Inhibitors of atypical protein kinase c and their use in treating hedgehog pathway-dependent cancers

Abstract

Methods of treating hedgehog pathway-dependent cancers are provided. Aspects of the methods include inhibiting hedgehog pathway-dependent cancer growth, proliferation, or metastasis promoted by hedgehog pathway signaling. In particular, methods of treating hedgehog pathway-dependent cancers with inhibitors of atypical protein kinase C iota are disclosed.

Description

[0001] Statement as to Federally Sponsored Research or Development

[0002] This invention was made with government support under contract AR054780 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The present invention relates to therapies for treating hedgehog pathway dependent cancers and disorders. In particular, the present invention relates to methods of treating hedgehog pathway dependent cancers and disorders with inhibitors of atypical protein kinase C (aPKC) iota. BACKGROUND

[0005] The Hedgehog (Hh) signaling pathway plays a critical role in development and tumorigenesis in metazoans. Three mammalian Hh genes have been identified: Sonic hedgehog (SHh), Desert hedgehog (DHh), and Indian hedgehog (IHh). These proteins are secreted proteins that act by antagonizing the receptor Patched (Ptchl or Ptch2 in humans). Ptch acts in part by antagonizing the activity of Smoothened (Smo), a G protein-coupled receptor that activates the transcription factor Gli. When Shh binds to Ptch, Ptch-mediated inhibition of Smo is relieved, allowing Smo to promote Gli-dependent transcription. During development, Hh-induced Smo activity promotes proliferation, migration, and differentiation of progenitor cells to pattern organ development. However, dysregulation of Hh pathway signaling, such as by Ptch inactivating mutations or Smo activating mutations, is associated with cancer (Toftgard, R. Hedgehog signaling in cancer. Cell Mol. Life Sci., 57: 1720-1731 (2000)). Tumor growth and maintenance of tumor epithelial cells requires induction of Hh target genes, and Hh pathway signaling is associated with tumor metastasis of many epithelial tumors. For example, initiation and expansion of basal cell carcinoma (BCC) requires high levels of Hh pathway signaling.

[0006] There remains a need for better methods to treat Hh pathway-associated cancers and diseases. SUMMARY

[0008] Methods of treating hedgehog pathway dependent cancers are provided. Aspects of the methods include inhibiting hedgehog pathway dependent cancer growth, proliferation, and / or metastasis promoted by hedgehog pathway signaling. In particular, methods of treating hedgehog pathway dependent cancers with inhibitors of aPKC iota are disclosed.

[0009] In one aspect, there is provided a method of treating a hedgehog pathway dependent cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising CRT0422839 or CRT0364436, or a pharmaceutically acceptable salt thereof.

[0010] In certain embodiments, the cancer comprises a constitutively active hedgehog pathway. In one embodiment, the cancer is basal cell carcinoma (BCC). In some embodiments, the cancer is metastatic.

[0011] Multiple treatment cycles can be administered to the subject over a period of time. For example, the subject can be administered treatment for at least 3 months, at least 6 months, at least 9 months, or at least 12 months or more. Preferably, the subject is administered multiple treatment cycles for a period of time sufficient to achieve at least partial tumor response or more preferably to achieve complete tumor response. In certain embodiments, the composition comprising CRT0422839 or CRT0364436, or a pharmaceutically acceptable salt thereof, is administered according to a daily dosing regimen or intermittently.

[0012] In certain embodiments, the method further comprises administering an additional anti-cancer therapy such as, but not limited to, surgery, chemotherapy, radiation therapy, immunotherapy, biotherapy, or a combination thereof.

[0013] In certain embodiments, the composition administered further comprises a pharmaceutically acceptable excipient.

[0014] In certain embodiments, the method further comprises administering a histone deacetylase (HDAC) inhibitor in combination with CRT0422839 or CRT0364436, or a pharmaceutically acceptable salt thereof. Exemplary HDAC inhibitors include hydroxamic acids such as vorinostat, belinostat, panobinostat, givinostat, dacinostat (LAQ824), and trichostatin A; sesquiterpene lactones such as eleutherobin, cyclic tetrapeptides such as trapoxin B; depsipeptides such as romidepsin, and benzamides such as entinostat (MS-275), tacedinaline (CI994), and mocetinostat. In one embodiment, the HDAC inhibitor is vorinostat.

[0015] In certain embodiments, the subject is a mammal, for example, a human or a non-human primate, a rodent, a farm animal, or a pet.

[0016] In certain embodiments, CRT0422839 or CRT0364436, or a pharmaceutically acceptable salt thereof, is administered in an amount sufficient to reduce viability of hedgehog pathway-dependent cancer cells in a subject.

[0017] In certain embodiments, CRT0422839 or CRT0364436, or a pharmaceutically acceptable salt thereof, is administered in an amount sufficient to reduce production of Gli 1 mRNA in hedgehog pathway-dependent cancer cells in a subject.

[0018] In certain embodiments, CRT0422839 or CRT0364436, or a pharmaceutically acceptable salt thereof, is administered in an amount sufficient to reduce growth and cell proliferation of hedgehog pathway-dependent cancer cells in a subject.

[0019] In another aspect, a method of inhibiting growth or proliferation of hedgehog pathway-dependent cancer cells is provided, the method comprising contacting the hedgehog pathway-dependent cancer cells with an effective amount of CRT0422839 or CRT0364436, or a pharmaceutically acceptable salt thereof.

[0020] In certain embodiments, the hedgehog pathway-dependent cancer cells are BCC cells.

[0021] In certain embodiments, the hedgehog pathway-dependent cancer cells comprise a constitutively active hedgehog pathway.

[0022] In certain embodiments, the hedgehog pathway-dependent cancer cells are in vivo or in vitro.

[0023] In certain embodiments, the hedgehog pathway-dependent cancer cells are mammalian (e.g., human or non-human primate, rodent, farm animal, or pet) cancer cells.

[0024] In certain embodiments, the method further comprises contacting the hedgehog pathway-dependent cancer cells with an HDAC inhibitor.

[0025] In another aspect, a composition comprising CRT0422839 or CRT0364436, or a pharmaceutically acceptable salt thereof, for use in treating a hedgehog pathway-dependent cancer is provided. In some embodiments, the composition further comprises an HDAC inhibitor. In one embodiment, the HDAC inhibitor is vorinostat.

[0026] On the other hand, compositions comprising CRT0422839 or CRT0364436 or pharmaceutically acceptable salts thereof are provided for the treatment of basal cell carcinoma. In some embodiments, the composition further comprises an HDAC inhibitor. In one embodiment, the HDAC inhibitor is vorinostat. Brief description of the attached diagram

[0028] The invention can be best understood by reading the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, by convention, the various features in the drawings are not to scale. Rather, for clarity, the dimensions of the various features have been arbitrarily enlarged or reduced. The drawings include the following figures.

[0029] Figures 1A-1D The effects of atypical protein kinase iota (aPKC1) inhibitors on BCC cell viability were demonstrated. The effects of compounds CRT0422839 and CRT0364436 on BCC viability were tested in the mouse BCC cell line (BSC1) and compared with results from the known peptide inhibitor PSI, previously described in Mirza et al. (JCI Insight (2017) 2(21):e97071). The effects of the aPKC inhibitor PSI (…) were demonstrated. Figure 1A ), CRT0329868 ( Figure 1B ), CRT0364436 ( Figure 1C ) or CRT0422839 ( Figure 1D The viability of BSCI cells after treatment with concentrations of 1 μm and 10 μm.

[0030] Figure 2A and 2B The effects of treatment with PSI, CRT0329868, CRT0422839, or CRT0364436 on GLI1 mRNA expression levels in the mouse BCC cell line (BSC1) were shown. GLI1 mRNA is a marker of Shh pathway output, and it was measured to further document the inhibition of BCC growth and Shh pathway signaling. Treatment with the compounds at concentrations of 1 μm and 10 μm for 6 hours was shown. Figure 2A ) or 24 hours Figure 2B The result after ).

[0031] Figures 3A-3B The aPKC1 inhibitor CRT0364436 / TEV-44229 was shown. Figure 3A ) and CRT0422839 / TEV-47448 ( Figure 3B The chemical structure and properties of ). Invention Details

[0033] Before the present methods and compositions are described, it is to be understood that this application is not limited to the particular methodology or compositions described, as such can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present application will be limited only by the appended claims.

[0034] Where a range of values is provided, unless otherwise stated the intervening values are expressly contemplated as being included in the range. For values which are less than one or one thousandth, or any other range less than one, the intervening values are zero or one-thousandth, etc. For ranges which include one or more intervening whole numbers, any other specified integers, or fractions thereof, intervening values are specifically contemplated. For ranges which include one or both of the same extreme values, intervening values excluding the one or both of that extreme value are also contemplated. The same principle applies to ranges having an upper and lower limit. In addition, any numerical range recited is intended to include all sub-ranges subsumed therein. For example, a range of "1 to 10" is intended to include all sub-ranges between and including the minimum value of 1 and the maximum value of 10, that is, all sub-ranges having a minimum component of 1 and a maximum component of 10. Also, all references to "or" can be construed as a statistical "or" rather than an exclusive "or", so, for example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and some combination thereof.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It will be understood that the disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0036] As will be apparent to those of ordinary skill in the art in light of the present disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present application. Any recited method can be carried out by

[0037] It must be noted that as used herein and in the appended claims, the singular form "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the inhibitor" includes reference to one or more inhibitors and equivalents thereof known to those skilled in the art, and so forth.

[0038] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publications. Further, the dates of publication provided can be different from the dates that appear on the publications that were provided, which can need to be independently confirmed.

[0039] The term "about," particularly with respect to a given quantity, means to cover variations of plus or minus five percent.

[0040] The terms "tumor," "cancer," and "neoplasia" are used interchangeably and refer to a cell or group of cells whose growth, proliferation, or survival is greater than that of a normal counterpart, e.g., a cell proliferative, hyperproliferative, or differentiative disorder. Typically, the growth is uncontrolled. The term "malignant" refers to invasion of nearby tissues. The term "metastasis" or secondary, recurrent, or recurrent tumor, cancer, or neoplasia refers to the spread or dissemination of a tumor, cancer, or neoplasia to other sites, locations, or regions in the body of a subject, where the site, location, or region is different from the primary tumor or cancer. Neoplasia, tumors, and cancers include benign, malignant, metastatic, and non-metastatic types, and include any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.) of neoplasia, tumor, or cancer, or a neoplasia, tumor, cancer, or metastasis that is progressing, worsening, stable, or in remission. In particular, the terms "tumor," "cancer," and "neoplasia" include carcinomas such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma.

[0041] The term "hedgehog pathway-dependent cancer" includes any cancer that is dependent on hedgehog pathway activation or associated with abnormal activation of the Hedgehog pathway such as, but not limited to, basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, small cell lung cancer, retinoblastoma, gastric and upper gastrointestinal cancer, osteosarcoma, pancreatic cancer, breast cancer, colon cancer, ovarian cancer, brain cancer, breast cancer, thyroid cancer, and prostate cancer.

[0042] "Antitumor activity" means a reduction in the rate of cell proliferation, and thus a reduction in the rate of growth of an existing tumor or a tumor that arises during treatment, and / or destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and thus a reduction in the overall size of a tumor during treatment. Such activity can be assessed using animal models, such as xenograft models of human renal cell carcinoma. See, e.g., Pulkkanen et al., In Vivo (2000) 14:393-400 and Everitt et al., Toxicol. Lett. (1995) 82-83:621-625 for descriptions of animal models.

[0043] A "therapeutically effective dose or amount" of an aPKC iota inhibitor (e.g., CRT0422839 or CRT0364436) means an amount that, as described herein, when an aPKC iota inhibitor is administered, or otherwise administered in combination with a histone deacetylase inhibitor (e.g., vorinostat), elicits a positive therapeutic response, such as anti-tumor activity. In addition, an "effective amount" of an aPKC iota inhibitor can inhibit the growth, proliferation, and / or metastasis of hedgehog pathway-dependent cancer cells, and / or reduce the production of Gli 1 mRNA in hedgehog pathway-dependent cancer cells, and / or reduce the viability of hedgehog pathway-dependent cancer cells.

[0044] The term "tumor response" as used herein means any measurable reduction or elimination of disease. Criteria for tumor response are based on the WHO Report Criteria [WHO Offset Publication, 48-World Health Organization, Geneva, Switzerland (1979)]. Ideally, all one- or two-dimensional measurable lesions should be measured at each assessment. When multiple lesions are present in any organ, such measurement can not be possible, and, in that case, the six most representative lesions should be selected, if any.

[0045] The term "complete response" (CR) as used herein means the complete disappearance of all clinically detectable malignant disease, as determined by at least two assessments at least 4 weeks apart.

[0046] The term "partial response" (PR) as used herein means a 50% or greater reduction in the sum of the products of the longest perpendicular diameters of all measurable disease, in the absence of any evidence of progressive disease and in the absence of any new lesions, as determined by at least two consecutive assessments at least 4 weeks apart. Assessments should show a partial decrease in size of lytic lesions, recalcification of lytic lesions, or a decrease in density of blastic lesions. Transient inflammation observed at metastatic disease sites is not uncommon. An individual lesion showing an increase in size does not necessarily disqualify a PR unless that increase is documented in two consecutive measurements at least 28 days apart.

[0047] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that can optionally be included in the composition and that does not cause a significant adverse toxicological response to the patient.

[0048] "Pharmaceutically acceptable salts" include, but are not limited to, salts of amino acids, salts prepared with inorganic acids such as chlorides, sulfates, phosphates, hydrogen phosphates, bromides, and nitrates, or salts prepared from corresponding inorganic acid forms of any of the foregoing, e.g., hydrochlorides, etc., or salts prepared with organic acids such as malates, maleates, fumarates, tartrates, succinates, ethyl succinates, citrates, acetates, lactates, methanesulfonates, benzoates, ascorbates, p-toluenesulfonates, palmoates, salicylates, and stearates, as well as estolates, gluconates, and lactobionates. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0049] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) increasing survival time; (b) decreasing the risk of death from a disease; (c) preventing a disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (d) inhibiting the disease, i.e., arresting its development (e.g., reducing the rate of disease progression); and (e) relieving the disease, i.e., causing regression of the disease.

[0050] "Substantially pure" generally refers to an isolated material (e.g., a compound, molecule, reagent) such that the material comprises a majority percentage of the sample in which it is found. Typically, a substantially pure component comprises 50% of a sample, preferably 80-85%, more preferably 90-95% of a sample.

[0051] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to any mammalian subject for whom diagnosis, prognosis, treatment, or therapy is desired, particularly humans. "Mammals" for purposes of treatment include all organisms classified as mammals, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some instances, the methods of the application find use in experimental animals, veterinary applications, and development of animal models of disease, including, but not limited to, rodents, including mice, rats, and hamsters; primates, and transgenic animals.

[0052] Methods

[0053] Methods of treating hedgehog pathway-dependent cancers with inhibitors of aPKC iota are disclosed. In some embodiments, inhibitors of aPKC iota are used in combination with inhibitors of HDAC1. Without being bound by a particular theory, aPKC phosphorylates the GLI1 transcription factor, leading to chromatin binding of GLI1 and activation of gene transcription leading to hedgehog pathway activation. GLI1 activity is controlled by regulation of its nuclear import. That is, GLI1 moves between its inactive nuclear lamina and its active nucleoplasm. The mechanism by which aPKC iota activates GLI1 appears to involve recruitment of HDAC1 to GLI1, where GLI1 is activated by HDAC1 -mediated deacetylation. Thus, inhibitors of aPKC iota and inhibitors of HDAC1 can be useful in treating hedgehog pathway-dependent cancers.

[0054] While the methods of the application are directed to treatment of existing tumors, it is recognized that the methods can be useful in preventing further tumor growth during a treatment period.

[0055] Inhibitors of atypical protein kinase C iota

[0056] As explained above, the methods of the application include administration of an inhibitor of aPKC iota. Exemplary inhibitors of aPKC iota include CRT0422839 (TEV-47448) and CRT0364436 (TEV-44229), or a pharmaceutically acceptable salt thereof.

[0057] CRT0422839 has the chemical formula:

[0058]

[0059] CRT0364436 has the chemical formula:

[0060]

[0061] Such inhibitors of aPKC iota have anti-tumor activity in treating hedgehog pathway-dependent cancers. In particular, these inhibitors of aPKC iota have the ability to inhibit growth, proliferation, and metastasis of hedgehog pathway-dependent cancer cells, reduce production of Gli 1 mRNA in hedgehog pathway-dependent cancer cells, and reduce the viability of hedgehog pathway-dependent cancer cells (see Examples).

[0062] Inhibitors of HDAC1

[0063] In certain embodiments, combination therapy is practiced with an aPKC iota inhibitor and an HDACl inhibitor. Exemplary HDACl inhibitors include hydroxamic acids such as vorinostat, belinostat, panobinostat, givinostat, dacinostat (LAQ824), and trichostatin A; sesquiterpene lactones such as depsipeptide; cyclic tetrapeptides such as trapoxin B; depsipeptides such as romidepsin, and benzamides such as entinostat (MS-275), trapoxin (CI994), and mocetinostat. In some embodiments, an inhibitor is used that selectively inhibits HDACl without affecting other HDAC classes, such as depsipeptide.

[0064] Inhibition of the Hedgehog signaling pathway

[0065] The use of aPKC iota inhibitors, alone or in combination with HDAC inhibitors, inhibits Hh pathway signaling as described herein. By inhibit, it is meant to reduce, suppress, decrease, attenuate, or antagonize the activity of the pathway. For example, it can be desirable to inhibit Hh pathway signaling with aPKC iota inhibitors and / or HDAC inhibitors in cells in which the Hh pathway is overactive or constitutively active, such as cancer cells, for example, in cells comprising an activating mutation in the Smo gene or an inactivating mutation in the Ptch or SUFU gene, or in cells in which pathway activators such as SHH / IHH ligand, SMO, or GLI1 / 2 are overexpressed. Other mutations that promote abnormal activation of the hedgehog signaling pathway are well known and can be readily determined by one of skill in the art. For a review of mutations associated with abnormal activation of hedgehog signaling, particularly mutations implicated in cancer, see, e.g., Pellegrini et al. (2017) Int. J. Mol. Sci. 18(11) pii: E2485, Bao et al. (2018) Mol. Nutr. Food Res. 62(1), Levanat et al. (2017) Curr. Pharm. Des. 23(1):73-94, Laukkanen et al. (2016) Anticancer Agents Med. Chem. 16(3):309-317, Suzman et al. (2015) Cancers (Basel). 7(4):1983-1993, Holikova et al. (2004) Int. J. Dermatol. 43(12):865-869, Wetmore (2003) Curr. Opin. Genet. Dev. 13(1):34-42, Bale (2002) Annu. Rev. Genomics Hum. Genet. 3:47-65, Lacour et al. (2002) Br. J. Dermatol. 146 Suppl 61:17-19, Wicking et al. (2001) Cancer Lett. 173(1):1-7, and Daya-Grosjean et al. (2005) Cancer Lett. 225(2):181-192; incorporated herein by reference.

[0066] In practicing the subject methods, the aPKC iota inhibitor, alone or in combination with the HDAC inhibitor, is provided to the cell in an effective amount, i.e., an amount effective to inhibit Hh pathway signaling. Biochemically, an effective amount or effective dose of the aPKC iota inhibitor and / or HDAC inhibitor is an amount sufficient to inhibit Hh pathway signaling in the cell by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, or 500% or more. In other words, the activity of the Hh signaling pathway in a cell contacted with an effective amount or effective dose of the aPKC iota inhibitor or HDAC inhibitor will be about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or will be about 0%, i.e., negligible, of the activity observed in a cell that has not been contacted with an effective amount / dose of the aPKC iota inhibitor and / or HDAC inhibitor. In other words, the Hh pathway signaling will be changed by about 0.5-fold or more, 1-fold or more, 2-fold or more, 5-fold or more, 8-fold or more, or 10-fold or more.

[0067] The amount of aPKC iota inhibitor or HDAC inhibitor that inhibits cellular activity can be determined in a variety of ways known to one of ordinary skill in the art of molecular biology. For example, the amount of phosphorylated transcription factor Gli in the cell can be measured by Western blotting; the amount of binding to Gli DNA target sequences can be measured by electrophoretic mobility shift assay (EMSA); the amount of expression of transcription factors normally activated by Hh signaling, such as ptchl, ptch2, hhipl, nhk2, and rab34, can be measured by, for example, measuring the RNA or protein levels of genes that are Gli transcriptional targets, or by transfecting / infecting cells with a nucleic acid vector comprising a Gli-responsive promoter operably linked to a reporter protein such as luciferase, EGFP, etc., and qualitatively or quantitatively measuring the amount of reporter protein produced. In this way, the inhibitory effect of the aPKC iota inhibitor or HDAC inhibitor can be demonstrated.

[0068] In a clinical sense, an effective dose of an aPKC iota inhibitor or HDAC inhibitor is one that, when administered for a suitable period of time, typically at least about one week, and possibly about two weeks, or longer, up to about 4 weeks, 8 weeks, or longer, will prove to alter symptoms associated with undesired activity of the Hh signaling pathway. For example, an effective dose of an aPKC iota inhibitor or HDAC inhibitor is one that, when administered for a suitable period of time, typically at least about one week, and possibly about two weeks, or longer, up to about 4 weeks, 8 weeks, or longer, will slow, stop, or reverse tumor growth and metastasis in a patient having cancer. Those of ordinary skill in the art will appreciate that an initial dose can be administered over this period of time, followed by maintenance doses, which in some cases will be at a reduced dose.

[0069] It is within the skill of one of ordinary skill in the art to calculate the effective amount or effective dose of an aPKC iota inhibitor or HDAC inhibitor to be administered, and will be routine to one of ordinary skill in the art. Not to be overlooked, the final amount to be administered will depend on a variety of factors, including the route of administration, the nature of the disorder or condition to be treated, and factors that differ between patients. The skilled clinician will be able to determine the effective amount of therapeutic agent to administer to a patient as needed to stop or reverse the progression of the disease condition. With the LD 50 Using ordinary skill, the skilled clinician will be able to optimize the dose of a particular treatment during routine clinical trials. Other useful information

[0070] The subject methods can be used to inhibit Hh pathway signaling - and thus cellular activities associated with Hh pathway signaling - in cells in vitro and in vivo. For example, any cell in which Hh pathway signaling is undesirable, such as a cancer cell in which uncontrolled Hh pathway signaling promotes proliferation or metastasis, can be contacted with an aPKC iota inhibitor and / or an HDAC inhibitor. The cell can be from any mammalian species, such as murine, rodent, canine, feline, equine, bovine, ovine, primate, human, etc.

[0071] If the subject method is performed in vitro, the cells can be from an established cell line or they can be primary cells, where "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that have been derived from a subject and that are permitted to grow in vitro for a limited number of culture passage (i.e., division) times. For example, a primary culture is one that can have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but fewer than the number of times sufficient to pass through a crisis phase. Typically, the primary cell lines of the present application are maintained in vitro for fewer than 10 passages.

[0072] If the cells are primary cells, they can be harvested from an individual by any convenient method. For example, the cells, e.g., blood cells, e.g., white blood cells, can be harvested by apheresis, leukopheresis, density gradient separation, etc. As another example, the cells, e.g., skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, nervous system tissue, etc., can be harvested by biopsy. A suitable solution can be used to disperse or suspend the harvested cells. Such a solution will typically be an isotonic salt solution, e.g., physiological saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal bovine serum or other naturally occurring factors, in combination with an acceptable low concentration of a buffer (typically 5-25 mM). Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately, or they can be stored for long periods, frozen, thawed, and used again. In this case, the cells will typically be frozen in 10% DMSO, 50% serum, 40% buffered media, or some other such solution as is commonly used in the art, to preserve the cells at such frozen temperatures, and thawed in a manner as is commonly known in the art for thawing frozen cultured cells.

[0073] The aPKC iota inhibitor or HDAC inhibitor can be dissolved in water or alcohol or a solvent such as DMSO or DMF and diluted into water or a suitable buffer prior to being provided to the cells.

[0074] To modulate Hh pathway signaling, a cell can be provided with an aPKC iota inhibitor or an HDAC inhibitor for a period of about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period of time from about 30 minutes to about 24 hours, which can be repeated at a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. The subject cell can be provided with one or more doses of the agent, e.g., one, two, three, or more than three, and the cell is allowed to incubate with the agent for a period of time, e.g., 16-24 hours, after which the medium is exchanged for fresh medium and the cell is further cultured.

[0075] Contact of a cell with an aPKC iota inhibitor or an HDAC inhibitor can occur in any medium and under any culture conditions that promote cell survival. For example, the cell can be suspended in any convenient suitable nutrient medium, such as Iscove's Modified DMEM or RPMI 1640 supplemented with fetal bovine serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols (particularly 2-mercaptoethanol), and antibiotics (e.g., penicillin and streptomycin). The culture can contain growth factors to which the cell responds. As defined herein, a growth factor is a molecule that is capable of promoting cell survival, growth, and / or differentiation in culture or in an intact tissue by specific action on a transmembrane receptor. Growth factors include polypeptide and non-polypeptide factors. Conditions that promote cell survival generally permit nonhomologous end joining and homologous recombination.

[0076] Cancerous cells of interest for study and treatment in the present application include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells, wherein the cancerous phenotype is promoted by Hh pathway signaling. In other words, Hh pathway signaling (and, in many cases, unregulated Hh pathway signaling) predisposes a cell in an individual to become cancerous, or induces or enhances a cancerous condition in an individual, e.g., tumor growth and metastasis. In many such cases, Hh pathway signaling is elevated in a tumor cell relative to the level of signaling observed in a healthy cell, e.g., by a factor of 2 or more, 3 or more, 4 or more, 6 or more, 8 or more, 10 or more, 20 or more, or 50 or more, above the level of Hh pathway signaling in a healthy cell. The level of Hh signaling can be measured by any convenient method, e.g., as known in the art or as described herein.

[0077] In some applications, aPKC iota inhibitors or HDAC inhibitors are employed to modulate Hh pathway signaling in vivo, for example, to inhibit tumor growth or metastasis to treat cancer. In these in vivo embodiments, the aPKC iota inhibitors or HDAC inhibitors are administered directly to an individual. The aPKC iota modulators can be administered by any of a number of well-known methods in the art as described below.

[0078] Formulations

[0079] The aPKC iota inhibitors (e.g., CRT0422839 or CRT0364436) or HDAC inhibitors can be incorporated into a variety of formulations. More particularly, the aPKC iota inhibitors or HDAC inhibitors can be formulated into pharmaceutical compositions by combination with suitable pharmaceutically acceptable carriers or diluents as would be known to one of ordinary skill in the art. A pharmaceutical formulation is a composition comprising one or more aPKC iota inhibitors and / or HDAC inhibitors in a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" can be a vehicle approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which the compound of the application is formulated for administration to a mammal. Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposomal dendrimers; liquids such as water and oils, including those of either vegetable or animal origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, saline; acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and coloring agents can be used. The pharmaceutical compositions can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Thus, administration of the aPKC iota inhibitors and / or HDAC inhibitors can be achieved by a variety of means including transdermal, intradermal, oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, and the like. The active agents can be systemic upon administration or can be localized through the use of regional administration, intramural administration, or the use of implants that exhibit a sustained release of the active agent at the site of implantation. The active agents can be formulated for immediate release or can be formulated for sustained release.

[0080] For some conditions, particularly central nervous system conditions, it can be desirable to formulate agents that cross the blood brain barrier (BBB). One strategy for drug delivery through the blood brain barrier (BBB) entails biochemical disruption of the BBB through osmotic means (such as mannitol or leukotrienes) or through the use of vasoactive substances (e.g., bradykinin). The potential to target specific agents to brain tumors using BBB opening is also an option. When the composition is administered by intravascular injection, the BBB-disrupting agent can be co-administered with the therapeutic composition of the present application. Other strategies for crossing the BBB can entail the use of endogenous transport systems, including clathrin-coated pit-1 -mediated transcytosis, carrier-mediated transporters (such as glucose and amino acid carriers), receptor-mediated transcytosis of insulin or transferrin, and active efflux transporters (such as p-glycoprotein). Active transport moieties can also be conjugated to the therapeutic compounds used in the present application to facilitate transport across the vascular endothelial wall. Alternatively, drug delivery of the therapeutic agent after the BBB can be by local delivery, for example, by intrathecal delivery, for example, through an Ommaya reservoir (see, e.g., U.S. Patent Nos. 5,222,982 and 5385582, incorporated herein by reference); by bolus injection, for example, through a syringe, for example, intravitreally or transcranially; by continuous infusion, for example, through a cannula insertion, for example, with counter-current flow (see, e.g., U.S. Application No. 20070254842, incorporated herein by reference); or by implantation of a device that has reversibly attached the agent (see, e.g., U.S. Application Nos. 20080081064 and 20090196903, incorporated herein by reference).

[0081] For inclusion in a medicament, the aPKC iota inhibitor and / or HDAC inhibitor can be obtained from a suitable commercial source. As a general matter, the total pharmaceutically effective amount of the aPKC iota inhibitor and / or HDAC inhibitor per dose of parenteral administration will be within a range that can be measured by a dose response curve.

[0082] The aPKC iota inhibitor-based therapy, with or without an HDAC inhibitor, i.e., the formulation for therapeutic administration, can be sterile. Sterility is readily achieved by filtration through sterile filtration membranes, e.g., 0.2 μιη membranes. Therapeutic compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Compositions comprising an aPKC iota inhibitor and / or an HDAC inhibitor can be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as aqueous solutions or as lyophilized formulations for reconstitution with a sterile aqueous solution. As an example of a lyophilized formulation, a 10 mL vial is filled with 5 ml of sterile filtered 1% (w / v) aqueous solution of the compound and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound using bacteriostatic water for injection. Alternatively, an aPKC iota inhibitor and / or an HDAC inhibitor can be formulated as a lotion for topical administration.

[0083] Pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are defined as vehicles, solvents, coatings, antibacterial and antifungal agents, isotonic and absoibable salts, preservatives, antioxidants, solubilizers, binders, excipients, disintegration, lubricants, sweetening, flavoring, or coloring agents approved by a regulatory agency of the Federal or a state government for use in animals or humans. Examples of such diluents include distilled water, buffered water, normal saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, pharmaceutical compositions or formulations can include other carriers, adjuvants or nontoxic, nontherapeutic, nonimmunogenic stabilizers, excipients, etc. The compositions can also include additional substances to approximate physiological conditions such as pH, buffering and salts, toxicity reducing agents, wetting and detergents.

[0084] The compositions can also contain any of a variety of stabilizing agents, such as antioxidants. Where the pharmaceutical composition comprises a polypeptide, the polypeptide can be complexed with a variety of well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, decrease its toxicity, enhance solubility or absorption). Examples of such modifying or complexing agents include sulfates, gluconates, citrates, and phosphates. The nucleic acids or polypeptides of the compositions can also be complexed with molecules that enhance their in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0085] Further guidance concerning formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249: 1527-1533 (1990).

[0086] The pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatments. Toxicity and therapeutic efficacy of active ingredients can be determined by standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Therapies that exhibit large therapeutic indices are preferred.

[0087] The data obtained from the cell culture and / or animal studies can be used in formulating a range of dosage for use in humans. The dosage of active ingredients typically falls within a range that includes an ED50 with minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[0088] The components used to formulate the pharmaceutical compositions are preferably of high purity and essentially free of potentially harmful contaminants (e.g., at least National Formulary (NF) grade, generally at least analytical grade, and more usually at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. In regards to a given compound that must be synthesized prior to use, the final product is generally substantially free of any potentially toxic agents that can be present during synthesis or purification, particularly any endotoxins. The compositions for parenteral administration are also sterile and substantially isotonic and prepared under GMP conditions.

[0089] In addition to other agents, aPKC iota inhibitors and / or HDAC inhibitors can be provided. For example, in methods of treating a cancer promoted by Hh pathway signaling, aPKC iota inhibitors and / or HDAC inhibitors can be co-administered with other known cancer therapies.

[0090] Administration

[0091] Hedgehog pathway-dependent cancers that can be treated according to the methods described herein include any cancer that is dependent on Hedgehog pathway activation or associated with abnormal or constitutive activation of the Hedgehog pathway, such as, but not limited to, basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, small cell lung cancer, retinoblastoma, gastric and upper gastrointestinal cancers, osteosarcoma, pancreatic cancer, breast cancer, colon cancer, ovarian cancer, brain cancer, breast cancer, thyroid cancer, and prostate cancer.

[0092] A therapeutically effective dose or amount of an aPKC iota inhibitor (e.g., CRT0422839 or CRT0364436) is administered alone or in combination with an HDAC inhibitor and / or optionally other anticancer agents. A "therapeutically effective dose or amount" of each of these agents means an amount that, when administered, results in a positive therapeutic response with respect to treatment of the individual's hedgehog pathway-dependent cancer. Of particular interest are amounts of these agents that provide an anti-tumor effect as defined herein. A "positive therapeutic response" means that the individual receiving treatment according to the application exhibits an improvement in one or more symptoms of the individual's hedgehog pathway-dependent cancer for which the individual is receiving therapy.

[0093] Thus, for example, a "positive therapeutic response" would be an improvement in the disease associated with the therapy (e.g., therapy with an aPKC iota inhibitor or combination therapy with an aPKC iota inhibitor and an HDAC inhibitor and / or optionally other anticancer agents), and / or an improvement in one or more symptoms of the disease associated with the therapy. Thus, for example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) reduction in tumor size; (2) reduction in the number of cancer cells; (3) inhibition (i.e., slowing to some extent and preferably stopping) of tumor growth; (4) inhibition (i.e., slowing to some extent and preferably stopping) of cancer cell infiltration into peripheral organs; (5) inhibition (i.e., slowing to some extent and preferably stopping) of tumor metastasis; and (6) relief to some extent of one or more symptoms associated with the cancer.

[0094] This therapeutic response can be further characterized in terms of the degree of improvement. Thus, for example, the improvement can be characterized as a complete response. A "complete response" refers to documentation of disappearance of all measurable or assessable signs and symptoms of disease confirmed by physical examination, laboratory, nuclear, and radiologic studies (i.e., CT (computed tomography) and / or MRI (magnetic resonance imaging)), and repeat of other non-invasive procedures on all initial abnormalities or positive sites at entry into the study. Alternatively, the improvement in the disease can be categorized as being a partial response. A "partial response" refers to a greater than 50% reduction in the sum of the products of the perpendicular diameters of all measurable lesions when compared to pretreatment measurements (partial response is not applicable to patients with only assessable responses).

[0095] In certain embodiments, aPKC iota inhibitors are administered in multiple therapeutically effective doses, alone or in combination with HDAC inhibitors and optionally other anti-cancer agents, according to a daily dosing regimen or intermittently. For example, the administration of a therapeutically effective dose can be one day per week, two days per week, three days per week, four days per week, or five days per week, and so on. "Intermittently" administration means that the administration of a therapeutically effective dose can be, for example, every other day, every two days, every three days, and so on. For example, in some embodiments, aPKC iota inhibitors, alone or in combination with HDAC inhibitors and / or optionally other anti-cancer agents, are administered twice per week or three times per week for an extended period of time, for example, for 1, 2, 3, 4, 5, 6, 7, 8... 10... 15... 24 weeks, and so on. "Twice per week" or "twice weekly" means that two therapeutically effective doses of the agent in question are administered to the subject within a 7-day period, starting on day 1 of the first week of dosing, with a minimum of 72 hours and a maximum of 96 hours between doses. "Three times per week" or "three times weekly" means that three therapeutically effective doses are administered to the subject within a 7-day period, with a minimum of 48 hours and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as "intermittent" therapy. According to the methods of the present invention, a subject can receive intermittent therapy (i.e., administration of a therapeutically effective dose twice per week or three times per week) for one or more week cycles until a desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as described below.

[0096] In certain embodiments, combination therapy with aPKC iota inhibitors and HDAC inhibitors and optionally other anti-cancer agents is administered. The aPKC iota inhibitor can be administered prior to, concurrently with, or subsequent to the HDAC inhibitor. If provided concurrently with the HDAC inhibitor, the aPKC iota inhibitor can be provided in the same or different composition. Thus, both agents can be presented to the individual by way of concurrent therapy. "Concurrent therapy" means administration to a human subject such that a therapeutic effect of the combination of substances is elicited in the subject receiving treatment. For example, concurrent therapy can be achieved by administration of at least one therapeutically effective dose of a pharmaceutical composition comprising an aPKC iota inhibitor and at least one therapeutically effective dose of a pharmaceutical composition comprising at least one HDAC inhibitor, according to a particular dosing regimen. Similarly, the aPKC iota inhibitor and / or the HDAC inhibitor and optionally other anti-cancer agents can be administered in at least one therapeutic dose. Administration of separate pharmaceutical compositions can be at the same time (i.e., concurrently) or at different times (i.e., sequentially, in either order, on the same day or on different days), as long as a therapeutic effect of the combination of these substances is elicited in the subject receiving treatment.

[0097] In certain embodiments, the aPKC iota inhibitor is administered for a brief period prior to administration of the HDAC inhibitor and continues for a brief period after the HDAC inhibitor treatment has ceased to ensure that the levels of the aPKC iota inhibitor in the subject during treatment are sufficient to inhibit the binding of GLI1 and HDAC1 and the activation of the GLI1 and hedgehog signaling pathway. For example, administration of the aPKC iota inhibitor to the subject can begin one week prior to the administration of the first dose of the HDAC inhibitor and continue for one week after the administration of the last dose of the HDAC inhibitor.

[0098] In other embodiments, the pharmaceutical composition comprising an agent such as an aPKC iota inhibitor and / or an HDAC inhibitor and / or optionally other anticancer agents is a sustained release formulation, or is a formulation that is administered using a sustained release device. Such devices are well known in the art and include, for example, transdermal patches and microimplantable pumps, which can provide drug delivery over time in a continuous, steady state manner at various dosages to achieve a sustained release effect with a non-sustained release pharmaceutical composition.

[0099] The pharmaceutical compositions comprising an aPKC iota inhibitor and / or an HDAC inhibitor and optionally other anticancer agents can be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art. Suitable routes of administration include parenteral administration, such as subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous (IV) or infusion, oral, pulmonary, nasal, topical, transdermal, intratumoral, and suppository. When the composition is administered by pulmonary delivery, the therapeutically effective dose is adjusted so that the soluble levels of the agent such as an aPKC iota inhibitor and / or an HDAC inhibitor in the bloodstream are equivalent to those obtained with a therapeutically effective dose administered parenterally (e.g., SC, IP, IM, or IV). In some embodiments, the pharmaceutical compositions comprising an aPKC iota inhibitor and / or an HDAC inhibitor and optionally other anticancer agents are administered by IM or SC injection, particularly by IM or SC injection locally to the tumor. In some embodiments, the aPKC iota inhibitor and / or an HDAC inhibitor and optionally other anticancer agents are administered topically, for example, on a patch or in a gel.

[0100] In some embodiments, the aPKC iota inhibitor and / or HDAC inhibitor and optionally other anti-cancer agent is administered by infusion or by local injection, for example by infusion at a rate of about 50 mg / h to about 400 mg / h, including about 75 mg / h to about 375 mg / h, about 100 mg / h to about 350 mg / h, about 150 mg / h to about 350 mg / h, about 200 mg / h to about 300 mg / h, about 225 mg / h to about 275 mg / h. Exemplary infusion rates can achieve a desired therapeutic dose, for example, about 0.5 mg / m 2 per day to about 10 mg / m 2 per day, including about 1 mg / m 2 per day to about 9 mg / m 2 per day, about 2 mg / m 2 per day to about 8 mg / m 2 per day, about 3 mg / m 2 per day to about 7 mg / m 2 per day, about 4 mg / m 2 per day to about 6 mg / m 2 per day, about 4.5 mg / m 2 per day to about 5.5 mg / m 2 per day. Administration (e.g., by infusion) can be repeated over a desired period, for example, repeated over a period of about 1 day to about 5 days or every few days, for example, once about 5 days, about 1 month, about 2 months, etc. The aPKC iota inhibitor and / or HDAC inhibitor can also be administered prior to, concurrent with, or after other therapeutic interventions such as surgical intervention to remove cancerous cells. The aPKC iota inhibitor and / or HDAC inhibitor can also be administered as part of a combination therapy in which at least one of immunotherapy, chemotherapy, radiotherapy, or biotherapy is administered to the subject.

[0101] Factors affecting the respective amounts of the various compositions to be administered include but are not limited to the mode of administration, the frequency of administration (i.e., daily or intermittent administration, such as twice or three times per week), the particular disease being treated, the severity of the disease, the history of the disease, whether the individual is being concurrently treated with another therapeutic agent, and the age, height, weight, health and physical condition of the individual being treated. Generally, a higher dosage is preferred for older individuals. Generally, a higher dosage is preferred for more severe cases of disease. Generally, a lower dosage is preferred for pregnant or lactating women, for younger individuals, and for less severe cases of disease. Usually, a higher dosage is preferred for patients who show an improved tolerance for the agent. In general, the dosage is to be increased when necessary up to a maximum of 400 mg / hour. It is understood that the dose will be adjusted to the body surface area of the subject.

[0102] The individual dose of the aPKC iota inhibitor and / or HDAC inhibitor and optionally other anticancer agent is generally not less than the amount required to produce a measurable effect on the subject, and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion ("ADME") of the aPKC iota inhibitor and / or HDAC inhibitor and optionally other anticancer agent and its metabolites, and thus based on the distribution of the composition in the subject. This includes consideration of the route of administration and dosage, which can be adjusted for local (for primarily local effects directly at the site of desired action), enteral (for systemic or local effects via the digestive tract when retained in part of the digestive tract), or parenteral (for systemic or local effects via a route other than the digestive tract) application. For example, administration of the aPKC iota inhibitor (e.g., CRT0422839 or CRT0364436) can be local or by injection, e.g., intravenous, intramuscular, or intratumoral injection or combinations thereof.

[0103] The distribution of the aPKC iota inhibitor in the subject and its corresponding biological activity is generally assessed from the proportion of the aPKC iota inhibitor present at the target of interest. For example, once the aPKC iota inhibitor is administered, it can accumulate with the glycoconjugate or other biological target, causing the substance to accumulate in cancer cells and cancerous tissue. Thus, a dosing regimen that administers the aPKCiota inhibitor so as to accumulate in the target of interest over time can be part of a strategy that permits lower individual doses. This can also mean, for example, that the dose of the aPKC iota inhibitor that is cleared more slowly in vivo can be reduced relative to the effective concentration calculated from in vitro assays (e.g., in vitro effective amounts approximate mM concentrations, relative to in vivo concentrations below mM).

[0104] As an example, the effective dose or dosing regimen can be calculated from the IC 50 of a given aPKC iota inhibitor for inhibiting aPKC kinase activity and / or GLI1 activation, and / or activation of the hedgehog pathway, and / or cell proliferation, and / or cell migration / invasion. 50 The precise measurement "IC 50 " means the concentration of drug required for 50% inhibition in vitro. Alternatively, the effective amount can be calculated from the EC 50 The precise measurement "EC

[0105] In general, the effective amount is generally no more than 200 times the calculated IC 50 . Typically, the effective amount is no more than 100 times the calculated IC 50about 75X, less than about 60X, 50X, 45X, 40X, 35X, 30X, 25X, 20X, 15X, 10X, even less than about 8X or 2X. In one embodiment, the effective amount is about IX to 50X of the calculated IC 50 about 2X to 40X, about 3X to 30X, or about 4X to 20X of the calculated IC 50 In other embodiments, the effective amount is the same as the calculated IC 50 and in certain embodiments, the effective amount is greater than the calculated IC 50

[0106] The effective amount typically does not exceed 100 times the calculated EC 50 For example, the amount of aPKC iota inhibitor administered is less than about 100X, less than about 50X, less than about 40X, 35X, 30X, or 25X, and many embodiments are less than about 20X, less than about 15X, even less than about 10X, 9X, 9X, 7X, 6X, 5X, 4X, 3X, 2X, or IX of the calculated EC 50 about 1X to 30X of the calculated EC 50 about 1X to 20X, or about 1X to 10X of the calculated EC 50 The effective amount can also be the same as or greater than the calculated EC 50 50 IC 50 can be calculated by inhibiting aPKC kinase activity and / or GLI1 activation, and / or cell proliferation and / or cell migration / invasion in vitro.

[0107] To achieve efficacy, the level of aPKC iota inhibitor must be above a certain level for a certain period of time. Efficacy is dose dependent, with higher levels of aPKC iota inhibitor contributing to greater anti-tumor effect. To minimize toxicity, the level of aPKC iota inhibitor can be kept below a certain level for a certain period of time and for a certain period of time ("rest period" allows for clearance of aPKC iota inhibitor). That is, the drug is kept below a certain level until a certain period of time before the next dose is given. Shorter rest periods between multiple doses result in greater toxicity.

[0108] ​​In certain embodiments, the method of treating a patient having a hedgehog pathway dependent cancer comprises a treatment cycle with an aPKC iota inhibitor alone or in combination with an HDAC inhibitor and / or optionally other anti-cancer agents, followed by a rest period in which no aPKC iota inhibitor and / or HDAC inhibitor is administered to allow the patient to "recover" from unwanted effects of the aPKC iota inhibitor and / or HDAC inhibitor. Multiple doses of the aPKC iota inhibitor and / or HDAC inhibitor can be administered according to a daily dosing regimen or intermittently followed by a rest period.

[0109] When a subject treated according to the foregoing dosing regimen exhibits a partial response or relapses after an extended period of remission, a subsequent course of treatment can be required to achieve complete remission of the disease. Thus, after a rest period of time off the first treatment period, the subject can receive one or more additional treatment periods comprising administration of an aPKC iota inhibitor alone or in combination with an HDAC inhibitor and optionally other anti-cancer agents. Such rest periods between treatment periods are referred to herein as discontinuation periods. It is recognized that the length of the discontinuation period depends on the degree of tumor response (i.e., complete versus partial) achieved during any previous treatment period with these therapeutic agents.

[0110] Use

[0111] One example of a cancer promoted by dysregulated Hh pathway signaling is basal cell carcinoma (BCC). BCC tumors have elevated Gli levels, and molecularly targeted drugs for BCC have focused on antagonizing Smo and reducing Gli mRNA. One such example is cyclopamine, a plant alkaloid that inhibits Smo. Model systems (in vitro and in vivo) show that cyclopamine effectively inhibits BCC, but clinical use of cyclopamine has shown serious side effects that can preclude its use. Another Smo antagonist that has shown good efficacy in metastatic BCC tumors is vismodegib. Other treatments include surgery, chemotherapy, immunotherapy (such as Euphorbia peplus, imiquimod, Aldara), and radiation therapy. In certain cases, BCCs are resistant to Smo antagonists because activating mutations in the Hh pathway are downstream or epistatic to Smo, or the cancer cells have developed resistance to Smo antagonists. The subject methods can be applied to such cancers.

[0112] Another example of a disorder promoted by dysregulated Hh pathway signaling is basal cell nevus syndrome (BCNS), also known as Gorlin syndrome, a rare multisystem disease marked by the development of dozens to hundreds of BCCs. Subjects with BCNS inherit a defective copy of PTCH1. BCNS is a rare disease with a prevalence of 1 case per 56,000-164,000 people in the population, and there is no effective and tolerable treatment. Thus, a drug that treats or prevents BCC tumors is of interest for subjects with BCNS.

[0113] The subject methods and compositions can be used to treat or prevent BCCs in two clinical populations: i) patients with hereditary BCC tumors, such as patients with basal cell nevus syndrome; and ii) patients with sporadic BCC tumors in the general population. In the United States, BCC is the most common cancer, with 1 million new cases diagnosed each year. Although BCCs are rarely fatal, their high incidence and frequent recurrence in affected individuals can lead to significant morbidity. Currently, the incidence of skin cancer is rising year by year, and the treatment of skin cancer places a huge burden on the national health service. Currently, there is no effective therapy for BCC prevention, as sunscreen has not been shown to reduce the development of BCCs in randomized controlled trials.

[0114] Another example of a cancer promoted by dysregulated Hh pathway signaling is medulloblastoma. Medulloblastoma is a highly malignant primary brain tumor that originates in the cerebellum or posterior fossa. Medulloblastoma is the most common malignant brain tumor, accounting for 14.5% of newly diagnosed cases. Medulloblastoma is usually formed near the fourth ventricle, between the brainstem and the cerebellum. Known therapies for medulloblastoma include chemotherapy, such as one or more of lomustine, cisplatin, carboplatin, vincristine, or cyclophosphamide, and vemurafenib. The subject methods can be applied to medulloblastomas that are resistant (or have developed resistance) to Smo antagonists.

[0115] Another example of a cancer promoted by dysregulated Hh pathway signaling is rhabdomyosarcoma. Rhabdomyosarcoma is a sarcoma (connective tissue cancer) in which the cancer cells are believed to arise from skeletal muscle progenitor cells. It can be found in any anatomic location. Most occur in areas that naturally lack skeletal muscle, such as the head, neck, and urogenital tract. Diagnosis of rhabdomyosarcoma relies on recognition of differentiation toward skeletal muscle cells. The proteins myoD1 and myogenin are transcription factor proteins that are normally present in developing skeletal muscle cells, which disappear after muscle maturation and become innervated. Thus, myoD1 and myogenin are normally absent from normal skeletal muscle and can serve as useful immunohistochemical markers for rhabdomyosarcoma. Treatments for rhabdomyosarcoma include chemotherapy, radiation therapy, and sometimes surgery.

[0116] Hedgehog pathway-dependent cancers in other tissues, including variants of Hedgehog pathway-dependent cancers in other tissues that are resistant to Smo antagonists, can also be treated by the subject methods. These include, for example, subtypes of small cell lung cancer, pancreatic cancer, colorectal cancer, ovarian cancer, and prostate cancer, all of which have been shown to respond to blockers of the hedgehog pathway.

[0117] Kit

[0118] Kits comprising one or more containers that hold compositions comprising at least one aPKC iota inhibitor (e.g., CRT0422839 or CRT0364436) and / or an HDAC inhibitor and / or optionally one or more other anti-cancer agents used to treat hedgehog pathway-dependent cancers are provided. The compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, including glass or plastic. The containers can have a sterile access port (for example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0119] The kits can further include a second container that contains a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials that are useful in the practice of the subject methods, such as other pharmaceutically-acceptable formulations, diluents, filters, needles, and syringes or other delivery devices. The delivery devices can be pre-filled with the compositions.

[0120] The kits can further include a package insert containing directions for use of the compositions comprising an aPKC iota inhibitor and / or an HDAC inhibitor to treat a hedgehog pathway-dependent cancer in a subject. The package insert can be an unapproved package insert draft or it can be an approved package insert approved by the Food and Drug Administration (FDA) or other regulatory agency. Alternatively, the instructions can be provided on a computer-readable medium (e.g., floppy disk, CD, DVD, flash drive, etc.) that has recorded information or they can be presented on a website address that can be accessed via the internet to use the information on the website. Any convenient method of providing instructions for treating a hedgehog pathway-dependent cancer in a subject can be present in the kits. Examples

[0121] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of what the inventors regard as their application nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0122] General methods in molecular and cellular biochemistry can be found in the following standard texts: Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0123] Example 1

[0124] Hedgehog pathway regulation in BCC cell models

[0125] Introduction

[0126] For survival and growth, basal cell carcinoma (BCC) requires high levels of hedgehog (HH) signaling (Chang et al. (2012) Arch. Dermatol. 148(11): 1324-1325, Atwood et al. (2015) Cancer Cell 27(3):342-353, Atwood et al. (2013) Nature 494(7438):484-488, Hutchin et al. (2005) Genes Dev. 19(2):214-223). Activation of the HH pathway involves binding of HH ligand to Patched-1, relieving inhibition of Smoothened (SMO). This leads to activation of the GLI family of transcription factors, ultimately promoting transcription of HH target genes, including Gli1 itself. The SMO inhibitor has recently been approved by the FDA for BCC treatment, but resistance has emerged as a significant problem (Chang et al., supra; Atwood et al. (2015), supra; Sekulic et al. (2012) N. Engl. J. Med. 366(23):2171-21791). Because BCCs consistently rely on the HH pathway for growth (Atwood et al. (2015), supra), resistant BCCs evolve to circumvent pharmacological blockade at the level of SMO using both intrinsic mutations in the pathway and non-canonical mechanisms of GLI activation (Atwood et al. (2015), supra; Atwood et al. (2013), supra;).

[0127] Recently, we identified non-canonical Overactivation of this non-canonical HH signaling pathway was identified as a potent mechanism of resistance in BCC (Atwood et al. (2013), supra;). aPKC phosphorylation of the GLI1 zinc finger domain leads to chromatin binding, gene transcription, and HH pathway activation downstream of SMO and Patched-1 input. Moreover, GLI promotes transcription of aPKC, creating another positive feedback loop with GLI. Overactivation of this non-canonical HH signaling pathway drives pathway activation and vismodegib escape in advanced BCC (Atwood et al. (2013), supra;). Small molecule inhibitors of aPKC, allosteric (Erdogan et al. (2006) J. Biol. Chem. 281(38):28450-28459) or orthosteric (Koo et al. (2013) Biochem. J. 451(2):329-342), are in development but have not yet been applied to treat BCC. et al. (2013) Biochem. J. 451(2):329-342), are in development but have not yet been applied to treat BCC.

[0128] Downstream of SMO, acetylation by p300 and subsequent deacetylation further modulate GLI proteins. Histone deacetylases 1 / 2 (HDAC1 / 2) deacetylate GLI1 / 2 at K518 and K757, respectively, are key steps in the nuclear maturation process of GLI transcription factors required for chromatin binding and gene transcription (Canettieri et al. (2010) Nat. Cell Biol. 12(2): 132-142). HDAC1 itself is a transcriptional target of GLI, creating a third positive feedback loop of HH signaling. Of particular interest, HDAC inhibition has been proposed for the treatment of many HH-driven cancers (Canettieri et al., supra; Coni et al. (2017) Sci Rep. 7:44079; Zhao et al. (2014) Pharmacol. Res. Perspect. 2(3):e00043; Coni et al. (2013) PLoS One 8(6):e65718). HDAC inhibitors block growth and promote apoptosis by altering histone-DNA complexes and changing the acetylation status of non-histone proteins (Falkenberg et al. (2014) Nat. Rev. Drug Discov. 13(9):673-691). Vorinostat, a class I / II HDAC inhibitor, is currently FDA-approved for the treatment of cutaneous lymphoma (Mann et al. (2007) Clin. Cancer Res. 13(8):2318-2322). Unfortunately, HDAC inhibition is hampered by its broad cytotoxic properties. De novo drug discovery remains challenging due to the lack of validated targets and the cost of clinical development (Hoelder et al. (2012) Mol. Oncol. 6(2): 155-176).

[0129] Here we show that aPKC iota inhibitors CRT0422839 and CRT0364436 modulate BCC cell viability and reduce GLI1 mRNA levels, consistent with GLI pathway modulation.

[0130] Results

[0131] To investigate the efficacy of aPKC iota inhibition on BCC in vitro, murine BCC cell lines were treated with increasing doses of CRT0422839 or CRT0364436. Treatment with CRT0422839 or CRT0364436 resulted in a dose-dependent reduction of BCC growth and viability Figure 1C and 1D ). BCC viability was compared to treatment with aPKC inhibitor PSI (Figure 1A ) and CRT0329868( Figure 1B ) treated viability (as previously described by Mirza et al. (Mirza et al. (JCI Insight (2017) 2(21) pii: e97071; hereby incorporated by reference in its entirety) were compared. In addition, treatment with CRT0422839 or CRT0364436 resulted in a dose-dependent reduction in Gli1 mRNA levels Figure 2A and 2B ).

[0132] Example 2

[0133] aPKC Biochemical kinase assay of iota

[0134] The ability of compounds to inhibit aPKC iota kinase activity was measured using the IMAP FP Progressive Binding System (Molecular Devices R8127) in 384-well black, non-binding, flat-bottom assay plates (Corning 3575). The assay mix (final volume = 10 μΐ) contained 20 mM Tris-HCL (pH 7.5), 150 μΜ ATP, 10 mM MgCl2, 0.01% Triton X-100, 250 μΜ EGTA, 1 mM DTT, 15 pM PKC iota (EMD Millipore 14-505), 100 nM FAM-PKC epsilon- pseudosubstrate (Molecular Devices RP7548), 0.1% DMSO, and various concentrations of test compounds CRT0422839 and CRT0364436. Compound dilutions (prepared in 100% DMSO) were added to the assay plates using a BioMek NX pin tool (Beckman Coulter) at 100 nl. The enzyme reaction was initiated by the addition of ATP (MilliporeSigma A7699) and the plates were then incubated in a 25 °C incubator for 1 hour. A 20 μΐ aliquot of IMAP detection reagent (1 :400 in 85% 1X Binding Buffer A and 15% 1X Binding Buffer B) was added to each well and then incubated at 25 °C for 2 hours. FP was then measured using a PerkinElmer Envision 2102 Multilabel Reader (PerkinElmer) using FP dual mirror, FP480 excitation filter, and P-pol 535 and S-pol 535 emission filters. Data analysis was performed using ActivityBase (IDBS). IC50 values were calculated by plotting percent inhibition versus log10 of compound concentration in XLFit 4 (IDBS) and fitting a 4-parameter logistic model (top and bottom constrained to 100 and 0, respectively).

[0135] The foregoing merely illustrates the principles of the application. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the application and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the application and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the application as well as specific examples thereof are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present application, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present application is embodied by the appended claims.

Claims

1. Use of a compound of Formula I: ###0001### or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a hedgehog pathway dependent cancer in a subject, wherein the cancer is basal cell carcinoma (BCC) and the compound mitigates hERG activity and reduces Gli 1 mRNA production.

2. The use of claim 1, wherein the cancer comprises a constitutively active hedgehog pathway.

3. The use of claim 1, wherein the cancer is metastatic.

4. The use of claim 1, wherein a plurality of treatment cycles are to be administered to the subject for a period of time sufficient to achieve at least partial tumor response.

5. The use of claim 4, wherein the period of time is at least 6 months.

6. The use of claim 5, wherein the period of time is at least 12 months.

7. The use of claim 1, wherein a plurality of treatment cycles are to be administered to the subject for a period of time sufficient to achieve complete tumor response.

8. The use of claim 1, wherein the medicament is to be administered to the subject according to a daily dosing regimen or intermittently.

9. The use of claim 1, wherein an additional anti-cancer therapy is to be administered to the subject.

10. The use of claim 9, wherein the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, immunotherapy, biotherapy, or a combination thereof.

11. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.

12. The use of claim 11, wherein a histone deacetylase (HDAC) inhibitor is also administered to the subject.

13. The use of claim 12, wherein the HDAC inhibitor is vorinostat.

14. The use of claim 13, wherein the subject is a mammal.

15. The use of claim 14, wherein the subject is a human.

16. The use of claim 1, wherein the medicament is to be administered in an amount sufficient to reduce basal cell carcinoma viability in the subject.

17. Use of a compound of Formula II: ###0002### or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting the growth or proliferation of basal cell carcinoma (BCC) cells, wherein Formula II inhibits basal cell carcinoma cell growth at 1 μΜ within 10 hours of treatment.

18. The use of claim 1, wherein Formula I reduces GLI1 mRNA levels to less than 50% of control GLI1 mRNA levels at 10 μΜ within six hours of treatment.

19. The use of claim 17, wherein the basal cell carcinoma cells are in vivo or in vitro.

20. The use of claim 17, comprising contacting the basal cell carcinoma cells with a histone deacetylase (HDAC) inhibitor.

21. The use of claim 20, wherein the HDAC inhibitor is vorinostat.

22. A composition comprising a pharmaceutically acceptable excipient and a compound of Formula I: ###0003### ​ ​ or Formula II a compound of Formula (I): or a pharmaceutically acceptable salt thereof, and a histone deacetylase (HDAC) inhibitor, for use in the treatment of basal cell carcinoma (BCC) and the HDAC inhibitor is vorinostat.

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