Beta-catenin nuclear transport inhibitors and methods using same

Abstract

The present disclosure relates to methods of treating, preventing, and / or ameliorating a disease or disorder involving excessive Wnt signaling in a subject in need thereof, the method comprising administering to the subject an inhibitor of β-catenin nuclear transport. In certain embodiments, the disease or disorder involving excessive Wnt signaling is cancer. The present disclosure further relates to pharmaceutical compositions comprising at least one inhibitor of β-catenin nuclear transport and a pharmaceutically acceptable carrier.

Description

[0001] TITLE OF THE INVENTION

[0002] Beta-Catenin Nuclear Transport Inhibitors and Methods Using Same

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Patent Application No. 63 / 714,573. filed October 31, 2024, which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under 2R01HL 124402 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The XML file named ”047162-7318WO1 - Sequence Listing.xml” created on October 30. 2025. comprising 15,285 bytes, is incorporated herein by reference in its entirety.

[0008] BACKGROUND

[0009] Wnt pathway members were originally identified as potent oncogenes and regulators of embryonic development. Excessive Wnt signaling has pronounced effects that depend on context. For example, in early embryonic development, increased Wnt signaling may lead to a secondary axis (i.e. “twinning” of the embry o), while in adult tissues, it can lead to excessive cell growth. In fact, aberrant upregulation of Wnt signaling has been implicated in a variety of cancers, including but not limited to colorectal cancers. 90% of which are caused by genetic alterations in Wnt pathway factors. Therefore, chemical inhibitors of Wnt signaling have tremendous therapeutic potential.

[0010] In Wnt signaling, P-catenin (CTNNB1) is a main signaling effector that relays the message from a Wnt ligand at the plasma membrane to transcription factors in the nucleus. As such, levels of -catenin are kept under control through a constitutively active degradation pathway. In the presence of a Wnt ligand, the degradation machinery is sequestered, resulting in an abundance of P-catenin which permits entry7to the nucleus where it drives the transcription of Wnt responsive genes. Despite intensive study, the mechanism of P-catenin translocation from the cytosol to the nucleus remains obscure.

[0011] Thus, there is a need in the art for inhibitors of P-catenin nuclear transport and methods thereof for the treatment of diseases or disorders involving excessive Wnt signaling. The present disclosure addresses this need.

[0012] BRIEF SUMMARY OF THE INVENTION

[0013] In one aspect, the disclosure provides a method of treating, preventing, and / or ameliorating a disease or disorder involving excessive Wnt signaling in a subject in need thereof. In certain embodiments, the method comprises administering to the subject an inhibitor of β-catenin nuclear transport.

[0014] In certain embodiments, the disease or disorder involving excessive Wnt signaling is cancer. In certain embodiments, the cancer is breast cancer, melanoma, prostate cancer, lung cancer, or colorectal cancer.

[0015] In certain embodiments, the inhibitor of P-catenin nuclear transport is a small molecule. In certain embodiments, the small molecule is selected from the group consisting

[0016]

[0017]

[0018] In certain embodiments, the inhibitor of P-catenin nuclear transport is a peptide. In certain embodiments, the peptide comprises an amino acid sequence which shares at least 85% sequence homology with SEQ ID NO: 1 or SEQ ID NO: 12.

[0019] In another aspect, the disclosure provides a pharmaceutical composition comprising at least one inhibitor of β-catenin nuclear transport and a pharmaceutically acceptable carrier.

[0020] BRIEF DESCRIPTION OF THE FIGURES

[0021] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

[0022] FIGs. 1 A-1D show that P-catenin localizes to the nucleus in a RanGEF dependent manner in S', cerevisiae. FIG. 1 A provides a representative deconvolved fluorescence image of x -catenin-GFP in a wild-type yeast strain that expresses Heh2-mCherry to label the nucleus, wherein white arrows indicate the nuclear compartment. FIG. IB provides quantification of mean nuclear to cytosolic fluorescence intensity from three independent replicates corresponding to FIG. 1A. FIG. 1C provides a representative deconvolved fluorescence image of x0-catenin-GFP in the RanGEF mutant mtrl-1) strain at room temperature or at 37 °C that co-expresses Heh2-mCherry as a nuclear envelope marker. FIG. ID provides shows the ratio of mean nuclear to cytosolic fluorescence intensity as measured in the wild-type or mtrl-1 strain from three independent replicates corresponding to FIG. 1C. p-values are from unpaired two-tailed t-test where ns is p>0.05. and ****p<0.0001 for FIG. IB and FIG. ID.

[0023] FIGs. 2A-2E show that the C-terminus of P-catenin contains a nuclear localization sequence (NLS). FIG. 2A: schematic diagram of Xenopus -catenin truncation constructs tested in the present disclosure. FIG. 2B: plot of the ratio of mean nuclear to cytosolic fluorescence intensity of Xenopus P-catenin GFP truncation constructs tested in a wild-ty pe yeast strain from three independent replicates; red bar indicates the mean value with the SD. FIG. 2C: deconvolved fluorescence image of the N-terminal deletion (141-782), ARM-repeats deletion (A141-664) or C-terminal deletion (1-664) of Xenopus p-catenin GFP in the wild-type strain; GFP and full length Xenopus P-catenin-GFP were used as controls. FIG.

[0024] 2D: deconvolved fluorescence images of the indicated fragments of Xenopus P-catenin GFP in the wild-type strain; white arrows indicate nuclear rim localization. FIG. 2E: deconvolved fluorescence images of indicated C-terminus fragments of Xenopus P-catenin GFP in the wild-type strain. Heh2-mCherry was co-expressed to label the nuclear membrane in FIGs.

[0025] 2C-2E.

[0026] FIGs. 3A-3B show sub-cellular localization of Xenopus P-catenin (665-745)-GFP in the S. cerevisiae mtrl-1 strain and HEK293T cells. FIG. 3A: deconvolved fluorescence image of Xenopus p-catenin-(664-745)-GFP in the wild type and RanGEF mutant mtrl-1) strain at room temperature (RT) or 37 °C that co-expresses Heh2-mChery as a nuclear envelope marker; white arrows indicate the nuclear compartment; the ratio of mean nuclear to cytosolic fluorescence intensity7from a single experiment. FIG. 3B: representative image of HEK293T cells expressing Xenopus P-catenin-(665-745). LaminBl was labeled to locate the nuclear envelope. GFP was used as a control. Ratio of nuclear to cytoplasmic intensities from two independent replicates are provided. / )- values are from unpaired two-tailed t-test where ns is p>0.05 and ****p<0.000I.

[0027] FIGs. 4A-4C show that residues 665-745 of P-catenin are required to induce secondary axes in Xenopus laevis. FIG. 4A: schematic diagram of Xenopus P-catenin constructs (SEQ ID NO:7). FIG. 4B: double axes were scored in st 19 embry os viewed dorsally with anterior to the top; data from three independent replicates depicted in histogram; p-values are from Fisher’s exact test where ns is p>0.05, p<0.05 (*), and 0.0021 (**). FIG. 4C: subcellular localization of x P-catenin-GFP, x[3-catenin (A665-745)-GFP or cNLS-xP-catenin (A665-745)-GFP in the dorsal blastopore lip of stage 10 Xenopus laevis embryos. cNLS-mCherry mRNA was co-injected to mark the nucleus.

[0028] FIGs. 5A-5F show that Kapl04 is specifically required for p-catenin nuclear localization in S. cerevisiae. FIG. 5 A: schematic of the Anchor Away assay mediated by the rapamycin induced dimerization of NTR-FRB and PmaI-FKBP12; Pmal is a plasma membrane ATPase. FIGs. 5B-5E: deconvolved fluorescence image of Xenopus P-catenin (665-782)-GFP treated with DMSO (vehicle) or rapamycin for 15 min in the no FRB, Kap95 (Karyopherin pi in human)-FRB, Kapl20 (importin 11 in human) and Kapl04 (KapP2 / Transportin 1 in human)-FRB. Heh2-mCherry was used as a nuclear envelope marker; white arrows indicate the nuclear compartment. FIG. 5F: plot showing the ratio of mean nuclear to cytosolic fluorescence intensity of Xenopus P-catenin (665-782)-GFP in the 10 NTR-FRB strains treated with DMSO or rapamycin. Red bar indicates the mean value with the SD. Experiments were performed three times, p-values are from unpaired two-tailed t-test where ns is p>0.05, and ****p<0.0001.

[0029] FIGs. 6A-6F show sub-cellular localization of Xenopus P-catenin (665-782)-GFP in Anchor Away strains Kapl08-FRB, Kapl21-FRB, Kapil 1-FRB, Kapl22-FRB, Kapl l4-FRB. and Kapl23-FRB, respectively, in S. cerevisiae. Representative deconvolved fluorescence image of xP-catenin (665-782)-GFP treated with DMSO (carrier) or rapamycin in the indicated NTR-FRB strain. Heh2-mCherry w as used as a nuclear membrane marker.

[0030] FIGs. 7A-7D show that P-catenin contains a PY-like NLS that is required for direct binding to TNPO1. FIG. 7A: conservation of amino acid sequences (SEQ ID NOs:8-l 1) that resembles a PY-NLS in the C-terminus of P-catenin. FIG. 7B: in vitro binding assay of purified recombinant TNPO1 to GST fusions of human P-catenin and human P-catenin containing the PM to AA mutations; proteins were separated by SDS-PAGE and stained with Coomassie Blue; GST alone was used as a negative control; * indicates TNPO1 bound to GST-hp-catenin. FIG. 7C: deconvolved fluorescence image of yeast cells expressing MBP(x3)-GFP tagged with the Xenopus P-catenin NLS (665-703) and also an NLS that contains the PM to AA mutation (top); untagged MBP(x3)-GFP was used as a control; plot of the ratio of mean nuclear to cytoplasmic fluorescence intensity from a single experiment (bottom). FIG. 7D: representative fluorescence image of HeLa cells expressing human P-catenin NLS (665-782) or the PM to AA mutant version (top); LaminBl was labeled to locate the nuclear membrane; GFP alone was used as a control; plot of the ratio of mean nuclear to cytoplasmic fluorescence intensity from three experiments (bottom), p-values are from unpaired two-tailed t-test where ns is p>0.05, and ****p<0.0001 for FIGs. 7C-7D.

[0031] FIG. 8 provides sequence lengths of Tnpol and Tnpo2 across species. Amino acid sequences of transportin 1 and transportin 2 were compared across four different species (S'. cerevisiae, human, mouse and Xenopus tropicalis).

[0032] FIGs. 9A-9D show that TNPO1 / 2 and the P-catenin NLS are required for Wnt signaling in vivo. FIG. 9A: depletion of tnpol and tnpo2 using two different pairs of nonoverlapping sgRNAs represses gfp expression inX tropicalis Tg(pbin7Lef-dGFP) embryos at stage 10; key used to quantify embry os with WMISH signal (blue - normal gfp signal, red -reduced gfp signal); un-injected (UIC) embry os were used as a negative control. FIG. 9B: siRNA mediated TNPO1 and / or TNPO2 knockdown reduces luciferase activity in mouse embryonic fibroblasts that harbor a stable integration of luciferase under the control of TCF / LEF promoters; Wnt signaling was activated by human P-catenin-GFP overexpression; control siRNA and GFP were used as negative controls; experiments were performed in triplicate. FIG. 9C: schematic diagram of three -catenin constructs used in the double axis assay in Xenopus laevis, ** indicates P687A, M688A substitutions (top left); dorsal views of X. laevis embry os with anterior to the top (bottom left); dotted lines indicate the embry onic axis and the white arrows indicate the head; histogram of the percent of embryos with secondary axes from three independent replicates. FIG. 9D: shows that the M9M peptide inhibits Wnt signaling; Wnt signaling was activated by Wnt3a (left), human P-catenin-GFP overexpression (middle and right) or cNLS-human P-catenin-GFP (right); no Wnt3a or GFP overexpression were used as negative controls; experiments were performed in triplicate, p-values are from Fisher's exact test FIG. 9A and FIG. 9C; unpaired two-tailed t-test (FIG. 9B and FIG. 9D) where ns is p>0.05, p<0.05 (*), 0.0021 (**), 0,0002 (***) and p<0.0001 (****)

[0033] FIGs. 10A-10E show Xenopus tropicalis tnpol and tnpo2 gene depletion by CRISPR / Cas9. FIG. 10A: schematic diagram of tnpol and tnpo2 sgRNA target sites. FIGs.

[0034] 10B-10E: ICE analysis of indel mutations at predicted target sites.

[0035] FIGs. 11A-1 IB provide Western blots of Tnpol / 2 and P-catenin from 3T3 TCF / LEF luciferase assays. Western blot demonstrating the efficacy of siRNA mediated Tnpol and Tnpo2 depletion in mouse embry onic fibroblast Wnt reporter cell lines.

[0036] FIGs. 12A-12B show Western blots of P-catenin from 3T3 TCF / LEF luciferase assays with M9M peptide (SEQ ID NO: 1) treatment; Western blot data for M9M peptide treatment in mouse embry onic fibroblast Wnt reporter cell lines; Wnt signaling is activated by Wnt3a (FIG. 12A) or human p-catenin (FIG. 12B). FIG. 12C: PY-NLS residues (underlined) in M9M peptide were mutated to alanine (underlined) to create M9M-A peptide (SEQ ID NO:2).

[0037] FIGs. 13A-13C show the Anchor Away cloning strategy' in S', cerevisiae. FIG. 13A: schematic diagram of FRB tagging to individual endogenous NTRs by homologous recombination. FIG. 13B: screening ofNTR-FRB strains by colony PCR. FIG. 13C: positive NTR-FRB strains from the colony PCR in FIG. 13B were further tested for cell grow th as some NTRs are essential for survival; no FRB and DMSO were used as negative controls.

[0038] FIGs. 14A-14F: Exemplary data for compound 10 (TEST-0000119). FIG. 14A: original HTRF using His- -catenin and biotinylated TNPO1. FIG. 14B: control HTRF with biotin-His compound. FIG. 14C: relative response (RU) as a function of time at varied concentrations (i.e., 1.6, 3.1, 6.3, 12.5, 25, and 50 pM). FIG. 14D: relative response (RU) as a function of time at varied concentrations (i.e., 0.2. 0.3, 0.5, 0.8, 1.2, 1.7, 2.6, 3.9, 5.9, 8.8, 13.2. 19.8. 29.6, and 44.4 pM). FIG. 14E: SPR data demonstrating Kd of about 1.9 pM. FIG.

[0039] 14F: SPR data using both 1:1 binding equation and Hill equation.

[0040] FIGs. 15A-15F: Exemplary' data for compound 2 (TEST-0000175). FIG. 15A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 15B: control HTRF with biotin-His compound. FIG. 15C: relative response (RU) as a function of time at varied concentrations i.e., 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, and 500 pM). FIG. 15D: relative response (RU) as a function of time at varied concentrations (i.e., 0.6, 0.9, 1.4, 2.1, 3.1, 4.6, 6.9, 10.4, 15.6, 23.4, 35.1, 52.7, 79, 118.5, 177.8, and 266.7 pM). FIG. 15E: SPR data demonstrating Kd of about 4 pM. FIG. 15F: SPR data using both 1:1 binding equation and Hill equation.

[0041] FIGs. 16A-16D: Exemplary data for compound 3 (TEST-0000173). FIG. 16A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 16B: control HTRF with biotin-His compound. FIG. 16C: relative response (RU) as a function of time at varied concentrations (i.e., 4.6, 6.9, 10.3, 15.4, 34.7, 78, 117.1. 175.6, 263.4, 395.1, 592.6, 888.9, 1333.3. and 2000 pM). FIG. I6D: SPR data using both 1:1 binding equation and Hill equation.

[0042] FIGs. 17A-17D: Exemplary' data for compound 1 (TEST-0000176). FIG. 17A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 17B: control HTRF with biotin-His compound. FIG. 17C: relative response (RU) as a function of time at varied concentrations (i.e., 4.6, 6.9, 10.3, 15.4, 23.1, 34.7, 52, 78, 117.1, 175.6, and 263.4 pM). FIG. 17D: SPR data using both 1:1 binding equation and Hill equation.

[0043] FIGs. 18A-18D: Exemplary data for compound 25 (TEST-0000142). FIG. 18A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 18B: control HTRF with biotin-His compound. FIG. 18C: relative response (RU) as a function of time at varied concentrations (z.e., 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, and 100 pM). FIG. 18D: relative response (RU) as a function of concentration.

[0044] FIGs. 19A-19D: Exemplary data for compound 22 (TEST-0000009). FIG. 19A: original HTRF using His-p-catenin and biotinylated TNPO1. FIG. 19B: control HTRF with biotin-His compound. FIG. 19C: relative response (RU) as a function of time at varied concentrations (z.e., 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, and 500 pM). FIG. 19D: relative response (RU) as a function of concentration.

[0045] FIGs. 20A-20D: Exemplary data for compound 20 (TEST-0000033). FIG. 20A: original HTRF using His-|3-catenin and biotinylated TNPO1. FIG. 20B: control HTRF with biotin-His compound. FIG. 20C: relative response (RU) as a function of time at varied concentrations (z.e., 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, and 500 pM). FIG. 20D: relative response (RU) as a function of concentration.

[0046] FIGs. 21A-21D: Exemplary data for compound 21 (TEST-0000115). FIG. 21 A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 21B: control HTRF with biotin-His compound. FIG. 21 C: relative response (RU) as a function of time at varied concentrations (z.e., 0.8, 1.6. 3.1, 6.3, 12.5, 25, 50, and 100 pM). FIG. 21D: relative response (RU) as a function of concentration.

[0047] FIGs. 22A-22D: Exemplary data for compound 12 (TEST-0000105). FIG. 22A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 22B: control HTRF with biotin-His compound. FIG. 22C: relative response (RU) as a function of time at varied concentrations (z.e., 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, and 100 pM). FIG. 22D: relative response (RU) as a function of concentration.

[0048] FIGs. 23A-23C: Exemplary data for compound 15 (TEST-0000072). FIG. 23 A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 23B: control HTRF with biotin-His compound. FIG. 23C: relative response (RU) as a function of time at varied concentrations (z.e., 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, and 100 pM).

[0049] FIGs. 24A-24B: Exemplary7data for compound 18 (TEST-0000055). FIG. 24A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 24B: control HTRF with biotin-His compound.

[0050] FIGs. 25A-25B: Exemplaiy data for compound 26 (TEST-0000151). FIG. 25A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 25B: control HTRF with biotin-His compound.

[0051] FIGs. 26A-26C: Exemplaiy data for compound 17 (TEST-0000067). FIG. 26A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 26B: control HTRF with biotin-His compound. FIG. 26C: relative response (RU) as a function of time at varied concentrations (z.e., 4.6, 6.9, 10.3, 15.4, 23.1, 34.7, 52, 78, 117.1, 175.6, 263.4. 395.1, 592.6, 888.9, 1333.3, and 2000 pM).

[0052] FIGs. 27A-27D: Exemplary data for compound 5 (TEST-0000145). FIG. 27A: original HTRF using His-P-catenin and biotinylated TNPO1. FIG. 27B: control HTRF with biotin-His compound. FIG. 27C: relative response (RU) as a function of time at varied concentrations (z.e., 0.9, 1.4. 2.1, 3.1, 4.6, 6.9. 10.4. 15.6, 23.4, 35.1, 52.7, 79, 118.5. 177.8, 266.7, and 400 pM). FIG. 27D: relative response (RU) as a function of concentration.

[0053] FIGs 28A-28C: Exemplary’ molecular modeling data for compound 21 (TEST-0000115) in a binding pocket of P-catenin with certain proposed interactions depicted. FIG.

[0054] 28A: exemplary binding model. FIG. 28B: alternate exemplary binding model. FIG. 28C: interaction map for the alternate exemplary binding model of FIG. 28B.

[0055] FIGs. 29A-29C: Exemplaiy data for compound 21 (TEST-0000115). FIG. 29A: AlphaLISA (protein-protein interaction disruption; ~30 pM or lower ICso) using His-P-catenin and biotinylated TNPO1. FIGs. 29B-29C: Percent effect ratio (FIG. 29B) and Percent negative control (FIG. 29C) HTRF assay (protein-protein interaction disruption; ~4.4 pM ICso) using His-P-catenin and biotinylated TNPO1.

[0056] FIGs. 30A-30B: Exemplary7molecular modeling data for the ( / ?)-enantiomer of compound 1 (TEST-0000176) in a binding pocket of P-catenin with certain proposed interactions depicted. FIG. 30A: exemplary binding model. FIG. 30B: interaction map for the exemplary binding model.

[0057] FIGs. 31A-31F: Exemplary data for compound 1 (TEST-0000176). FIG. 31A: SPR data indicating binding to P-catenin (Kj ~ 19 pM); compound was tested from lOOOpM-2.3pM (1.5x dilution). Top concentrations (1000, 666.7, 444.4, 296.3, 197.5 pM), showed a high reference binding and are excluded from the sensograms. FIG. 3 IB: Graph depicting 1:1 binding. FIG. 31C: Graph depicting Hill equation. FIG. 31D: AlphaLISA (protein-protein interaction disruption) using His-P-catenin and biotinylated TNPO1. FIGs. 31E-31F Percent effect ratio (FIG. 31E) and Percent negative control (FIG. 31F) HTRF assay (protein-protein interaction disruption; ~19 pM IC50) using His-P-catenin and biotinylated TNPO1.

[0058] FIGs. 32A-32B: Exemplary molecular modeling data for compound 18 (TEST- 0000055) in a binding pocket of P-catenin with certain proposed interactions depicted. FIG.

[0059] 32A: exemplary binding model. FIG. 32B: interaction map for the exemplary binding model.

[0060] FIGs. 33A-33C: Exemplary data for compound 18 (TEST-0000055). FIG. 33A: AlphaLISA (protein-protein interaction disruption) using His- -catenin and biotinylated TNPO1. FIGs. 33B-33C: Percent effect ratio (FIG. 33B) and Percent negative control (FIG.

[0061] 33C) HTRF assay (protein-protein interaction disruption; ~4 pM IC50) using His-P-catenin and biotinylated TNPOI.

[0062] FIGs. 34A-34B: Exemplary molecular modeling data for compound 10 (TEST-0000119) in a binding pocket of P-catenin with certain proposed interactions depicted. FIG.

[0063] 34A: exemplary binding model. FIG. 34B: interaction map for the exemplary binding model.

[0064] FIGs. 35A-35E: Exemplary data for compound 10 (TEST-0000119). FIG. 31A: SPR data indicating binding to P-catenin (KJ ~ 2-4 pM). FIG. 35B: Graph depicting 1: 1 binding. FIG. 35C: Graph depicting Hill equation. FIGs. 35D-35E Percent effect ratio (FIG. 35D) and Percent negative control (FIG. 35E) HTRF assay (protein-protein interaction disruption; ~65 pM IC50) using His-P-catenin and biotinylated TNPOI.

[0065] FIG. 36: Schematic depiction of aXenopus oocyte microinjection method.

[0066] FIGs. 37A-37E: In vivo studies of Xenopus embryos with administration of TEST-0000176. FIG. 37A: Image of uninjected control and H2O vehicle only. 37B: Image of uninjected control and EtOH vehicle only. FIG. 37C: Image of H2O vehicle injection only and abnormal embryos derived from oocytes administered IX concentration of TEST-0000176 (z.e., 3 / 56 embryos evaluated). FIG. 37D: Image of H2O vehicle injection only and abnormal embryos derived from oocytes administered 2X concentration of TEST-0000176 (z.e., 3 / 49 embryos evaluated). FIG. 37E: Image of uninjected controls. FIG. 37F: Image of uninjected control and abnormal embryos derived from oocytes administered 6X concentration of TEST-0000176 (z.e., 2 / 67 embryos evaluated).

[0067] FIGs. 38A-38C: In vivo studies 0? Xenopus embryos with administration of TEST-0000115. FIG. 38A: Image of EtOH vehicle injection only and abnormal embryos derived from oocytes administered IX concentration of TEST-0000115 (z.e., 1 / 37 embryos evaluated). FIG. 38B: Image of EtOH vehicle injection only and abnormal embryos derived from oocytes administered 2X concentration of TEST-0000115 (z.e., 5 / 30 embryos evaluated). FIG. 38C: Image of uninjected control and abnormal embry os derived from oocytes administered 6X concentration of TEST-0000115 (z.e., 10 / 54 embryos evaluated).

[0068] FIGs. 39A-39C: In vivo studies of Xenopus embryos with administration of TEST-0000119. FIG. 39A: Image of EtOH vehicle injection only and abnormal embryos derived from oocytes administered IX concentration of TEST-0000119 (z.e., 2 / 34 embryos evaluated). FIG. 39B: Image of EtOH vehicle injection only and abnormal embryos derived from oocytes administered 2X concentration of TEST-0000119 (z.e., 3 / 25 embryos evaluated). FIG. 39C: Image of uninjected control and abnormal embryos derived from oocytes administered 6X concentration of TEST-0000119 (z.e., 15 / 46 embryos evaluated).

[0069] DETAILED DESCRIPTION OF THE INVENTION

[0070] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0071] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.

[0072] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting: information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0073] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0074] Description

[0075] The present disclosure describes an unconventional approach to address the longstanding question of P-catenin nuclear transport by exploiting a heterologous model system, the budding yeast Saccharomyces cerevlsiae, which contains conserved nuclear transport machinery. In contrast to prior work, the present disclosure demonstrates that P-catenin accumulates in the nucleus in a Ran dependent manner, which, without wishing to be bound by theory, suggests the use of a nuclear transport receptor (NTR). Systematic and conditional inhibition of NTRs revealed that only Kapl04, the orthologue of Kap-P2 / Transportin-1 (TNPO1), was required for P-catenin nuclear import.

[0076] The present disclosure further provides evidence that direct binding between TNP01 and P-catenin is mediated by a conserved ammo acid sequence which resembles a PY NLS. Using the Xenopus secondary axis and TCF / LEF reporter assays described herein, it has been demonstrated that the results observed in yeast may be directly translated to vertebrates. By elucidating the NLS in p-catenin and its cognate NTR, the present disclosure provides new therapeutic targets for a host of human diseases caused by excessive Wnt signaling.

[0077] The present disclosure further describes the fortuitous identification of the P-catenin NTR as TNPO1, as it is one of the few NTRs where the NLS-NTR interaction is resolved to the atomic level, which is significant for the purposes of rational drug design. This knowledge base has established a consensus amino acid sequence (PY-NLS) that enabled the identification of the P-catenin NLS and the key PM residues required for TNP01 binding. Further, it has led to the generation of novel high affinity peptides that, as shown here, can be used to inhibit Wnt signaling in TCF / LEF luciferase mouse fibroblast cell lines, demonstrating proof of principle that inhibiting TNP01 could be a viable therapeutic strategy.

[0078] The present disclosure further describes certain exemplary compounds which inhibit the P-catenin / TNPOl interaction. In certain embodiments, inhibition of the P-catenin / TNPO 1 prevents nuclear transport of P-catenin and downstream effects thereof. In certain embodiments, inhibition of the P-catenin / TNPOl using the compounds of the disclosure is useful for treating, preventing, and / or inhibiting diseases or disorders associated with aberrant Wnt signaling, including but not limited to colorectal cancer.

[0079] Definitions

[0080] The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0081] A disease or disorder is “ameliorated” or "alleviated" if the severity or frequency of at least one sign or symptom of the disease or disorder experienced by a patient is reduced.

[0082] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0083] The term “excessive” as used herein, refers to a substance or process which occurs in an amount or to a degree that is greater than what is normally observed. In the context of Wnt signaling, “excessive” indicates that the amount of a Wnt signaling ligand, or a process which occurs as a result thereof, is present, or occurs, in an amount or to a degree which is greater than what is normally observed. In certain embodiments, excessive Wnt signaling prevents constitutive degradation of cytosolic P-catenin. In certain embodiments, accumulation of P-catenin in the nucleus is indicative of excessive Wnt signaling.

[0084] The term "independently selected from" as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X1, X2, and X3are independently selected from noble gases" would include the scenario where, for example, X1, X2, and X3are all the same, where X1, X2, and X3are all different, where X1and X2are the same but X3is different, and other analogous permutations.

[0085] The term “inhibit” as used herein, means to reduce a molecule, a reaction, an interaction, a gene, an mRNA. and / or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.

[0086] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the invention, and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0087] The terms “mediated” or “mediated by” as used herein, refer to control exerted over a process or event by one or more necessary elements. A process or event is mediated by a component when involvement of said component is necessary to achieve the process or event. For example, formation of C is mediated by elements A and B if said process (e g. formation of C) necessitates an interaction between the aforementioned elements.

[0088] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or earner, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0089] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and / or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and clathrates thereof.

[0090] The terms “pharmaceutically effective amount” and “effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.

[0091] The term “prevent,” “preventing,” or “prevention” as used herein means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. Disease, condition and disorder are used interchangeably herein.

[0092] By the term “specifically binds” as used herein, is meant a molecule, such as an antibody, which recognizes and binds to another molecule or feature, but does not substantially recognize or bind other molecules or features in a sample.

[0093] As used herein, the terms “subject” and “individual” and “patient” can be used interchangeably and may refer to a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.

[0094] The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%. or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wl% or less. The term "substantially free of can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0095] The terms "‘treat,’' “treating,” and “treatment,” refer to one or more therapeutic or palliative measures described herein. The methods of “treatment” employ administration to a subject, in need of such treatment, a composition, for example, a subject afflicted with a disease or disorder, or a subject who has one or symptoms of such a disease or disorder, in order to cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0096] Compounds

[0097] In one aspect, the disclosure provides exemplary’ compounds which inhibit the [1-catenin / TNPOl interaction, thereby inhibiting nuclear transport of P-catenin, and any downstream effects thereof. Certain exemplary compounds of the disclosure which are suitable for disrupting the P-catenin / TNPOl interaction are provided in Table 1.

[0098] Table 1. Exemplary compounds of the disclosure

[0099]

[0100]

[0101]

[0102]

[0103] Methods

[0104] The present disclosure provides a method of treating, preventing, and / or ameliorating a disease or disorder involving excessive Wnt signaling in a subject in need thereof, the method comprising administering to the subject an inhibitor of [3-catenin nuclear transport.

[0105] In certain embodiments, the disease or disorder involving excessive Wnt signaling is cancer. In certain embodiments, the cancer is selected from the group consisting of breast cancer, melanoma, prostate cancer, lung cancer, and colorectal cancer. In certain embodiments, the cancer is colorectal cancer.

[0106] In certain embodiments, [3-catenin nuclear transport is mediated by TNPO1. In certain embodiments, abrogation of a binding interaction between |3-catenin and TNPO1 inhibits [3-catenin nuclear transport. In certain embodiments, the [3-catenin nuclear transport inhibitor is a TNPO1 inhibitor.

[0107] In certain embodiments, the inhibitor is a small molecule. In certain embodiments, the

[0108]

[0109] small molecule isH. In certain embodiments, the small molecule is

[0110]

[0111] . In certain embodiments, the small molecule

[0112]

[0113] certain embodiments, the small molecule is

[0114]

[0115] . In certain embodiments, the

[0116] small molecule

[0117]

[0118] certain embodiments, the small molecule is

[0119]

[0120] certain embodiments, the small molecule is

[0121]

[0122]

[0123] ,

[0124]

[0125]

[0126] certain embodiments, the small molecule is

[0127]

[0128] ., le is

[0129]

[0130] In certain embodiments, the small molecule is OH In certain embodiments, the small

[0131]

[0132] ,

[0133] embodiments, the small molecule i

[0134]

[0135] certain embodiments, the small

[0136]

[0137] In certain embodiments, the inhibitor is a peptide. In certain embodiments, the peptide comprises an amino acid sequence which shares 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence homology wih SEQ ID NO: 1. In certain embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the peptide comprises an amino acid sequence which shares 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence homology wih SEQ ID NO: 12. In certain embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 12.

[0138] In certain embodiments, the subject is administered at least one additional agent useful for the treatment, prevention, and / or amelioration of a disease or disorder involving excessive Wnt signaling.

[0139] In certain embodiments, the disease or disorder is involving excessive Wnt signaling is cancer.

[0140] In certain embodiments, the administration to the subject is by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes

[0141] In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.

[0142] Salts

[0143] The compounds described herein may form salts with acids or bases, and such salts are included in the present invention. The term '‘salts’’ embraces addition salts of free acids or bases that are useful within the methods of the invention. The term “pharmaceutically acceptable salt’’ refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. In certain embodiments, the salts are pharmaceutically acceptable salts. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity', which have utility' in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods of the invention.

[0144] Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic. 4- hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, sulfanilic, 2-hydroxyethanesulfonic, trifluoromethanesulfonic, p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic, - hydroxybutyric, salicylic, galactaric. galacturonic acid, glycerophosphonic acids and saccharin (e.g.. saccharinate. saccharate). Salts may be comprised of a fraction of one. one or more than one molar equivalent of acid or base with respect to any compound of the invention.

[0145] Suitable pharmaceutically acceptable base addition salts of compounds of the invention include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as. for example, N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (or N- methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

[0146] Combination Therapies

[0147] In one aspect, the compounds of the invention are useful within the methods of the invention in combination with one or more additional agents useful for treating a disease or disorder involving excessive Wnt signaling. These additional agents may comprise compounds or compositions identified herein, or compounds (e.g., commercially available compounds) known to treat, prevent, or ameliorate the symptoms of a disease or disorder involving excessive Wnt signaling.

[0148] Pharmaceutical Compositions and Formulations

[0149] The present disclosure provides a pharmaceutical composition comprising at least one inhibitor of β-catenin nuclear transport and a pharmaceutically acceptable carrier.

[0150] In certain embodiments, the inhibitor is a peptide. In certain embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:1.

[0151] In certain embodiments, the pharmaceutical composition further comprises at least one additional agent useful for the treatment, prevention, and / or amelioration of a disease or disorder involving excessive Wnt signaling. In certain embodiments, the disease or disorder involving excessive Wnt signaling is cancer. In certain embodiments, the cancer is selected from the group consisting of breast cancer, melanoma, prostate cancer, lung cancer, and colorectal cancer.

[0152] The invention provides pharmaceutical compositions comprising at least one compound of the invention or a salt or solvate thereof, which are useful to practice methods of the invention. Such a pharmaceutical composition may consist of at least one compound of the invention or a salt or solvate thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the invention or a salt or solvate thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one compound of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0153] In certain embodiments, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In other embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng / kg / day and 1,000 mg / kg / day.

[0154] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0155] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

[0156] In certain embodiments, the compositions of the invention are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.

[0157] The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[0158] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.

[0159] As used herein, a “unit dose’7is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g, about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0160] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0161] In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier.

[0162] Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0163] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity' may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0164] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and / or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, ‘’additional ingredients” include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.

[0165] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid.

[0166] The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%. or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary' chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelflife of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0167] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil. fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or / 7-propyl para-hydroxybenzoates. ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

[0168] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0169] Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, ionic and non-ionic surfactants, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0170] A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0171] Methods for impregnating or coating a material with a chemical composition are know n in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.

[0172] Administration / Dosing

[0173] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0174] Administration of the compositions of the present invention to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 0.01 mg / kg to 100 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0175] The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day. once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as. but not limited to, type and severity of the disease being treated, and type and age of the animal.

[0176] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0177] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0178] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.

[0179] In certain embodiments, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age. disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.

[0180] Compounds of the invention for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7.000 mg, about 40 pg to about 6,500 mg, about 80 pg to about 6,000 mg, about 100 pg to about 5,500 mg, about 200 pg to about 5,000 mg, about 400 pg to about 4,000 mg. about 800 pg to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in-between.

[0181] In some embodiments, the dose of a compound of the invention is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2.000 mg, or less than about 1.000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg. or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0182] In certain embodiments, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

[0183] The term “container” includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.

[0184] Administration

[0185] Routes of administration of any of the compositions of the invention include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal. transmucosal (e.g. sublingual, lingual, (trans)buccal, (trans )urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonaiy, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0186] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

[0187] Oral Administration

[0188] For oral application, particularly suitable are tablets, dragees, liquids, drops, capsules, caplets and gelcaps. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic, generally recognized as safe (GRAS) pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.

[0189] Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U. S. Patents Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. The capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

[0190] Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

[0191] Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin from animal-derived collagen or from a hypromellose, a modified form of cellulose, and manufactured using optional mixtures of gelatin, water and plasticizers such as sorbitol or glycerol. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0192] For oral administration, the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents; fillers; lubricants; disintegrates; or wetting agents. If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY ® film coating systems available from Colorcon, West Point. Pa. (e.. OPADRY® OY Type. OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY® White, 32K18400). It is understood that similar type of film coating or polymeric products from other companies may be used.

[0193] A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients.

[0194] Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface-active agents include, but are not limited to, sodium laury l sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, com starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to. magnesium stearate, stearic acid, silica, and talc.

[0195] Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are ty pically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a “granulation.” For example, solvent-using “wet” granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.

[0196] Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (z.e., having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e., drug) by forming a solid dispersion or solid solution.

[0197] U. S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt.

[0198] The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds useful within the methods of the invention, and a further layer providing for the immediate release of one or more compounds useful within the methods of the invention. Using a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

[0199] Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); nonaqueous vehicles (e.g, almond oil, oily esters or ethyl alcohol); and preservatives (e.g, methyl or propyl para-hydroxy benzoates or sorbic acid). Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

[0200] Parenteral Administration

[0201] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to. administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques. Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to. suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (z.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0202] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a nontoxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0203] Topical Administration

[0204] An obstacle for topical administration of pharmaceuticals is the stratum comeum layer of the epidermis. The stratum comeum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells. One of the factors that limit the penetration rate (flux) of a compound through the stratum comeum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.

[0205] Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w / w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may-further comprise one or more of the additional ingredients described herein.

[0206] Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

[0207] One acceptable vehicle for topical delivery' of some of the compositions of the invention may contain liposomes. The composition of the liposomes and their use are known in the art ( / .e., U. S. Patent No. 6.323,219).

[0208] In alternative embodiments, the topically active pharmaceutical composition may be optionally combined with other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers, buffering agents, preservatives, and the like. In other embodiments, a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum comeum with respect to a composition lacking the permeation enhancer. Various permeation enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known to those of skill in the art. In another aspect, the composition may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum comeum, and thus allows increased transport across the stratum comeum. Various hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art.

[0209] The topically active pharmaceutical composition should be applied in an amount effective to affect desired changes. As used herein “amount effective” shall mean an amount sufficient to cover the region of skin surface where a change is desired. An active compound should be present in the amount of from about 0.0001% to about 15% by weight volume of the composition. For example, it should be present in an amount from about 0.0005% to about 5% of the composition; for example, it should be present in an amount of from about 0.001% to about 1% of the composition. Such compounds may be synthetically-or naturally derived.

[0210] Buccal Administration

[0211] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may contain, for example. 0.1 to 20% (w / w) of the active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, may have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein. The examples of formulations described herein are not exhaustive and it is understood that the invention includes additional modifications of these and other formulations not described herein, but which are known to those of skill in the art.

[0212] Rectal Administration

[0213] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of. for example, a supposi tory, a retention enema preparation, and a solution for rectal or colonic irrigation.

[0214] Suppository' formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (z.e., about 20°C) and which is liquid at the rectal temperature of the subject (z.e., about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.

[0215] Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to. antioxidants, and preservatives.

[0216] Additional Administration Forms

[0217] Additional dosage forms of this invention include dosage forms as described in U. S. Patents Nos. 6,340,475, 6,488.962, 6,451,808. 5,972,389, 5,582,837, and 5,007,790.

[0218] Additional dosage forms of this invention also include dosage forms as described in U. S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03 / 35041, WO 03 / 35040, WO 03 / 35029, WO 03 / 35177, WO 03 / 35039, WO 02 / 96404, WO 02 / 32416, WO 01 / 97783, WO 01 / 56544, WO 01 / 32217, WO 98 / 55107, WO 98 / 11879, WO 97 / 47285, WO 93 / 18755, and WO 90 / 11757.

[0219] Controlled Release Formulations and Drug Delivery Systems

[0220] In certain embodiments, the compositions and / or formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

[0221] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

[0222] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

[0223] In certain embodiments of the invention, the compounds useful within the invention are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0224] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours.

[0225] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

[0226] The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

[0227] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

[0228] As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

[0229] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples descnbed herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size / volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g.. nitrogen atmosphere, and reducing / oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[0230] It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0231] Materials and Methods

[0232] Xenopus

[0233] Xenopus tropicalis and Xenopus laevis were maintained and cared for in accordance to the Yale University Institutional Animal Care and Use Committee (IACUC) protocols. In vitro fertilization was performed as per standard protocols.

[0234] Saccharomyces cerevisiae strains

[0235] Yeast strains were grown at 30 °C. unless indicated otherwise in YPD medium (1% Bacto yeast extract, 2% bactopeptone, 2% glucose, 0.05% adenine sulfate). Transformation of yeast was carried out using standard protocols.

[0236] Mammalian cells

[0237] HEK293T and HeLa cells were maintained and cultured with DMEM medium + 10% Fetal Bovine Serum (FBS) + 1% Penicillin and Streptomycin (PS) in a T-75 flask. The engineered 3T3 mouse fibroblast cell line that stably expresses the TCF / LEF luciferase transgene was maintained with DMEM medium + Cell Growth Medium Concentrate (Enzo life sciences). Further experimental procedures for the 3T3 cell line can be found under TCF / LEF luciferase assay method section. Upon 70-80% cell confluency, cells were transfected with plasmids using jetPRIME (Polyplus-transfection) following the manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde / PBS and further processed for immunofluorescence 24-48 hours post-transfection. Plasmid, mRNA, siRNA, CRISPR and M9M peptide

[0238] Xenopus p-catenin-GFP (Addgene #16839), Xenopus cNLS-P-catenin-GFP (Addgene #16838), GST-human P-catenin (Addgene #24193), and NLS-mCherry (Addgene #49313) plasmids were obtained from Addgene. GST-transportin 1 (TNPO1) and Gal-MBP(x3)-GFP plasmids were provided from UTSW and University of Groningen, respectively. Gibson Assembly (New England Biolabs) was used to generate GFP tagged Xenopus p-catenin truncation constructs following the manufacturer’s instructions. Both the human and Xenopus P-catenin P687A, M688A variants were generated using Q5 site-directed mutagenesis (New England Biolabs) following the manufacturer’s instructions. Subsequently, the P-catenin constructs were sub-cloned into the pRS406 vector containing an ADH1 promotor for yeast studies or a pCS2+ vector for mammalian / Xenopus studies. mRNAs were generated using the SP6 mMessage machine kit (Thermo Fisher Scientific) and RNA clean & concentrator kit (Zymo Research) following the manufacturer’s instructions. siRNAs were obtained which were directed against mouse TNPO1 (sl08857), mouse TNPO2 (sl02754) and a control siRNA (4390843) from Thermo Fisher Scientific. To generate CRISPR sgRNAs, the EnGen sgRNA synthesis kit (NEB) was used following the manufacturer’s instructions with the following targeting sequences tnpol sgRNA#l (5’-GGCATGGGGGCCACCTCTTG-3’) (SEQ ID NO:3), tnpol sgRNA#2 (5’- GGGTTACGTTTGTCCTCAAG-3’) (SEQ ID NO:4), tnpo2 sgRNA #1 (5’- GGGCGTTTAGCCGCGTTCTA-3’) (SEQ ID NO: 5), and tnpo2 sgRNA #2 (5 - GGCGTCATGGATGAGTCCGA-3 ) (SEQ ID NO:6) (designed using CRISPRscan). CRISPR experiments in wildtype or transgenic Xenopus tropicalis were performed as previously described in Bhattacharya et al. (Dev. Biol. 2015, 408:196-204). CRISPR gene editing efficiency was assessed using Synthego ICE (ice.synthego.com) as previously described in Sempouet al. (Front. Physiol. 2018, 9:1705). The M9M peptide (SEQ ID NO:1) (GGSYNDFGNYNNQSSNFGPMKGGNFGGRFEPYANPTKR) and the M9M-A peptide (SEQ ID NO:2) (GGSYNDFGNYNNQSSNAAAAKGGNFGGAFEAAANPTKR) were synthesized by LifeTein.

[0239] Yeast and mammalian fl-catenin sub-cellular localization by microscopy

[0240] Expression plasmids containing full length and fragments of the P-catenin-GFP coding sequence under the control of the ADFI1 promoter w ere transformed into the W303, Heh2-mCherry:: NAT (BWCPL1314) strain. A yeast colony that incorporated the plasmid sequence w as cultured and mounted onto a coverslip for live imaging on a DeltaVision wide- field microscope (GE Healthcare) with a CoolSnapHQ2CCD camera. Yeast fluorescent images were deconvolved using the iterative algorithm sofWoRx. 6.5.1 (Applied Precision, GE Healthcare). [3-catenin-GFP transfected HeLa or HEK293T cells were mounted on Pro-Long Gold coated coverslip for imaging on a Zeiss Axio Observer microscope. All fluorescent images were analyzed with Fiji software. For quantification, the oval selection tool was used to draw a circle in both the nuclear and cytosolic regions on the same image plane to measure florescence intensity.

[0241] Secondary axis assays and fi-catenin sub-cellular localization

[0242] Xenopus laevis embry os were injected with a mixture of either 200 pg of Xenopus or human -catenin-GFP mRNA and cNLS-mCherry mRNA in one of four cells (targeting the ventral side). Embryos were assessed for a secondary axis via stereomicroscopy at stage 17-19. For the P-catenin localization experiments by fluorescence, Stage 10 embry os were fixed in 4% paraformaldehyde / PBS at 4 °C overnight on a nutator. Embry os were washed in lx PBS + 0.1% TritonX-100, and the dorsal blastopore lips were sectioned with a razor blade and mounted on Pro-Long Gold (Invitrogen) coated coverslip before imaging on a Zeiss 710 confocal microscope.

[0243] The Anchor Away assay

[0244] To employ the Anchor Away approach, a yeast strain was used that harbors a FKBP12 fusion of the endogenous plasma membrane H+-ATPase (PMA1 gene), a Heh2-mCherry fusion to mark the nuclear envelope, and a mutated TORI gene (HHY110: HEH2-mCherry:: KAN, PMAl-2xFKBP12, fprl:: NAT tor 1-1). In this strain, individual, endogenous NTRs are tagged with the FRB domain at the C-terminus by homologous recombination of a PCR product that contains an FRB sequence, a 3x HA epitope, and a selective marker, HIS3. flanked by a 60 bp homology7arm of endogenous NTR coding sequence (FIG. 13 A).

[0245] Integration of an FRB sequence is confirmed by colony PCR using a gene specific forward and a plasmid specific reverse primer and rapamycin induced cell death for essential NTRs (FIGs. 13B-13C for colony PCR primers). Subsequently, an expression plasmid containing the coding sequence for xP-catenin (665-782)-GFP under the control of the ADH1 promoter was transformed into the Anchor Away line. These lines were treated with 1 mg / ml of rapamycin (5-15 minutes of incubation) or vehicle alone (DMSO) before imaging. TCFLEF Luciferase Assay

[0246] A 3T3 mouse fibroblast cell line that has a stable integration of the luciferase reporter gene under the Wnt responsive TCF / LEF promoters were used for this assay (Enzo life sciences). Cells were maintained with DMEM medium + Cell Growth Medium Concentrate (Enzo life sciences). Prior to the transfection, cells were seeded on a 24 well plate in media containing DMEM + Cell Assay Medium Concentrate (Enzo life sciences). Subsequently, cells were transfected with siRNA (25 pmol) first, then GFP or human [3-catenin-GFP DNA (0.5 pg) for 48 hours and 24 hours, respectively using JetPRIME per the manufacturer’s instructions. Luciferase was quantified using the Luciferase Assay System (Promega) and the Promega Glomax luminometer according to the manufacturer’s instructions. For the M9M peptide experiment, cells were transfected with an M9M or M9M-A peptide dose ranging from 0.635 pg

[0247] to 0.5 pg using ProteoJuice Protein transfection following the manufacturer’s instructions for 20 hours and Wnt signaling was activated either by Wnt3a ligand (50 ng / ml) or human P-catenin-GFP DNA (0.5 pg) for 16-24 hours.

[0248] In vivo TCF / LEF GFP in situ hybridization

[0249] Heterozy gous Xenopus tropicalis Tg(pbin7Lef-dGFP) were crossed with wild-ty pe A Iropicalis. Fertilized embryos were injected with sgRNAs targeting Inpol and inpo2 and Cas9 protein at one-cell stage and collected at stage 10 for in situ hybridization as previously-described in Khoka et al. (Dev. Dyn. 2002, 225:499-510). Digoxigenin-labeled anti-sense GFP probe was used to detect GFP transcript expression. Progeny that did not carry the transgene were used as a negative control.

[0250] Western blotting

[0251] 3T3 mouse fibroblast cells were lysed in RIPA buffer to harvest protein samples. Protein levels are normalized and immunoblots were carried out in Bolt 4%-12% Bis-Tris plus gels following standard protocols.

[0252] In vitro binding experiment

[0253] pGEX-6Pl, pGEX-human -catenin or pGEX-human transportin 1 were transformed into the BL21 E. coli strain and cultured in LB with antibiotics to mid-log phase (ODeoo 0.6-0.8). To induce expression of the recombinant proteins (GST alone, GST-hp-catenin and GST-hTNPOl), IPTG was added at a final concentration of 1 mM for 3 hours. All cultures were harvested in 50 mL batches and stored at -20°C until further use. Glutathione Sepharose (GT) beads (Millipore Sigma) were washed and equilibrated in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCh, 10% glycerol, 0.05% NP-40, 1 mM DTT and protease inhibitor cocktail mix (Millipore Sigma)). Bacterial pellets were resuspended with the ice cold lysis buffer, sonicated and spun down at 4°C at 30,000 x g for 20 minutes. The supernatant was collected into a new 50 ml conical tube and incubated with 200 pl of GT bead slurry for 1 hour at 4°C. Subsequently, the GST-GT bead slurry was collected and washed with lysis buffer (excluding the protease cocktail mix). The GST tag was removed from hTNPOl using proTEV Plus Protease (Promega), and the protease enzyme was further removed from hTNPOl protein by Ni-NTA Magnetic Beads (NEB) per the manufacturer's instructions. hNTPOl protein was incubated with GT beads preloaded with GST fusion protein (GST alone or GST-hp-catenin) for 1 hour at 4°C. The beads were washed with the lysis buffer and eluted with SDS-PAGE sample buffer. Protein samples w ere separated by SDS-PAGE and detected with Coomassie (BioRad).

[0254] Quantification and Statistical Analysis

[0255] Statistical significance was defined as p<0.05 (*), 0.002 (**), 0.0002 (***), and 0.0001 (****). The double axis assay and in situ data were analyzed by Fisher's exact tests. Otherwise, unpaired two-tailed Student’s t tests or a one way ANOVA test was used to determine significance of mean ratio of nuclear to cytosolic fluorescence intensity in GraphPad Prism 8.4.3.

[0256] EXAMPLES

[0257] Various embodiments of the present application can be better understood by reference to the following Examples which are offered by w ay of illustration. The scope of the present application is not limited to the Examples given herein.

[0258] Example 1: P-catenin accumulates in the yeast nucleus

[0259] Investigating the P-catenin nuclear import mechanism in yeast relies on the premise that a minimal, conserved P-catenin transport machinery exists in this organism. Therefore, P-catenin accumulation in the yeast nucleus was examined first.

[0260] In principle, three mechanisms of xp-catenin nuclear import are possible: 1) xp-catenin-GFP is imported by an NTR, 2) xP-catenin-GFP piggybacks on an unknown binding partner that is itself imported by an NTR or 3) xP-catenin-GFP has an intrinsic ability- to cross the NPC free of NTRs.

[0261] The localization of aXenopus [3-catenin-GFP (x[3-catenin-GFP) was assessed in a wildtype yeast strain expressing an endogenously tagged nuclear envelope membrane protein, Heh2-mCherry, to help visualize the nuclear boundary. Enrichment of xP-catenin-GFP in the nucleus compared to GFP alone (FIG. 1A). To quantify this steady state distribution, the nuclear enrichment of xP-catenin-GFP was measured by relating the mean GFP fluorescence in the nucleus (N) and cytoplasm (C) (FIG. IB). The xP-catenin-GFP had a mean N: C ratio of ~1.6, which was significantly higher than GFP alone (1.1). As xP-catenin-GFP is 119 kDa, it w ould be unable to easily pass through the NPC diffusion barrier suggesting that xp-catenin-GFP can access a facilitated nuclear transport mechanism through the NPC.

[0262] Example 2: P-catenin import requires a functional Ran pathway

[0263] To rale out crossing of P-catenin w ithout NTRs, the dependency of xP-catenin-GFP nuclear accumulation on a functional Ran gradient was evaluated, which would specifically impact NTR-mediated transport. Therefore. P-catenin-GFP localization was assessed in the mtrl-1 mutant strain, which is a temperature sensitive, loss of function allele in the gene encoding the yeast Ran-GEF (SRM1IMTRLPRP20). As Ran-GEF exchanges GDP for GTP on Ran in the nucleus, it is essential for the functioning of the nuclear transport system. At room temperature, xP-catenin-GFP is localized in the nucleus with a N: C ratio similar to the wild type strain (-1.6) (FIG. 1 C). In striking contrast, growth at 37 °C, which is non-permissive for Mtrlp function, results in the re-distribution of xP-catenin-GFP such that it is evenly distributed between the nucleus and cytoplasm ith N: C ratios identical to GFP alone (1.1) (FIGs. 1C-1D). Importantly, xP-catenin-GFP localization was not affected by the elevated temperature as wild-type cells show-ed N: C ratios of -1.6 even at 37 °C. Thus, nuclear accumulation of xP-catenin-GFP is dependent on a functioning Ran GTPase system raising the possibility' that it requires a NTR-mediated pathway to accumulate in the nucleus.

[0264] Example 3: Identification of nuclear localization elements of P-catenin

[0265] Having established that P-catenin import requires a functional Ran pathway, the sequence elements of P-catenin that confer nuclear localization were subsequently investigated. P-catenin can be divided into three domains: a central domain which is rich in armadillo (ARM) repeats and two flanking unstructured domains (FIG. 2A). Constructs were systematically generated w herein each of these domains w as individually' deleted and the localization of each construct in yeast was examined. Removal of the C -terminus significantly reduced nuclear enrichment of xP-catenin-(l-664)-GFP (mean N: C values of 1.4) compared to constructs lacking either the N [xP-catenin-(141-782)-GFP| or ARM (A141-664) domains, which accumulated in the nucleus at levels similar to the full length protein (mean N: C of 1.6) (FIGs. 2A-2C). These data suggested that the C-temiinus contains sequence elements required for nuclear accumulation. Additionally, the C -terminus of P-catenin (xP-catenin-(665-782)-GFP) was sufficient to confer nuclear accumulation of GFP to levels comparable to the full length protein (mean N: C 1.6) (FIGs. 2A-2B and FIG. 2D). Of note, both the ARM repeats (xP-catenin-(141-664)-GFP) and the N-terminus of P-catenin (xP-catenin-(l-141)-GFP) conferred some nuclear localization of GFP but to a considerably lesser extent than the C-terminus (mean N: C of ~1.3) (FIGs. 2A-2B and FIG. 2D).

[0266] Additionally, the ARM repeats had some affinity for the nuclear periphery (FIG. 2D).

[0267] Overall, while there may be several elements of xP-catenin that, in isolation, can target to the nucleus, the C-terminus contains a sequence that was both necessary and sufficient for nuclear accumulation at levels comparable to the full length protein. These data suggested that the C-terminus of xP-catenin contained an NLS, which was further mapped to amino acids 665-745 (FIGs. 2A-2B and FIG. 2E).

[0268] Further support that nuclear accumulation of xP-catenin-(665-745)-GFP was dependent on the Ran pathway was provided using the mtrl-1 strain (FIG. 3 A). To ensure that this sequence did not confer binding to a yeast-specific factor, localization of xP-catenin-(665-745)-GFP in HEK293T cells, a human embryonic kidney cell line was similarly evaluated. In line with the yeast results, xP-catenin-(665-745)-GFP showed higher levels of nuclear accumulation compared to GFP alone (FIG. 3B).

[0269] Next, the function of the P-catenin NLS in the context of Wnt signaling was investigated using the secondary axis assay in Xenopus. Overexpression of Wnt effectors including P-catenin induces a secondary axis in Xenopus embry os. By injecting a moderate dose (200 pg) of xP-catenin mRNA, secondary' axes develop in roughly half of the embry os (FIGs. 4A-4B) compared to none in the un-injected controls (UIC). Deletion of the coding sequence for the NLS |xP-catenin-(A665-745)-GFP], resulted in a significant reduction of the number of embry os with secondary axes (FIGs. 4A-4B). To ensure that this loss of function is due to the inhibition of P-catenin nuclear import, the classical NLS of the SV40 Large T-antigen (cNLS) was added, which is imported by Kap-a / pi. to the N-terminus of P-catenin [cNLS-xP-catenin-(A665-745)-GFPj.

[0270] It was found that cNLS-xP-catenin-( 665-745)-GFP induced secondary axes similarly to the full length P-catenin (FIG. 4B), which is consistent with the supposition that xP-catenin-(A665-745)-GFP was still functional for Wnt signaling but lacked the nuclear localization element. Supporting this supposition, the xP-catenin-GFP in these Xenopus embryos was imaged and it was observed that while xP-catenin-GFP localizes to the cell membrane and nucleus, xP-catenin-(A665-745)-GFP shows a reduction in nuclear accumulation, which is rescued by the addition of the cNLS (FIG. 4C).

[0271] Example 4: Kap 104 mediates the nuclear import of xP-catenin-(665-782)-GFP in yeast Next, to define the NTR responsible for xP-catenin-GFP nuclear import, the Anchor-Away approach was used to systematically inhibit 10 budding yeast NTRs, all of which have orthologues in human cells (Table 2). This strategy takes advantage of the rapamycin-induced dimerization of a FK506 binding protein (FKBP12) with the FKBP-rapamycin binding (FRB) domain (FIG. 5A). In this system, NTR-FRB fusions are expressed in a strain harboring FKBP12 fused to a highly abundant plasma membrane protein (Pmal) (FIG. 5A). The addition of rapamycin leads to the rapid (~15 min) trapping of the NTRs at the plasma membrane.

[0272] Table 2. List of human NTR genes and yeast orthologs

[0273]

[0274]

[0275] It was systematically tested whether the addition of rapamycin (or the DMSO carrier alone) impacted the nuclear accumulation of xP-catenin-(665-782)-GFP in each of the 10 NTR-FRB strains. Plasma membrane trapping of the KapPl orthologue, Kap95-FRB, did not impact nuclear localization of xP-catenin-(665-782)-GFP (FIG. 5B). Indeed, trapping 9 of the 10 NTRs, including the Imp-11 orthologue, Kapl20, had no overt influence on xP-catenin-(665-782)-GFP nuclear localization (FIGs. 5B-5C and FIGs. 6A-6F). In contrast, a remarkable inhibition of nuclear accumulation was observed specifically when KaplO4-FRB (orthologue of KapP2 / Transportin-l(TNPOl)) was anchored away (FIGs. 5B-5C). These data support a model in which Kapl04 specifically mediates the nuclear import of xP-catenin-(665-782)-GFP in the yeast system.

[0276] Example 5: P-catenin-NLS is directly recognized by TNPO1

[0277] Having established that Kapl04 mediates P-catenin nuclear transport in yeast, the xP-catenin-(665-782) protein sequence was compared to established TNPO1 NLS sequences. By close inspection, the xP-catenin amino acid sequence (665-703) resembles a PY-NLS and is conserved across vertebrate species (FIG. 7A). It has been demonstrated that the binding affinity of TNPO1 to the PY-NLS is most dependent on proline (P) and tyrosine (Y) amino acids (or P and methionine (M) in the context of P-catenin). To test the importance of the P and M amino acids for the function of the xP-catenin NLS, the codons were mutated to two tandem alanine (A) residues, and the impact of these changes on the ability' of the xP-catenin-(665-703) to import a GFP fusion to 3 maltose binding proteins was evaluated. MBP(x3)-GFP is constitutively excluded from the nucleus due to its large molecular weight (149 kDa) (FIG. 7C). Fusion of the xP-catenin-NLS can confer nuclear localization of this large fusion protein. Further, this localization is dependent on the PM motif as substitution of PM with AA abolishes nuclear localization (FIG. 7C). These amino acids were also critical for human (h)P-catenin-(665-782)-GFP nuclear accumulation in human cell lines as the PM to AA substitution reduced the mean N: C ratios from 2.5 to 1.5 (FIG. 7D). Thus, the PM sequence in the P-catenin NLS is required for nuclear import in yeast and human cells.

[0278] To confirm that the P-catenin-NLS is directly recognized by TNPO1, recombinant TNPO1 and GST fusions of human p-catenin (GST-hp-catenin) and human p-catenin were generated containing the PM-AA mutations (GST-hp-catenin P687A. M688A). These GST fusions (and GST alone) were immobilized on GT Sepharose beads and tested binding to purified TNP01. Specific binding of TNPO1 to the GST-h -catenin was observed (FIG. 7B), which was disrupted by the PM to AA mutations in the NLS. When taken together, these data establish a model in which TNP01 can import P-catenin through a direct interaction with its PY-like NLS.

[0279] Example 6: TNPO1 / 2 are required for Wnt signaling

[0280] To explore the function of TNPO1 -mediated import of P-catenin in vertebrates, two different Wnt signaling assays were applied: 1) a TCF / LEF reporter and 2) Xenopus secondary axis development. First, once P-catenin enters the nucleus, it binds to the TCF / LEF complex to activate transcription of Wnt responsive genes. A well-established reporter assay (commonly known as TOPFLASH) places the TCF / LEF DNA binding element upstream of a reporter such as GFP or luciferase. In A tropicalis, the Tg(phin7Lef-dGFP) line has seven tandem TCF / LEF DNA binding sites upstream of GFP and is an effective reporter of Wnt signaling. Heterozy gous transgenic animals were crossed with a wildty pe animal such that half of the resultant progeny had the transgene. In vertebrates, the tnpol gene is duplicated (tnpol and tnpo2 and both paralogs have nearly identical sequence and function (FIG. 8). Therefore, sgRNAs targeting both tnpol and tnpo2 with Cas9 protein were injected at the one cell stage and raised embry os to stlO before fixing them. Because GFP fluorescence is undetectable at these early stages, whole mount in situ hybridization (WMISH) was used to visualize GFP transcripts as an assay' for Wnt reporter activation and used sibling embiyos without the transgene as a WMISH negative control. When both tnpol and tnpo2 were depleted using FO CRISPR, significantly more embryos had weak expression of the GFP transgene compared to un-injected control embryos (FIG. 9 A). This result was specific as a second set of non-overlapping sgRNAs gave comparable results (FIG. 9 A). Importantly, deleterious gene modification was detected at the appropnate targeted sites using Inference of CRISPR Edits (ICE) analysis (FIGs. 10A-10E).

[0281] Next, the function of mouse Tnpol / 2 was tested in a stable transgenic mouse fibroblast cell line in which luciferase was expressed under the control of TCF / LEF DNA binding elements. To activate Wnt signaling, a full length human -catenin-GFP was transfected that increased luciferase signal 7.7 fold over transfection of GFP alone (FIG. 9B). Then the luciferase activity' was measured in transgenic fibroblasts transfected with hp-catenin-GFP in which transcripts of TNPO1 or TNPO2 (alone or simultaneously) were depleted using specific siRNAs. Compared to control siRNA, depletion of either TNPO1 or TNPO2 led to a 34% reduction in luciferase signal (FIG. 9B). By targeting both transcripts. the luciferase signals were reduced by 64% (FIG. 9B). The production of hp-catenin-GFP and the specific reduction of TNP01 / 2 was observed by Western blot (FIGs. 11A-1 IB). Thus, TNP01 / 2 are required for robust Wnt signaling.

[0282] As reduction of TNP01 / 2 would affect the localization of all of their cargos, the specific impact of inhibiting P-catenin nuclear import was evaluated next by testing the function of the PY-AA mutant in the Xenopus secondary axis assay. The number of secondary axes induced by the wildtype h -catenin mRNA to a PM to AA mutated version (P687A, M688A) were compared. A significant reduction in the number of secondary axes induced by the PM-AA mutant P-catenin was observed (FIG. 9C). Addition of cNLS to the N-terminus of the PM-AA mutant rescued induction of secondary' axes. Without wishing to be bound by theory’, these data suggest that the PM-AA mutant fails to enter the nucleus to activate Wnt signaling (FIG. 9C).

[0283] Example 7: The P-catenin- TNPO1 interaction is a viable target for Wnt signaling inhibition

[0284] Having established that TNPO1 binds directly to a PY-like NLS and imports P-catenin into the nucleus, direct perturbation of the P-catenin-TNPO 1 interaction was considered as a viable therapeutic strategy7. Thus, M9M, a TNP01 peptide inhibitor which binds with high affinity to the TNP01 NLS binding site, was considered for evaluation. The M9M peptide was evaluated for potential to inhibit Wnt signaling in the mouse fibroblast TCF / LEF luciferase reporter cell line. Transfection of the M9M peptide reduced luciferase activity’ in a dose dependent manner, regardless of whether activation was induced with a Wnt ligand (Wnt3a) or by co-transfection with human P-catenin (FIG. 9D and FIGs. 12A-12B).

[0285] The M9M peptide is a chimera of the NLSs of hnRNP M and Al. To test specificity, the understanding of the key7amino acids that confer binding to TNP01 was leveraged in each individual NLS to design a control M9M-A peptide. M9M-A contains 7 amino acid substitutions that would abrogate binding to TNPO 1. The M9M-A peptide only reduced luciferase activity by 23%. compared to 53% by M9M at a similar dosage (FIG. 9D).

[0286] The M9M peptide inhibits the nuclear import of a multitude of TNP01 / 2 cargos, thus, it was deemed critical to ensure that the M9M-mediated inhibition of Wnt signaling was specifically due to the reduced nuclear import of P-catenin. Therefore, a human p-catenin was transfected with a cNLS. which would be imported by Kap-ot / Kap-P 1 and thus be insensitive to M9M inhibition. The cNLS-human P-catenin could drive the luciferase reporter to levels comparable to human P-catenin but the M9M peptide only reduced this signal by -10% (FIG.

[0287] 9D). Thus, these data support the conclusion that the M9M peptide’s impact on Wnt signaling is due, at least in part, to the inhibition of P-catenin nuclear transport via TNPO1 / 2. Together, Wnt signaling can be inhibited by blocking TNPO1 mediated import of P-catenin either by mutating the P-catenin NLS or competitive inhibition with the M9M peptide.

[0288] Example 8: Exemplary HTRF and SPR data for certain compounds of the disclosure In one aspect, the disclosure provides exemplary data demonstrating the utility of certain compounds described herein for inhibiting the interaction between P-catenin and TNPO1, including homogenous time-resolved fluorescence (HTRF) assay data and surface plasmon resonance (SPR) assay data. Exemplary compounds for which HTRF and / or SPR data are provided include compound 10 (FIGs. 14A-14F), 2 (FIGs. 15A-15F), 3 (FIGs. 16A-16F), 1 (FIGs. 17A-17D), 25 (FIGs. 18A-18D), 22 (FIGs. 19A-19D), 20 (FIGs. 20A-20D), 11 (FIGs. 21A-21D), 12 (FIGs. 22A-22D), 15 (FIGs. 23A-23C), 18 (FIGs. 24A-24B), 26 (FIGs. 25A-25B), 17 (Figs. 26A-26C), and 5 (FIGs. 27A-27D); See also Table 3. In one aspect, in vitro and / or in vivo data which suggests or demonstrates that a compound at least partially inhibits the P-catenin / TNPOl is indicative of a utility of said compound for disrupting P-catenin nuclear transport, and any downstream effects thereof (e.g., Wnt signaling). Thus, in one aspect, compounds which inhibit the P-catenin / TNPOl interaction are suitable for treating, preventing, and / or ameliorating diseases or disorders associated with aberrant Wnt signaling, including but not limited to colorectal cancer.

[0289] Table 3. Exemplary' P-catenin-TNPOl protein-protein interaction (PPI) interference data

[0290]

[0291]

[0292] The HTRF and / or SPR assays utilized to identify and / or confirm the activity of the compounds are described herein.

[0293] fi-catenin / TNPOl Homogenous Time-Resolved Fluorescence (HTRF) Assay

[0294] PGEX-4T3-h. TNPO1 -biotin Recombinant protein from GenScript

[0295] TNPO1 was produced as a GST fusion protein and biotinylated using an avi-tag. Protein was purified from the supernatant of cell lysate using Ni column + TEV protease cleavage + Superdex 200 column (while we did not get the exact details from GenScript. we suspect that TEV protease had a His tag, and Ni column was used to remove TEV protease after GST was cleaved off).

[0296] 6xHis MBP B-catenin recombinant protein (three different preparations)

[0297] (1) his B-catenin 663-703 (9 mg / ml); (2) his B-catenin 138-781, (4.4 mg / ml); (3) his B-catenin 663-703 (7.6 mg / ml)

[0298] HTRF biochemical assay reagents

[0299] Biotin-TNPOl is captured using streptavidin labeled with an HTRF energy Acceptor or Donor, whereas 6XHis B-catenin is captured using an anti-His Mab labeled with an HTRF energy Acceptor or Donor.

[0300] Detection combinations employed

[0301] Four different detection combinations were employed, as depicted in Table 4.

[0302] Importantly, of the four HTRF detection combinations tested, only two combinations shows significant increase over the no protein background, thus representing a critical assay optimization step. Table 4. HTRF Acceptor / Donor Combinations

[0303]

[0304] Assay optimization Test 1

[0305] Protein-protein cross-titration was performed using four different combinations of the donor and acceptor reagents. The proteins were tested at concentrations of 300, 30, 3 and 0 nM under four HTRF detection combinations (Table 4). Of the four HTRF detection combinations tested, only two combinations showed any increase over no protein background: HTRF MAb Anti-6HIS Tb cryptate DONOR paired with either Streptavidin-XL665 or Streptavidin-d2 ACCEPTOR. Of the three tested B-catenin protein preps, the B-catenin aal38-781 prep had the highest signal above “no B-catenin background”. The signal became saturated at 30 nM B-catenin. At 300 nM B-cat, the “hook” effect typically seen at high protein concentrations due to detection reagent saturation was demonstrated. 30 or 3 nM biotin-TNPOl showed the highest signal above no TNPO1 background. 300 nM TNPO1 had low signal due to the “hook” effect. Of the three his B-cat protein preps, only the 6X his B-cat 138-781 prep showed signal above no 6X his B-cat background signal. The signal became saturated at 30 nM B-cat. At 300 nM B-cat, the “hook” effect was demonstrated. The condition with the highest signal over background was 3 nM biotin-TNPO l and 3 nM his-B-catenin 138-781 with the HTRF detection combination of MAb Anti-6HIS Tb cryptate DONOR paired with Streptavidin-d2 ACCEPTOR.

[0306] Assay Optimization Test 2

[0307] Protein-protein cross-titration centered around 3 nM of protein was performed using the HTRF detection combination of MAb Anti-6HIS Tb cryptate DONOR paired with Streptavidin-d2 ACCEPTOR. The proteins were cross-titrated at 30, 10, 3.3, 1.1, 0.37, 0 nM concentrations. The B-catenin protein showed low background signal in the absence of TNPO1. Concentrations above 10 nM demonstrated the hook effect and as such were not recommended. The TNPO1 protein showed some background signal in the absence of B-catenin. No hook effect was seen at the tested concentrations. The final protein concentrations were selected based on two considerations: highest signal-to-background (S / B) ratio with protein concentrations on the linear part of the dose curve (before reaching the hook effect). Assay Optimization Test 3

[0308] The purpose of the third test was to test for specificity of protein-protein interaction detection by blocking binding of one protein to the other using “unlabeled” B-catenin. Competing protein: recombinant full length human B-catenin protein which is tagged with GST but does not have 6x his tag (Abeam ab63175). Concentration tested: 100 nM (which is 50 times higher than the 2 nM concentration of the 6x his-B-catenin). Result: the “untagged” B-catenin completely blocked the HTRF signal and fully competed with the tagged B-catenin, confirming the specificity of protein-protein interaction detection.

[0309] Assay Optimization Test 4

[0310] The purpose of the fourth test to test for specificity of protein-protein interaction detection by using hbNLS (EQ-60) B-catenin NLS peptide (N-EDKPQDYKKRLSVELTSSLFRTEPMAWNETADLGLDIGAQ-C) (SEQ ID NO: 12). Peptide concentrations tested: 16-point dose response with 2-fold dilutions, 60 mM top and 0.02 nM bottom final assay concentrations). HTRF signal inhibition was observed at the higher peptide concentrations. The amount of inhibition was 60% at 60 mM peptide and 44% at 30 mM, so the peptide did not completely block binding of TNPO to -catenin, however the inhibition was dose-dependent.

[0311] Optimized HTRF Assay Conditions

[0312] Reagents

[0313] • Assay Buffer: HTRF PPI Terbium detection buffer Revvity#61DB10RDF

[0314] • PGEX-4T3-h. TNPOl-biotin Recombinant protein from GenScript

[0315] • 6xHis MBP B-catenin 138-781 Recombinant protein

[0316] • HTRF Mab Anti-6His-Tb cryptate Gold. Cisbio 61HI2TLF

[0317] • HTRF Streptavidin-d2. Cisbio 610SADLF

[0318] • Assay plate: Coming 4513 low volume white low binding 384-well plate

[0319] HTRF Assay Protocol

[0320] • Dispense 20 nL of 1000X compound or DMSO (negative, vehicle control) to dry assay plate using Echo acoustic dispenser (Labcyte, now Beckman).

[0321] • Dispense 5 mL / well of 8 nM his-B-catenin stock using Multidrop Combi reagent dispenser (Thermo Fisher Scientific). Quick spin. Incubate 15 minutes at room temperature to allow compounds to bind. Final concentration of B-catenin in the assay is 2 nM.

[0322] • Dispense 5 mL / well of 40 nM TNPO1 stock using Multidrop Combi reagent dispenser (Thermo Fisher Scientific). Incubate 1 hour at room temperature. Final concentration of TNPO1 in the assay is 10 nM.

[0323] • Dilute Anti-6His-Tb cryptate Gold and Streptavidin-d2 together (1:40 dilution in assay buffer). Dispense 10 mL / well using Multidrop Combi reagent dispenser (Thermo Fisher Scientific).

[0324] • Incubate 1 hour at room tempature.

[0325] • Read HTRF (337 excitation, 665 and 620 emission) using PHERAStar FSX microplate reader (BMG Labtech).

[0326] Exemplary Compound Screen Utilizing HTRF Assay of the Disclosure

[0327] In one non-limiting exemplary screen, 191 compounds and P-catenin NLS peptide (Lifetein) were screened in inter-plate duplicates at a single concentration. Based on the stock concentration, compounds were screened in either 100 rnM final (mostly fragments) or 20 mM final (mostly small molecules). Lifetein -cat NLS peptide was screened at 60 mM final concentration. Each screening plate had 32 replicate wells of the negative and 32 replicate wells of the positive controls (negative control: P-catenin and TNPO incubated with 0.1% DMSO vehicle control in place of compounds; positive control: “no P-catenin’" wells).

[0328] Signal to background S / B (mean of the high-signal positive control divided by the mean of the low-signal negative control). Average S / B = 1.8 observed. Coefficient of variation CV (calculated for both positive and negative control, based on the mean and standard deviations (stdev) values of the control replicates, using (stdev / mean)*100 formula).

[0329] 2.5% average CV of the positive and 7.8% average CV of the negative control observed. Z’ factor (calculated based on the mean and stdev of both control populations, accounts for both assay window and variability). Average Z’=0.41 observed. Data QC: while the variability in this assay is low, the assay window is also low, which affects the Z’. Overall, the screening stats are acceptable.

[0330] HTRF 665 / 615 ratio was monitored as the main screening readout. Test compounds data was normalized to the mean of the negative control samples (set as 0 %Effect) and the mean of the positive control samples (set as 100 %Effect). Individual 665 and 615 fluorescence were also captured and monitored to flag compounds potentially interfering with the HTRF readout. Based on the replicate data for 665 / 615 ration, 665 fluorescence and 615 fluorescence data, 29 compounds were selected for follow-up assays.

[0331] fi-catenin / TNPOl Surface Plasmon Resonance (SPR) Assay

[0332] Surface Plasmon Resonance (SPR) was used to detect potential direct binding interactions of compounds (small molecules and fragments) with P-catenin. The Biacore 8K SPR system from Cytiva Life Sciences was used for these experiments. SPR experiments use a sensor chip made of glass with a thin layer of metal such as gold. The protein, referred to as ligand, gets immobilized on the chip. Following ligand immobilization, solution carrying the compound (referred to as analyte) is then infused over the sensor chip. The change in mass caused by the association or dissociation of the analyte on the sensor chip surface is recorded as a sensogram, and the change is measured in Response Units (RU).

[0333] Each sensor chip has 8 channels allowing for 8 parallel injections to occur simultaneously. Further, each channel has 2 flow cells. Flow cell 2 is the active surface used for the interaction analysis as it has the protein immobilized. Flow cell 1 is the reference surface which lacks immobilized protein and is used as a reference to detect non-specific compound binding.

[0334] Assay Optimization Summary

[0335] For all assay optimizations, 6xHis MBP B-catenin recombinant protein was used: his B-catenin (4.4 mg / ml). The same set of 16 compounds (mix of small molecules and fragments) were tested.

[0336] Assay Optimization Test 1

[0337] The NiHC200M NTA chip from XanTec bioanalytics was used for the assay. This is a high affinity7poly -NTA chip resulting in highly stable affinity-based immobilization of the His-tagged P-catenin. p-catenin was immobilized on the active surface (flow cell 2) using 1x dilution of the 10x HBS-P+ buffer from Cytiva (0. IM HEPES, 1.5 M NaCl, 0.5% v / v Surfactant P20). 30 pg / ml of the protein (diluted in lx HBS-P+ buffer) was injected at a flow rate of 10 pl / min for 10 mins on flow cell 2 for efficient immobilization. After immobilization, compounds were tested for their interaction with P-catenin. A final DMSO concentration of 2% was maintained and, accordingly, DMSO was also added to the running buffer (lx HBS-P+) to maintain a 2% final DMSO concentration. Small molecules were tested at a concentration range of 200 pM - 0.8 pM, while fragments were tested at concentration range of 1000 pM - 4 pM, in a Multi Cycle format, wherein each injection of an analyte concentration is done in a separate cycle, and a single SPR curve is generated per analyte per concentration. The compound association time was set at 120s, the dissociation time was set at 60 s. A constant compound flow rate of 30 pl / min was maintained. After the run, Biacore Insight Evaluation Software was used to QC the data and to analyze any potential binding of the compounds to P-catenin. One of the QC parameters is “Reference binding”. It quantifies binding of the compound (analyte) to the reference surface in the dissociation phase. Compounds that stay bound to the reference surface in the dissociation phase are considered “sticky” (e.g., high non-specific reference binding in the absence of the immobilized protein) and cause an increase in RU values without saturating SPR sensograms. This increase is not due to compound binding to the protein in the association phase, but due to the compound accumulating on the chip surface after every injection. These values were very' high (beyond the permissible limits) for most of the tested compounds. As a result, Test 1 was not optimal to determine the binding of compounds to P-catenin.

[0338] Assay Optimization Test 2

[0339] To overcome the high reference binding displayed by the compounds in Test 1, Assay optimization Test 1 was repeated, except that the final DMSO concentration was increased to 5%. A high reference binding was still observed for the compounds tested, and as a result Test 2 was not optimal to determine the binding of compounds to P-catenin.

[0340] Assay Optimization Test 3

[0341] In this test, CM5 chip (Cytiva) was used to immobilize P-catenin. CM5 chip has a carboxymethylated dextran matrix. The protein (ligand) is covalently bound to the sensor chip surface via carboxyl groups on the dextran. Amine coupling was utilized to covalently capture P-catenin on the chip surface. Amine coupling utilizes the functional group -NH2 present in the ligand. As a first step, pH scouting was performed to determine the optimal pH and ionic strength for ligand immobilization. P-catenin was diluted to 10 pg / ml and 20 pg / ml in 10 mM acetate buffers of different pH (4.0, 4.5, 5.0, 5.5). The diluted protein was injected over pre-activated chip to determine which one of the eight conditions resulted in a high response.

[0342] Based on the pH scouting test, 20 pg / ml of the protein diluted in 10 mM acetate buffer at pH 4.5 showed efficient protein immobilization and was selected for the binding experiments. After protein immobilization, compounds were tested for their interaction with p-catenin. A final DMSO concentration of 2% was maintained and, accordingly, DMSO was also added to the running buffer (lx PBS-P+) to maintain a 2% final DMSO concentration.

[0343] Small molecules were tested at a concentration range of 100 pM - 0.8 pM, while fragments were tested at a concentration range of 500 pM - 4 pM, in a Multi Cycle format, wherein each injection of an analyte concentration is done in a separate cycle and a single SPR curve is generated per analyte per concentration. The compound association time was set at 120s, the dissociation time was set at 60 s. A constant flow rate of 30 pl / min was maintained.

[0344] After the run. Biacore Insight Evaluation Software was used to QC the data and to analyze any potential binding of the compounds to P-catenin. Changing chip chemistry' and shifting to CM5 chip were successful in overcoming the high reference binding for all compounds tested.

[0345] Example 9: In vivo evaluation of certain exemplary compounds in ti Xenopus model In one aspect, the disclosure describes the evaluation of certain compounds of the disclosure for inhibition of head-tail axis formation, indicative of P-catenin inhibition, in a Xenopus model. Depletion of P-catenin eliminates head and back structures in the embryo (z.e., ventralizes embryo), resulting in loss of head, brain, spinal cord, somites (muscles), wherein only ventral tissue is observed with complete depletion. In certain embodiments, addition of P-catenin results in frogs with two heads. Thus, P-catenin is critical for formation of the head-tail axis in Xenopus.

[0346] In one aspect, the compounds of the disclosure are suitable for inhibiting head-tail axis formation in vivo using Xenopus embryos. The in vivo study described herein — microinjection of compounds of the disclosure m ' Xenopus — was performed in a manner consistent with procedures described in the literature, including Khohka et al. (Dev. Dyn., 2002, 225(4):499-510; https: / / doi<dot>org / 10.1002 / dvdy,10184), which is incorporated herein by reference in its entirety. See FIG. 36 for depiction of Xenopus oocyte microinjection.

[0347] In this study, solutions of compound (e.g., TEST-00017, TEST-0000115, and TEST-0000119) were prepared having a concentration of 80 pM (i.e., IX), 160 pM (i.e., 2X), or 480 pM (i.e.. 6X), with certain embryos administered 10 nM or 20 nM formulations to assess dose-responsiveness. In certain embodiments, in vivo studies using compound TEST-0000176 resulted in minimal head loss indicative of minimal P-catenin elimination (FIGs. 37A-37F). No significant dose-responsiveness was not observed in embryos administered the compound at concentrations of 10 nM and 20 nM. In certain embodiments, dose-dependent head loss, and thus P-catenin elimination, was observed with administration of compound TEST-0000115 (FIGs. 38A-38C). In certain embodiments, dose-dependent head loss, and thus P-catenin elimination, was observed with administration of compound TEST-0000119 (FIGs. 39A-39C).

[0348] Sequence Listing

[0349] SEQ ID NO: 1 M9M GGSYNDFGNYNNQSSNFGPMKGGNFGGRFEPYANPTKR SEQ ID NO: 2 M9M-A GGSYNDFGNYNNQSSNAAAAKGGNFGGAFEAAANPTKR SEQ ID NO: 3 tnpol sgRNAl

[0350] GGCATGGGGGCCACCTCTTG SEQ ID NO: 4 tnpol sgRNA2

[0351] GGGTTACGTTTGTCCTCAAG SEQ ID NO: 5 tnpo2 sgRNAl

[0352] GGGCGTTTAGCCGCGTTCTA SEQ ID NO: 6 tnpo2 sgRNA2

[0353] GGCGTCATGGATGAGTCCGA

[0354] SEQ ID NO: 7 NLS SV40 large T antigen

[0355] PKKKRKV SEQ ID NO: 8 human mouse chicken NLS RMSEDKPQDYKKRLSVELTSSLFRTEPMAWNETADLGLDIGAQ SEQ ID NO: 9 zebrafish NLS RMSEDKPQDYKKRLSVELTSSLFRTEPMTWNETGDLGLDIGAQ SEQ ID NO: 10 xenopus NLS RMSEDKPQDYKKRLSVELTSSLFRTEPMPWNEAADLGLDIGAQ SEQ ID NO: 11 P687AM688A RMSEDKPQDYKKRLSVELTSSLFRTEAAPWNEAADLGLDIGAQ SEQ ID NO: 12 hbNLS (EQ-60) B-catenin NLS peptide EDKPQDYKKRLSVELTSSLFRTEPMAWNETADLGLDIGAQ

[0356] SEQ ID NO: 13

[0357] AGCATGGGGGCCACCTCTTG SEQ ID NO: 14

[0358] TGGTTACGTTTGTCCTCAAG SEQ ID NO: 15

[0359] GAGCGTTTAGCCGCGTTCTA SEQ ID NO: 16

[0360] GGCGTCATGGATGAGTCCGA

[0361] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features show n and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be w ithin the scope of embodiments of the present application.

[0362] Enumerated Embodiments

[0363] The following exemplary embodiments are provided, the numbering of w hich is not to be construed as designating levels of importance:

[0364] Embodiment 1 provides a method of treating, preventing, and / or ameliorating a disease or disorder involving excessive Wnt signaling in a subject in need thereof, the method comprising administering to the subject an inhibitor of β-catenin nuclear transport.

[0365] Embodiment 2 provides the method of Embodiment 1, wherein the disease or disorder involving excessive Wnt signaling is cancer.

[0366] Embodiment 3 provides the method of Embodiment 2, wherein the cancer is selected from the group consisting of breast cancer, melanoma, prostate cancer, lung cancer, and colorectal cancer.

[0367] Embodiment 4 provides the method of Embodiment 3, wherein the cancer is colorectal cancer. Embodiment 5 provides the method of any one of Embodiments 1-4, wherein β-catenin nuclear transport is mediated by TNPO1.

[0368] Embodiment 6 provides the method of any one of Embodiments 1-5, wherein the inhibitor is a small molecule.

[0369] Embodiment 7 provides the method of Embodiment 6, wherein the small molecule

[0370]

[0371]

[0372] Embodiment 8 provides the method of any one of Embodiments 1-5, wherein the inhibitor is a peptide.

[0373] Embodiment 9 provides the method of Embodiment 8, wherein the peptide comprises an amino acid sequence which shares at least 85% sequence homology with SEQ ID NO: 1 or SEQ ID NO: 12.

[0374] Embodiment 10 provides the method of any one of Embodiments 1-9, wherein the subject is administered at least one additional agent useful for the treatment, prevention, and / or amelioration of a disease or disorder involving excessive Wnt signaling.

[0375] Embodiment 11 provides the method of Embodiment 10, wherein the disease or disorder is involving excessive Wnt signaling is cancer.

[0376] Embodiment 12 provides the method of any one of Embodiments 1-11. wherein the administration to the subject is by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes.

[0377] Embodiment 13 provides the method of any one of Embodiments 1-12, wherein the subject is a mammal.

[0378] Embodiment 14 provides the method of Embodiment 13, wherein the mammal is a human.

[0379] Embodiment 15 provides a pharmaceutical composition comprising at least one inhibitor of β-catenin nuclear transport and a pharmaceutically acceptable carrier. Embodiment 16 provides the pharmaceutical composition of Embodiment 15, wherein the inhibitor is a small molecule or peptide.

[0380] Embodiment 17 provides the pharmaceutical composition of Embodiment 16, wherein the small molecule is selected from the group consisting of:

[0381]

[0382]

[0383] Embodiment 18 provides the pharmaceutical composition of Embodiment 16, wherein the peptide comprises an amino acid sequence which shares at least 85% sequence homology with SEQ ID NO: 1 or SEQ ID NO: 12.

[0384] Embodiment 19 provides the pharmaceutical composition of any one of Embodiments 15-18, further comprising at least one additional agent useful for the treatment, prevention, and / or amelioration of a disease or disorder involving excessive Wnt signaling.

[0385] Embodiment 20 provides the pharmaceutical composition of Embodiment 19, wherein the disease or disorder involving excessive Wnt signaling is cancer.

[0386] Embodiment 21 provides the pharmaceutical composition of Embodiment 20, wherein the cancer is selected from the group consisting of breast cancer, melanoma, prostate cancer, lung cancer, and colorectal cancer.

[0387] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A method of treating, preventing, and / or ameliorating a disease or disorder involving excessive Wnt signaling in a subject in need thereof, the method comprising administering to the subject an inhibitor of β-catenin nuclear transport.

2. The method of claim 1, wherein the disease or disorder involving excessive Wnt signaling is cancer.

3. The method of claim 2, wherein the cancer is selected from the group consisting of breast cancer, melanoma, prostate cancer, lung cancer, and colorectal cancer.

4. The method of claim 3, wherein the cancer is colorectal cancer.

5. The method of any one of claims 1-4, wherein β-catenin nuclear transport is mediated by TNPO1.

6. The method of any one of claims 1-5, wherein the inhibitor is a small molecule.

7. The method of claim 6, wherein the small molecule selected from the group consisting of:

8. The method of any one of claims 1-5, wherein the inhibitor is a peptide.

9. The method of claim 8, wherein the peptide comprises an amino acid sequence whichshares at least 85% sequence homology with SEQ ID NO: 1 or SEQ ID NO: 12.

10. The method of any one of claims 1-9. wherein the subject is administered at least one additional agent useful for the treatment, prevention, and / or amelioration of a disease or disorder involving excessive Wnt signaling.

11. The method of claim 10. wherein the disease or disorder is involving excessive Wnt signaling is cancer.

12. The method of any one of claims 1-11, wherein the administration to the subject is by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes.

13. The method of any one of claims 1-12, wherein the subject is a mammal.

14. The method of claim 13, wherein the mammal is a human.

15. A pharmaceutical composition comprising at least one inhibitor of β-catenin nuclear transport and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, wherein the inhibitor is a small molecule or peptide.

17. The pharmaceutical composition of claim 16, wherein the small molecule is selected from the group consisting of:

18. The pharmaceutical composition of claim 16, wherein the peptide comprises an amino acid sequence which shares at least 85% sequence homology with SEQ ID NO:1 or SEQ ID NO: 12.

19. The pharmaceutical composition of any one of claims 15-18, further comprising at least one additional agent useful for the treatment, prevention, and / or amelioration of a disease or disorder involving excessive Wnt signaling.

20. The pharmaceutical composition of claim 19, wherein the disease or disorder involving excessive Wnt signaling is cancer.

21. The pharmaceutical composition of claim 20, wherein the cancer is selected from the group consisting of breast cancer, melanoma, prostate cancer, lung cancer, and colorectal cancer.

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