncogenic signaling pathways in human colon cancer in other ways. They express TdTomato from the Rosa locus, which constitutively fluoresces red when activated in tumors. The shAPC construct allows for doxycycline-dependent knockdown of APC. Using this system, the effect of APC knockdown can be shown in the context of the GSK3α and β locus, in direct contrast to GSK3 drug inhibition in the context of genetic APC loss. This further extends the concept of synthetic vulnerability by inhibition or knockdown of GSK3α and β at APC, but does so without the use of GSK3 inhibitors, showing that APC knockdown in the context of GSK3α and β loss can mediate this effect as well (Figures 10A-10C).

[0215] Similar results were observed when a similar experiment was performed using a spleen injection model of metastatic tumor growth instead of peritoneal carcinomatosis. Briefly, GSK3ABKPT shAPC organoids were dissociated and 250,000 cells were injected into the spleen of mice. The spleen was removed after 5 minutes of perfusion, and the tumor was allowed to grow in the liver of the mice with or without doxycycline. The mice were maintained on either doxycycline-containing or control diet until they reached their endpoint (as defined by euthanasia criteria) or died. There was a significant difference in the survival of mice in those treated with doxycycline, which is shown in the Kaplan-Meier plot in Figure 11.

[0216] Example 10: Enhanced safety and efficacy compared to 5-fluorouracil One of the most important aspects of this treatment modality is the possibility of very limited side effects and potentially improved normal cell growth while simultaneously treating and killing colon cancer. In humans, the chemotherapy, 5-fluorouracil, is the backbone of colon cancer treatment but has significant toxic side effects. Mice tolerate higher doses of 5-fluorouracil on a per body weight per day basis compared to humans. When high doses of 5-fluorouracil are administered to mice, they lose 20% of their total body weight in one week (Figure 12A). This resulted in tumor clearance, which is quantified in Figures 12B-12D. This is in direct contrast to the improved inhibition and treatment effect of tumor burden identified by using a nanoparticle formulation of LY2090314 (Figures 5A-5C). In addition to the improved overall clinical efficacy of LY2090314 in treating and reducing tumor burden, the mice showed minimal weight change and were all active and healthy (represented in Figure 12E).

[0217] Example 11: Further small molecule inhibitors To further generalize the approach of using various GSK3 inhibitors, we identified two other compounds that induce Wnt responses in colonic organoids via GSK3 inhibition. Specifically, SAR502250 and AZD2858 show the ability to grow normal wild-type organoids and promote Wnt responses based on TOP-TdTomato reporter activity. Using primary wild-type colonic crypts, the ability of SAR502250 to promote wild-type organoid growth was evaluated using a resazurin metabolism-based assay. LY2090314 is more potent than SAR502250, but SAR502250 can independently enable wild-type organoid growth (Figure 13A).

[0218] SAR502250 also inhibited colon cancer growth as shown in Figure 13B, where the drug dose response on colon cancer organoids (AKPVT with mutations in APC, KRASG12D, P53, and red fluorescence) was 2x10 -5 Even M clearly shows a milder inhibition than LY2090314. Viability reduction was assessed using a resazurin-based metabolism / proliferation assay.

[0219] AZD2858 also showed a similar ability to grow wild-type organoid cultures. When AZD2858 was applied to wild-type organoids, the drug clearly demonstrated the ability to support normal colonic organoid growth at low nanomolar concentrations (Figure 13C). Furthermore, this was confirmed to be on-target Wnt signaling using the TOP / TdTomato reporter after its genetic incorporation into wild-type organoids.

[0220] Example 12: Combination treatment using R-spondin and LY2090314 Here, we showed that a GSK3 inhibitor (LY2090314) had a synergistic effect when combined with a Wnt agonist, increasing the overall ability to inhibit tumor organoid growth (Figure 14).

[0221] As shown in the heat map below, concentrated RSPO1 alone has a moderate effect on inhibiting cancer organoids alone (bottom row).However, in the presence of low nanomolar concentrations of LY2090314, the effect of RSPO1 on inhibiting colon cancer organoids can be greater than the additive results achieved by each component alone: ​​the reduction in viability of organoids is greater than that of each of the above drugs alone, decreasing by more than 40%.

[0222] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0223] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A composition for increasing Wnt signaling activity for use in a method of treating cancer in a subject in need of such treatment, wherein the cancer comprises cells having one or more mutations in the adenomatous polyposis coli (APC) gene, the method comprising: administering to the subject an effective amount of the composition to increase Wnt signaling activity in the cancer cells of the subject to reduce cancer cell proliferation and / or reduce cancer cell viability in the subject; The composition for increasing Wnt signaling activity comprises a glycogen synthase kinase 3 (GSK-3) inhibitor and / or purified Wnt protein.

2. A composition for increasing Wnt signaling activity for use in a method of treating an adenoma in a subject in need of treatment thereof, wherein the adenoma comprises cells having one or more mutations in the adenomatous polyposis coli (APC) gene, the method comprising: administering to the subject an effective amount of the composition to increase Wnt signaling activity in the adenoma cells of the subject to reduce adenoma cell proliferation and / or reduce adenoma cell viability in the subject; The composition for increasing Wnt signaling activity comprises a glycogen synthase kinase 3 (GSK-3) inhibitor and / or purified Wnt protein.

3. A composition for increasing Wnt signaling activity for use in a method for increasing colon tissue regeneration in a subject in need thereof, the method comprising: administering to the subject an effective amount of the composition to increase Wnt signaling activity in normal cells of the subject and to increase proliferation or survival of the normal cells; The composition for increasing Wnt signaling activity comprises a glycogen synthase kinase 3 (GSK-3) inhibitor and / or purified Wnt protein.

4. The composition of any one of claims 1 to 3, wherein said amount of said composition does not reduce the proliferation and / or viability of healthy cells in said subject.

5. The GSK-3 inhibitor has the structure: 【Chemistry 15】 The composition according to any one of claims 1 to 3, comprising:

6. The composition of any one of claims 1 to 3, wherein the GSK-3 inhibitor is LY2090314, SAR502250, AZD2858, or an analog, derivative, or prodrug thereof.

7. 4. The composition of any one of claims 1 to 3, wherein the GSK-3 inhibitor is encapsulated within and / or associated with a delivery vehicle that increases the serum half-life of the GSK-3 inhibitor compared to the serum half-life of an equivalent amount of the GSK-3 inhibitor alone.

8. 8. The composition of claim 7, wherein the delivery vehicle is a nanoparticle or microparticle selected from the group consisting of a liposome, a polymeric particle, a virus-like particle, and a protein nanostructure.

9. The composition of claim 8, wherein the polymeric nanoparticles comprise poly(lactic-co-glycolic acid) (PLGA) and / or polyhydroxyalkanoate.

10. The composition of claim 1 , wherein the cancer cells are characterized by increased Wnt signaling activity compared to normal control cells.

11. 10. The composition of claim 1, wherein the cancer cells have more than one mutation in the adenomatous polyposis coli (APC) gene.

12. 2. The composition of claim 1, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, peritoneal carcinomatosis, pancreatic cancer, adenocarcinoma, ovarian cancer, multiple myeloma, and sarcoma of the pancreas, bone, bladder, brain, breast, cervix, esophagus, kidney, liver, lung, nasopharynx, prostate, skin, stomach, and uterus.

13. The composition of claim 1 , wherein the cancer is colon cancer.

14. The composition of any one of claims 1 to 3, wherein the composition comprises a pharmaceutically acceptable excipient for administration to the subject.

15. The composition of any one of claims 1 to 3, wherein the composition is administered to the subject by a route selected from the group consisting of intravenous, intramuscular, intravascular, intrapericardial, intrathecal, intracapsular, intraorbital, intracardiac, intraperitoneal, subcutaneous, intraarticular, subarachnoid, intraspinal, and oral.

16. 4. The composition of any one of claims 1 to 3, wherein the composition is administered to the subject in combination with one or more additional active agents selected from the group consisting of chemotherapeutic agents, anti-infective agents, and combinations thereof.

17. The composition of any one of claims 1 to 3, wherein the composition is administered in combination with one or more immune checkpoint modulators selected from the group consisting of a PD-1 antagonist, a PD-1 ligand antagonist, and a CTLA4 antagonist.

18. The composition according to any one of claims 1 to 3, characterized in that the composition is administered in combination with adoptive T cell therapy and / or a cancer vaccine.

19. The composition according to any one of claims 1 to 3, wherein the composition is administered in combination with surgery or radiation therapy.

20. 10. The composition of claim 1, wherein the subject has had surgery to remove cancer and the composition is administered to reduce or prevent the growth of cancer cells in the subject and / or enhance the growth of normal tissue in the subject.

21. 1. A composition for use in a method of enhancing Wnt signaling pathway activity in cancer cells having one or more mutations in the adenomatous polyposis coli (APC) gene, the composition comprising a GSK-3 inhibitor and / or a purified Wnt protein, the method comprising contacting the cancer cells with an effective amount of the GSK-3 inhibitor and / or the purified Wnt protein to increase Wnt signaling in the cells.

22. 22. The composition of claim 21, wherein the method reduces the proliferation and / or viability of the cancer cells with reduced toxicity to normal cells.

23. 22. The composition of claim 21, wherein the cell has more than one mutation in the APC gene.

24. 22. The composition of claim 21, wherein the GSK-3 inhibitor is LY2090314.

25. A dosage form for injection or oral administration comprising a GSK-3 inhibitor or a Wnt protein encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in cancer cells of a subject, reduce cancer cell proliferation and / or reduce cancer cell viability in said subject.

26. A dosage form for injection or oral administration comprising a GSK-3 inhibitor or a Wnt protein encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in adenoma cells of a subject, reduce adenoma cell proliferation and / or reduce adenoma cell viability in the subject.

27. A dosage form for injection or oral administration comprising a GSK-3 inhibitor or Wnt protein encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in normal cells of a subject and increase colon tissue regeneration in the subject.

28. 28. The dosage form of any one of claims 25 to 27, wherein the amount effective to increase Wnt signaling activity in cancer cells does not reduce proliferation and / or viability of healthy cells in the subject.

29. 28. The dosage form of any one of claims 25 to 27, wherein the GSK-3 inhibitor is selected from the group consisting of LY2090314, SAR502250, AZD2858, and combinations thereof.

30. 26. The dosage form of claim 25, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, carcinomatosis, pancreatic cancer, and adenocarcinoma.

31. 26. The dosage form of claim 25, wherein the effective amount is effective to reduce tumor size.

32. 10. A kit comprising a GSK-3 inhibitor encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in cancer cells of a subject and to reduce cancer cell proliferation and / or reduce cancer cell viability in the subject, and instructions for use as described in claim 1.

33. A kit comprising a GSK-3 inhibitor encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in adenoma cells of a subject and to reduce adenoma cell proliferation and / or reduce adenoma cell viability in the subject, and instructions for use as described in claim 2.

34. A kit comprising a GSK-3 inhibitor encapsulated in nanoparticles in an amount effective to increase Wnt signaling activity in normal cells of a subject and to increase colon tissue regeneration in the subject, and instructions for use as described in claim 3.

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