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  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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  • A61P27/02—Ophthalmic agents
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/06—Peri-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/06—Peri-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/10—Spiro-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
  • C07D487/14—Ortho-condensed systems

Definitions

• the presently disclosed subject matter relates to compositions and methods for the treatment of ophthalmic diseases mediated by activation of calcium/calmodulin-dependent kinase kinase 2 (CaMKK2), an intermediate kinase that regulates cellular effector functions in a number of cell types, especially immune cells.
• CaMKK2 calcium/calmodulin-dependent kinase kinase 2
• the primary focus of the presently disclosed subject matter relates to inflammatory diseases of the eye, ocular adnexae, and external tissues (eyelids, orbit).
• These diseases include anterior segment (or“front of the eye”) inflammatory diseases as well as posterior segment (or“back of the eye”) diseases in which activation of CaMKK2 in immune cells initiates, mediates, or modulates disease activity; and in which inhibition of CaMKK2 in target immune cells might represent a potential therapeutic or disease-modifying strategy.
• ocular surface inflammatory diseases including but not limited to ocular graft versus host disease, ocular cicatricial pemphigoid, vernal keratoconjunctivitis, allergic eye disease, meibomian gland dysfunction, aqueous tear deficiency (common dry eye disease), comeal scarring, and conjunctival scarring and fibrosis;
• uveitis and other inflammatory diseases of the eye including but not limited to keratitis, scleritis, LTDis, iridocyclitis, intermediate uveitis, pars planitis, posterior uveitis, choroiditis, chorioretinitis, retinitis, or panuveitis of noninfectious, infectious, or idiopathic etiologies
• 3)“back of the eye” retinal diseases which
• CaMKK2 Calcium/calmodulin-dependent protein kinase kinase 2
• CaMKK2 is an intracellular intermediate kinase that is ubiquitously expressed in many cell types.
• the activity of CaMKK2 is particularly increased within certain“activated” cells, including cells of the immune system, especially activated T lymphocytes and infiltrating macrophages (1-5).
• CaMKK2 appears to be an amplifier of effector functions for“activated” cells more than a maintainer of homeostasis. Accordingly, in experimental studies, mice null for CaMKK2 demonstrate no major phenotype, including no evidence of immunodeficiency.
• CaMKK2 is one of the most important calcium responsive kinases, and its activity is regulated by cytoplasmic calcium and calmodulin levels. Once active, CaMKK2 phosphorylates several substrates. In addition to auto-phosphorylating and activating other CaMKK2 molecules, CaMKK2 directly phosphorylates calcium/calmodulin- dependent protein kinase I (CaMKI), calcium/calmodulin-dependent protein kinase IV (CaMKIV), and adenosine monophosphate-activated protein kinase (AMPK) (FIGS. 1 and 2). Phosphorylation of these substrates amplifies multiple downstream signaling cascades, modulating multiple cellular effector functions.
• CaMKI calcium/calmodulin-dependent protein kinase I
• CaMKIV calcium/calmodulin-dependent protein kinase IV
• AMPK adenosine monophosphate-activated protein kinase
• CaMKK2 activity in T cells Via phosphorylation of its substrates CaMKI, CamKIV, and AMPK, CaMKK2 regulates a number of specific effector functions in T cells (FIG. 1).
• CaMKI phosphorylation of its substrates CaMKI, CamKIV, and AMPK
• CaMKK2 regulates a number of specific effector functions in T cells (FIG. 1).
• calcium is the major secondary messenger system, irrespective of activation via T cell receptor or amplification by co- stimulatory signals and/or IL-2 (6), resulting in activation of calmodulin and CaMKK2.
• downstream activation of AMPK regulates cellular bioenergetics, proliferation, and cytokine production (1, 5).
• CaMKI regulates T cell migration and adherence (7).
• CaMKIV is important in T cell cytokine effector responses.
• CaMKK2 also amplifies activation of PTK2B (protein tyrosine kinase beta), which mediates the p38 MAPK pathway, crucial for production of many effector cytokines (3).
• PTK2B protein tyrosine kinase beta
• knockout of CaMKK2 diminishes T cell-mediated inflammation in a number of inflammatory diseases, such as experimental graft versus host disease (GVHD) (2, 8-11).
• GVHD experimental graft versus host disease
• CaMKK2 activity in macrophages Via phosphorylation of its substrates CaMKI, CamKIV, and AMPK, CaMKK2 is also a major regulator of macrophage function (FIG. 2). For example, activation of AMPK turns on a whole host of gene transcription important for reparative function in nonclassical macrophages, and specific inhibition of CaMKK2 inhibits transition of monocyte-to-nonclassical macrophages via prevention of AMPK activation (12). As in T cells, CaMKK2 also amplifies activation of PTK2B (protein tyrosine kinase beta), which mediates the p38 MAPK pathway, crucial also for production of many macrophage-derived effector cytokines (3).
• PTK2B protein tyrosine kinase beta
• CaMKK2 inhibition decreases production of pro -inflammatory cytokine that have been implicated in tissue injury and destruction.
• Increased activity of CaMKK2 is observed in macrophages previously exposed to activating stimuli (e.g. LPS)(lipopolysaccharide) (4).
• Knockout of CaMKK2 impairs the ability of macrophages to adhere and extend membrane processes, resulting in reduced macrophage accumulation and diminished cytokine release in response to such activating stimuli (4).
• OSIDs and uveitic diseases are well established as disorders with significant ocular morbidity and vision loss, and inflammatory cells (especially macrophages) are also known to contribute to the severity and visual morbidity of posterior segment diseases (13-17). All of these conditions can include both diseases in which immune cells, especially T cells and macrophages, are the primary mediators of disease (i.e. these cells infiltrate ocular tissues and trigger local injury as a primary disease process) and diseases in which immune cells are secondary pathologic mediators in response to a primary ocular disease process.
• CaMKK2 functions as an amplification circuit; knockout diminishes inflammation but does not induce immunosuppression (4).
• CaMKK2 represents an attractive therapeutic target in ocular inflammatory diseases when taken together with the established role of CaMKK2 in T cell and macrophage function.
• preclinical data from experimental mouse models of ocular inflammatory disease demonstrate that targeted inhibition of CaMKK2 ameliorates disease severity, with reduced infiltration of T cells and macrophages (See Examples 4 and 5 of this application).
• the presently disclosed subject matter relates to compositions and methods for novel small molecule inhibitors of calcium / calmodulin-dependent protein kinase kinase 2 (CaMKK2), an intermediate kinase that has increased activity within cells of the immune system, including activated T cells and macrophages.
• CaMKK2 calcium / calmodulin-dependent protein kinase kinase 2
• Novel small molecules have been designed with high specificity and efficacy for inhibition of CaMKK2, and these molecules retain hydrophilicity and high aqueous solubility.
• SMICs small molecule inhibitors of CaMKK2
• these SMICs are ideally suited for treatment of ophthalmic diseases via topical or subconjunctival routes of ocular administration.
• FIG. 1 is a schematic illustrating CaMKK2 regulation of T cell effector responses, and how CaMKK2 amplification of multiple downstream pathways mediates many proinflammatory effector mechanisms.
• FIG. 2 is a schematic illustrating CaMKK2 regulation of macrophage effector responses, and how CaMKK2 amplification of multiple downstream pathways mediate both proinflammatory and profibrotic effector mechanisms.
• FIG. 3A is a schematic illustrating the pathobiology of ocular graft versus host disease after allogeneic hematopoietic stem cell transplantation (HSCT) (i.e. bone marrow transplant).
• HSCT allogeneic hematopoietic stem cell transplantation
• FIG. 3B is a hematoxylin and eosin (H&E) micrograph image depicting histology of immune cell (T cell and macrophage) infiltration of conjunctiva in OGVHD.
• FIG. 3C is a clinical slit-lamp biomicro scopic photograph illustrating a clinical presentation of severe OGVHD.
• FIG. 4A and 4B are Western blots illustrating inhibition of CaMKK2 enzymatic activity by (A) CaMKK2 auto-phosphorylation and (B) AMPK phosphorylation, by vehicle, small molecule compounds EYE301-EYE305, and tool compound STO-609, in cultured HEK293 cells.
• FIG. 4C and 4D are bar graphs illustrating the densitometry analysis of Western blots, demonstrating inhibitory activity of CaMKK2 of C) CaMKK2 auto-phosphorylation and D) AMPK phosphorylation, by vehicle, small molecule compounds EYE301-EYE305, and tool compound STO- 609, in cultured HEK293 cells.
• FIG. 5 is a bar graph illustrating measurement of pro-inflammatory cytokines in a mixed lymphocyte reaction, which can be used to screen functional inhibitory capacity of small molecule inhibitors of CaMKK2 (SMICs) in vitro (i.e.“GVHD in a dish”).
• SMICs small molecule inhibitors of CaMKK2
• FIG. 6 is an illustration of a mouse model of graft-versus-host disease, based on bone marrow transplantation, with concurrent adoptive transfer of splenic T cells, from C57B1/6 into BALB/c mice.
• FIG. 7A is a slit lamp biomicro scopic photograph of the external eye of a mouse after receiving a“safe” blood marrow transplant (i.e. no concurrent T cell adoptive transfer).
• FIG. 7B is a slit lamp biomicroscopic photograph of the external eye of a mouse, depicting ocular graft versus host disease (OGVHD) following an allogenic blood marrow transplant with concurrent adoptive transfer of splenic T cells.
• OGVHD ocular graft versus host disease
• FIG. 8 is a scatter plot of OGVHD eye pathology scores of“GVHD” BMT mice (with concurrent adoptive transfer of splenic T cells), as compared to minimal OGVHD severity in“safe” BMT mice.
• FIG. 9A and 9B illustrates ocular surface fluorescein staining after A) control“safe” BMT and B) GVHD BMT (with concurrent adoptive transfer of splenic T cells).
• FIG. 9C is a plot of the ocular surface fluorescein staining scores of control safe BMT mice and GVHD BMT mice.
• FIG. 10A-10D are images showing histopathologic evidence of T cell (A and B) and macrophage (C and D) infiltration into the corneal stroma, bulbar, and tarsal conjunctiva.
• FIG. 11A is a slit lamp biomicroscopic photograph of the eye of a control-treated mouse with OGVHD.
• FIG. 11B is a slit lamp biomicro scopic photograph of a STO-609 treated mouse, with minimal signs of OGVHD following treatment.
• FIG. 11C is a scatter plot of the eye pathology scores from allogeneic BMT + splenic T cell transfer, following local ocular treatment with either vehicle control or STO-609.
• FIG. 12 Candidate SMICs demonstrate similar potency to STO-609 in vitro.
• HEK-293 cells were treated with STO-609, candidate SMICs, or vehicle control prior to stimulation with lmM ionomycin, a known activator of CaMKK2.
• Cell lysates were then probed with phospho-specific antibodies to CaMKK2 and AMPK.
• B-actin was used as a loading control.
• Graphs show quantification of densitometry with corrections for loading variation.
• FIG. 13 Mice underwent local ocular administration of the tool compound inhibitor of CaMKK2, STO-609, lead SMIC compounds EY1006.A001.B001 and EY1001.A002.B001, prednisolone starting at day 14 post-BMT and continuing for two weeks. Both lead SMICs EY1006.A001.B001 and EY1001.A002.B001 reduced severity of clinical OGVHD findings with efficacy similar to STO-609. By contrast, vehicle control-treated eyes had lid margin swelling and scarring with lash and periocular fur loss, eyelid crusting, chemosis, abnormal tear film, and keratopathy.
• STO-609 and lead SMICs were superior to vehicle and prednisolone, in preventing signs of OGVHD (p ⁇ 0.05 for STO-609, EY1006.A001.B001, EY1001.A002.B001 vs vehicle or prednisolone). Mild disease (score 0-5); moderate disease (score 6-10); severe disease (score > 10).
• the term "about”, when referring to a value or to an amount of mass, weight, time, volume, concentration, and/or percentage can encompass variations of, in some embodiments +/- 20%, in some embodiments +/-l0%, in some embodiments +/- 5%, in some embodiments +/- 1%, in some embodiments +/- 0.5%, and in some embodiments +/-0.l%, from the specified amount, as such variations are appropriate in the disclosed packages and methods.
• CaMKK2 calcium/calmodulin kinase kinase 2
• CaMKK2 is an enzyme encoded by the CAMKK2 gene (18) and was first proposed to be a key mediator of central nervous system appetite control in 2008, as it was shown to be present in centers of the brain controlling satiety (19). More recently, CaMKK2 has been shown to be an important regulatory kinase in macrophages and T cells (FIGS.
• the term“disease” as used herein includes various diseases, disorders, symptoms, conditions, and/or indications.
• CaMKK2 belongs to the Serine/Threonine protein kinase family, and to the Ca +2 /calmodulin-dependent protein kinase subfamily (18). Further, CaMKK2 is regulated by cytoplasmic calmodulin levels and inflammatory stimuli. In active T cells, calcium is the major secondary messenger system, irrespective of activation via T cell receptor or amplification by co stimulatory signals and/or IL-2 (6).
• CaMKK2 phosphorylates calcium/calmodulin-dependent kinase I and IV (CaMKI and CaMKIV) and adenosine monophosphate- activated protein kinase (AMPK), which regulate numerous T cell functions (1, 2, 5, 6, 8, 10), as shown in FIG. 1.
• CaMKI regulates T cell migration and adherence (6)
• CaMKIV is important in T cell cytokine effector responses (2, 6, 8, 10).
• AMPK regulates T cell bioenergetics, proliferation, and cytokine production (1, 5, 6). Activation of these kinases and downstream signaling pathways promote T cell activation and subsequent inflammation.
• CaMKK2 also amplifies activation of protein tyrosine kinase beta (PTK2B) (3, 4), which mediates many pro inflammatory and fibrogenic effector systems. Importantly, CaMKK2 functions as an amplification circuit such that knockout diminishes inflammation but does not induce immunosuppression (3, 4).
• PTK2B protein tyrosine kinase beta
• CaMKK2 is also a major regulator of macrophage function, as shown in FIG. 2. Activation of AMPK by CaMKK2 turns on a whole host of gene transcription important for reparative function in nonclassical macrophages (3, 4, 12), and specific inhibition of CaMKK2 inhibits transition of monocyte-to-nonclassical macrophages via prevention of AMPK activation (12). CaMKK2 also amplifies activation of PTK2B (protein tyrosine kinase beta), which mediates the p38 MAPK pathway, crucial also for production of many macrophage-derived effector cytokines (3, 4). Thus, CaMKK2 inhibition decreases production of pro-inflammatory cytokine that have been implicated in tissue injury and destruction.
• PTK2B protein tyrosine kinase beta
• OSIDs ocular surface inflammatory diseases
• ocular graft versus host disease ocular cicatricial pemphigoid
• vernal keratoconjunctivitis allergic eye disease, meibomian gland dysfunction, aqueous tear deficiency (common dry eye disease), corneal scarring, and conjunctival scarring and fibrosis
• uveitis and other inflammatory diseases of the eye including but not limited to keratitis, scleritis, LTDis, iridocyclitis, intermediate uveitis, pars planitis, posterior uveitis, choroiditis, chorioretinitis, retinitis, or panuveitis of noninfectious, infectious, or idiopathic etiologies
• 3)“back of the eye” retinal diseases which include but are not limited to dry age-related macular degeneration, neovascular age-
• OGVHD ocular graft versus host disease
• FOG. 3 model ocular inflammatory disease
• SMICs novel small molecule inhibitors of CaMKK2
• both T cells and macrophages are known to play a pathogenic role in OGVHD (14, 23); and the disease can treated by ocular drug administration (23).
• OGVHD occurs in over 60% of patients undergoing allogeneic hematopoietic stem cell transplant (HSCT) (i.e. bone marrow transplant) (13, 15).
• HSCT allogeneic hematopoietic stem cell transplant
• OGVHD In OGVHD, donor T cells encounter recipient transplantation antigens within ocular tissue, leading to T cell activation and cytokine production (FIG. 3A) (14, 23). This triggers recruitment and infiltration of donor macrophages, additional“autoimmune” T cells, with secondary contribution of neutrophils and B cells (14, 23) (FIG. 3B). These infiltrating inflammatory cells serve as“effectors” of tissue damage.
• OGVHD manifests as findings of aqueous tear deficiency, Meibomian gland dysfunction, keratopathy, and in severe cases, conjunctival scarring, lid margin scarring, and corneal ulceration (FIG. 3C) (13, 24, 25).
• OGVHD shares clinical and pathologic overlap with more common OSIDs (i.e. common dry eye, Meibomian gland dysfunction, others), and with other ocular inflammatory diseases (i.e. various forms of uveitis) (26-29), experimental preclinical models of OGVHD represent ideal systems to study the therapeutic potential of drugs that target and inhibit CaMKK2.
• the small molecule STO-609 is a well characterized and specific inhibitor of CaMKK2 (30). Though the biophysical properties of STO-609 have limited its potential as a therapeutic, the molecular interaction of STO-609 with CaMKK2 provides important insights into specific characteristics that enable inhibition of CaMKK2 activity.
• the planar, rigid STO-609 molecule fits into a narrow pocket within the kinase domain, competing with and preventing ATP binding.
• the binding pocket interactions between STO-609 and CaMKK2 are predominantly hydrophobic in nature (i.e., especially at the CaMMK2 peptide interacting residues Ile 171, Vall79, Alal92, Val249, Phe267, Gly273, Pro274, and Leu3l9).
• two key hydrogen bonding interactions help accommodate STO-609 binding within the active site - (i) the interaction between the STO-609 amide oxygen and Val270; and (ii) the interaction between the carboxylic acid of bound STO-609 and Asp330. Further, an active site molecule of water forms a hydrogen bond bridge between the carboxylic acid of STO-609 and Glu236. Collectively, these molecular interactions enable STO-609 inhibitory capacity of CaMKK2. STO-609 has Ki for CaMKK2 ⁇ 30 nM and IC50 ⁇ 40 nM in HeLa cells and IC50 of about 100 nM in macrophages and T cells (31).
• STO-609 displays 6-fold less activity for CaMKKl and >80-fold selectivity for CaMKK2 over CaMKI, CaMKII, CaMKIV, MLCK, PKC, PKA, p42, MAPK, VEGFR1, and PDGFR (30).
• STO-609 suffers from several limitations that render it useful only as a tool compound.
• the current synthetic route universally employed to synthesize STO-609 at scales of about 100 mg or more yields a pair of isomeric bromides that comprise as much as 40-50% of the mixture, as shown below.
• STO-609 exhibits poor solubility (i.e., the maximum concentration of STO- 609 in DMSO is 10 mM with sonication) such that in vivo and in vitro work with the compound leads to precipitation.
• STO-609 also exhibits poor oral bioavailability, which limits its development as a therapeutic and restricts its use as a tool compound for laboratory studies. Further, various off-target effects have been suspected, including activation of aryl hydrocarbon receptors (32).
• the inventors were able to design formulations for local ocular (i.e., periocular and topical) application of STO-609. Additionally, with knowledge of STO-609 orientation in the CaMKK2 binding pocket, the inventors were able to determine which regions of STO-609 are best suited for structural alteration and addition of groups to the molecular framework that could enhance aqueous solubility.
• the presently disclosed subject matter further comprises pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and/or derivatives of the compounds of Formulas (I) and (II).
• pharmaceutically acceptable refers to generally recognized for use in animals, such as (but not limited to) humans, and that are not biologically or otherwise undesirable in a subject.
• the benzimidazole urea scaffold of the compound of Formula (I) maintains a flat, rigid aromatic structure, similar to STO-609, as shown below.
• compositions of Formulas (I) and (II) (and all current and proposed benzimidazole urea series compounds disclosed herein) produce a single compound.
• the production of non-binding isomers does not occur, enhancing both efficiency and fidelity of expected synthetic products even for large-scale synthesis.
• the presently disclosed subject matter further includes synthetically accessible structural analogs of Formulas (I) and (II), such as (but not limited to) the compounds of subseries Formulas (III) through (V), and to pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or derivatives thereof, wherein R comprises an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.
• alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain, or combination thereof, which may be fully saturated, mono- or polyunsaturated (i.e., alkenyl) and can include di- and multivalent radicals (e.g., alkylene), having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons).
• saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec -butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
• An unsaturated alkyl group is one having one or more double bonds or triple bonds.
• alkenyl groups examples include, but are not limited to, vinyl, 2-propenyl, 2-isopentenyl, 2-(butadienyl), 2,4- pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
• the alkyl group may be substituted or unsubstituted; for example with one or more halogens, e.g., trifluoromethyl.
• heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom.
• A“heteroatom” includes oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), selenium (Se) and silicon (Si), wherein N and S may optionally be oxidized, and N may optionally be quaternized.
• a heteroatom(s) may be placed at any chemically acceptable position including an interior position, the position at which the alkyl group is attached to the remainder of the molecule (the proximal end), or at the distal end (e.g., for heteroalkylene groups).
• Examples include but are not limited to:— C(0)R',— C(0)NR',— NR'R",— OR',— SR', and/or— S0 2 R', and— CN. Up to two heteroatoms may be consecutive, such as, for example,— CH 2— NH— OCH 3 .
• cycloalkyl and“heterocycloalkyl” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of“alkyl” and“heteroalkyl,” respectively.
• cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
• heterocycloalkyl include, but are not limited to, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, and tetrahydrothienyl.
• the cycloalkyl or heterocycloalkyl group may be substituted or unsubstituted.
• aryl by itself or in combination with another term, means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon group, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together or linked covalently.
• a fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring.
• heteroaryl by itself or in combination with another term, means, unless otherwise stated an aryl group (as defined above) containing one to four heteroatoms (as defined above).
• the term“heteroaryl” includes fused ring heteroaryl groups, which are multiple rings (e.g., 5 and/or 6-membered rings) fused together wherein at least one of the fused rings is a heteroaromatic ring.
• a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
• Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, purinyl, benzothiazolyl, benzimidazolyl, indolyl, isoquinolyl, quinoxalinyl, 5-quinoxalinyl, and quinolyl.
• the aryl or heteroaryl group may be substituted or unsubstituted, for example with a halogen.
• the aryl or heteroaryl group may be mono-, di- or tri-substituted.
• the presently disclosed subject matter includes benzimidazole cyclic (thio)urea subseries compounds, such as (but not limited to) the compounds of Formulas (VI) and (VII), and to pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or derivatives thereof, wherein X is oxygen (O) or sulfur (S), and n is 1 or 2.
• esters refers to any chemical compound derived by the reaction of an oxoacid (an organic acid that contains oxygen) with a hydroxyl compound, such as an alcohol. Esters are usually derived from an organic acid in which at least one hydroxyl (-OH) group is replaced by an -O-alkyl (alkoxy) group. Most commonly, esters are formed by condensing a carboxylic acid with an alcohol.
• prodrug refers to a compound that exhibits no significant pharmacological activity unless it is converted to a pharmacologically active parent compound.
• pharmaceutically useful prodrugs are compounds that upon administration to an individual are converted in vivo to the corresponding pharmacologically active parent compound.
• suitable prodrugs can also be converted to the pharmacologically active parent drug in vitro in the presence of an exogenously provided converting activity, such as (but not limited to) a converting enzyme.
• suitable benzimidazole thio(urea) ester prodrug compounds can include those of Formula (VIII), and to pharmaceutically acceptable salts, solvates, hydrates, or derivatives thereof, wherein X is O or S, and R is an ester prodrug.
• the presently disclosed subject matter further includes benzimidazole thio(urea) carboxamide subseries compounds.
• carboxamide refers to a moiety comprising a carbon, nitrogen, and oxygen atom bonded in the configuration shown as Formula A. Specifically, the carbon atom is bonded to a carbon atom in a radical to which the carboxamide moiety is bonded. Further, the nitrogen atom is bonded to the carbonyl carbon and is also bonded to two other atoms, at least one of which is selected from a hydrogen atom or a carbon atom of another radical to which the carboxamide moiety is bonded.
• suitable benzimidazole thio(urea) carboxamide compounds can include (but are not limited to) compounds of Formulas (IX) through (XI), and to pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or derivatives thereof, wherein X is O or S; R is alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups.; and Ri and R 2 are the same of different alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups.
• the presently disclosed subject matter includes benzimidazole aliphatic and alicyclic subseries compounds.
• aliphatic refers to an organic compound characterized by a straight or branched chain structure, or closed ring structure that includes saturated carbon bonds and optionally one or more unconjugated unsaturated bonds, such as a carbon-carbon double bond.
• alicyclic refers to an organic compound that includes a closed ring structure comprising saturated carbon bonds and optionally one or more unconjugated carbon-carbon double bonds.
• suitable benzimidazole aliphatic and alicyclic subseries compounds can include the compounds of Formulas (XII) through (XV), and to pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or derivatives thereof, wherein n is 1, 2, or 3; R is alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups; and Ri and R 2 are the same of different alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups.
• the presently disclosed subject matter includes aryl and hetaryl benzimidazole urea compounds, such as the compounds of Formula (XVI), and to pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or derivatives thereof.
• the presently disclosed subject matter includes benzimidazole (thio)urea subseries compounds that include an sp 3 hybridized carbon atom.
• An sp 3 hybridized carbon atom refers to a carbon atom that forms four bonds to four substituents placed in a tetragonal fashion around the carbon atom.
• Suitable benzimidazole (thio)urea subseries compounds that include an sp 3 hybridized carbon atom include (but are not limited to) compounds of Formula (XVII), and to pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or derivatives thereof, wherein X is O or S.
• the presently disclosed subject matter further includes synthetically accessible structural analogs of Formulas (I) and (II), such as (but not limited to) the compounds of subseries Formulas (XVIII) and (XIX), and to pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or derivatives thereof, wherein Ri comprises H or COOCH 3 , and R 2 is CFhCN, CH 2 COOC 2 H 5 , or CHiCOPh.
• the compound has the structure:
• the compound has the structure:
• the compound may have groups that will ionize and become charged under physiologic conditions having the structure:
• the presently disclosed subject matter provides a pharmaceutical composition
• a pharmaceutical composition comprising, consisting of, or consisting essentially of a compound as described herein and a pharmaceutically acceptable carrier.
• suitable carriers can include (but are not limited to) water, aqueous solution, polymer (such as hydroxypropyl methylcellulose), petrolatum, mineral oil, castor oil, carboxymethyl cellulose, organic liquid lipid, polyvinyl alcohol, hydroxypropyl cellulose, hyaluronic acid, glycerin, polyethylene glycol, polysorbate 80, povidone, and/or dextran.
• the disclosed compositions can be present in the carrier in an amount of from about 0.5-20 weight%, such as about 0.5-10%, 0.5-9%, 0.5-8%, 0.5-7%, 0.5-6%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, or 0.5- 1%, based on the total weight of the composition.
• the disclosed compositions can optionally comprise one or more buffers, tonicity agents, preservatives, and/or chelating agents. Suitable buffers include (but are not limited to) acetate, borate, carbonate, citrate, and/or phosphate buffers.
• Suitable tonicity agents that can be used to adjust the disclosed compositions to a desired isotonic range can include (but are not limited to) glycerin, mannitol, sorbitol, sodium chloride, and/or other electrolytes.
• Suitable preservatives that can be used to prevent bacterial contamination include (but are not limited to) polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, and/or thimerosal.
• Suitable chelating agents that can be used to enhance preservative effectiveness include (but are not limited to) edetate salts, such as edetate disodium, edetate calcium disodium, edetate trisodium, and/or edetate dipotassium.
• edetate salts such as edetate disodium, edetate calcium disodium, edetate trisodium, and/or edetate dipotassium.
• the invention also includes all suitable isotopic variations of a compound of the invention.
• An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature.
• isotopes that can be incorporated into a compound of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as 3 ⁇ 4 (deuterium), 3 H (tritium), 13 C, 14 C, 15 N, 17 0, 18 0, 32 P, 33 P, 33 S, 34 S, 35 S, 36 S, 18 F, 36 Cl, 82 Br, 123 I, 124 I, 129 I and 131 I, respectively.
• Certain isotopic variations of a compound of the invention for example, those in which one or more radioactive isotopes such as 3 H or 14 C are incorporated, are useful in drug and/or substrate tissue distribution studies.
• Tritiated and carbon-l4, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability.
• Substitution with positron emitting isotopes, such as U C, 18 F, 15 0 and 13 N, can be useful in Positron Emission Topography (PET) studies.
• substitution with isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances.
• Isotopic variations of a compound of the invention can generally be prepared by conventional procedures known by a person skilled in the art such as by the illustrative methods or by the preparations described in the examples hereafter using appropriate isotopic variations of suitable reagents.
• the isotope-labeled compounds contain deuterium (3 ⁇ 4), tritium ( 3 H) or 14 C isotopes.
• Isotope-labeled compounds of this invention can be prepared by the general methods well known to persons having ordinary skill in the art.
• Such isotope-labeled compounds can be conveniently prepared by carrying out the procedures disclosed in the Examples disclosed herein and Schemes by substituting a readily available isotope-labeled reagent for a non-labeled reagent.
• compounds may be treated with isotope-labeled reagents to exchange a normal atom with its isotope, for example, hydrogen for deuterium can be exchanged by the action of a deuteric acid such as D2SO4/D2O.
• deuterium may be also incorporated into a compound using methods such as through reduction such as using L1AID4 or NaBD 3 , catalytic hydrogenation or acidic or basic isotopic exchange using appropriate deuterated reagents such as deuterides, D2 and D2O.
• methods such as through reduction such as using L1AID4 or NaBD 3 , catalytic hydrogenation or acidic or basic isotopic exchange using appropriate deuterated reagents such as deuterides, D2 and D2O.
• deuterated reagents such as deuterides, D2 and D2O.
• PCT publications, WO2014/169280; WO2015/058067; U.S. Pat. Nos. 8,354,557; 8,704,001 and US Patent Application Publication Nos.; 2010/0331540; 2014/0081019; 2014/0341994; 2015/0299166 the methods are hereby incorporated by reference.
• the pH of the disclosed compositions can be about 4 to 8, such as about 4.5-7.5, 4.5-6.5, or 4.5-5.5.
• the presently disclosed subject matter is directed to a method of modulating CaMKK2 activity in a cell.
• the method comprises, consists of, or consists essentially of administering an effective amount of a compound as provided herein to the cell, such that the CaMKK2 activity is modulated.
• the term“administering” as used herein refers to the dosage of a compound or composition, such as a single dose or multiple doses of the disclosed compounds.
• the method used to administer includes multiple routes of delivery, especially topical delivery to the eye.
• Additional routes of delivery for any number of disease indications include but are not limited to subconjunctival delivery, sub-Tenon’s delivery, intracameral delivery, intravitreal delivery, suprachoroidal delivery, punctal delivery, retrobulbar delivery, intravenous delivery, subcutaneous delivery, intramuscular delivery, oral delivery, inhalational delivery, and intrathecal delivery.
• compositions provided herein can be administered topically to the skin, orifices, or mucosa.
• topical administration includes (intra)dermal, conjunctival, intracorneal, intraocular, ophthalmic, and transdermal administration.
• compositions provided herein can be formulated in any dosage forms that are suitable for topical administration for local or systemic effect, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, irrigations, sprays, suppositories, bandages, dermal patches.
• the topical formulation of the pharmaceutical compositions provided herein can also comprise liposomes, micelles, microspheres, nanosystems, and mixtures thereof.
• Pharmaceutically acceptable carriers and excipients suitable for use in the topical formulations provided herein include, but are not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.
• the pharmaceutical compositions provided herein can be provided in the forms of ointments, creams, and gels.
• Suitable ointment vehicles include oleaginous or hydrocarbon vehicles, including lard, benzoinated lard, olive oil, cottonseed oil, and other oils, white petrolatum; emulsifiable or absorption vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointment; water- soluble ointment vehicles, including polyethylene glycols of varying molecular weight; emulsion vehicles, either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid (see, Remington: The Science and Practice of Pharmacy, supra). These vehicles are emollient but generally require addition of antioxidants and preservatives.
• Suitable cream base can be oil-in-water or water-in-oil.
• Cream vehicles may be water- washable, and contain an oil phase, an emulsifier, and an aqueous phase.
• the oil phase is also called the“internal” phase, which is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol.
• the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
• the emulsifier in a cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.
• Gels are semisolid, suspension-type systems. Single -phase gels contain organic macromolecules distributed substantially uniformly throughout the liquid carrier. Suitable gelling agents include crosslinked acrylic acid polymers, such as carbomers, carboxypolyalkylenes, CARBOPOL®; hydrophilic polymers, such as polyethylene oxides, polyoxyethylene- polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums, such as tragacanth and xanthan gum; sodium alginate; and gelatin.
• dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring.
• the presently disclosed subject matter is directed to a method of inhibiting CaMKK2 activity in a target cell.
• the method comprises, consists of, or consists essentially of administering an effective amount of a compound as described herein to the cell such that the CaMKK2 activity is inhibited.
• the presently disclosed subject matter is directed to a method for treating an ocular indication in a subject.
• the method comprises, consists of, or consists essentially of administering to the subject an effective amount of a compound as described herein such that the ocular indication is treated.
• Typical ocular diseases include but are not limited to anterior segment or front-of-the-eye diseases (i.e., corneal and/or conjunctival diseases, such as aqueous tear deficiency, meibomian gland dysfunction, or OGVHD).
• Diseases can also include uveitis and other inflammatory diseases of the eye (i.e., crizis, iridocyclitis, intermediate uveitis, posterior uveitis, or panuveitis, of noninfectious, infectious, or idiopathic etiologies).
• Diseases can also include posterior segment or back-of-the-eye diseases (i.e., retinal and/or choroidal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, or retinal degeneration / dystrophy).
• the ocular indication comprises one or more ocular diseases characterized by T cell and /or macrophage-mediated inflammation.
• the disease is characterized by increased CaMKK2 activity.
• the“effective amount” (or“therapeutically effective amount”) of a composition comprises an amount sufficient to effect beneficial or desirable biological and/or clinical results.
• subject includes animals, such as mammals. Suitable subjects can include (but are not limited to) primates, cows, sheep, goats, dogs, cats, horses, rabbits, rats, mice, and the like. In some embodiments, the subject is a human.
• the term“treated” or“treatment” as used herein refers to arresting or ameliorating a disease, disease, or at least one of the clinical symptoms of a disease or disease; reducing the risk of acquiring a disease, disease, or at least one of the clinical symptoms of a disease or disease; reducing the development of a disease, disease or at least one of the clinical symptoms of the disease or disease; and/or reducing the risk of developing a disease or disease or at least one of the clinical symptoms of a disease or disease.
• Treatment can also refer to inhibiting a disease or disease, either physically (e.g., stabili ation of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both, or inhibiting at least one physical parameter that may not be discernible to the subject.
• treated or “treatment” can refer to delaying the onset of the disease or disease or at least symptoms thereof in a subject that may be exposed to or predisposed to a disease or disease, even though that subject does not yet experience or display symptoms of the disease or disease.
• the presently disclosed subject matter is directed to a method of treating cancer in a subject.
• the method comprises, consists of, or consists essentially of administering an effective amount of a compound as described herein to the subject such that the cancer is treated.
• the cancer is characterized by increased CaMKK2 activity within cancer cells or within infiltrating or accessory cells (i.e. vascular cells, immune cells, etc.) within or related to the cancer.
• the presently disclosed subject matter is directed to a method of treating a subject with a satiety-control disease.
• the method comprises, consists of, or consists essentially of administering an effective amount of a compound as described herein to a subject such that the satiety-control disease is treated.
• Table 1 illustrates the initial 5 compounds synthesized, each of which is representative of a novel subclass.
• HEK293 cells were cultured to sub confluence in RPMI-40 complete media and 1% fetal bovine serum (FBS), incubated at 37°C in 5% C0 2 . Cultured cells were then switched to serum-free media overnight prior to initiation of experiments. Cells were then pre-treated with one of the five SMICs (EY301, EY302, EY303, EY304, and EY305, all at 10 mM concentration), STO-609 (10 mM), or vehicle control for 2 hours incubated at 37°C in 5% C0 2 .
• FBS fetal bovine serum
• a one-way mixed lymphocyte reaction (MLR) (FIG. 5) will be used to screen for functional inhibitory capacity of EY301-EY305.
• Spleen cells of C57BL/6 mice will be used as response cells, and the spleen cells of BALB/c mice (irradiated with Co60, 3000 rads) will be used as stimulator cells.
• the two types of cells are then mixed with equal volumes and concentrations and incubated at 37°C in 5% C0 2 in low serum conditions for 24 hours, in the presence of 1) vehicle control; 2-6) one each of the five SMICs (EY301-EY305), or 7) STO-609, (at varying concentration 10 mM, 3 mM, 1 mM, 0.3 mM, and 0.1 mM for each SMIC or STO-609). Each concentration / condition will be performed in triplicate.
• the supernatant will then be recovered and concentrated for analysis by enzyme-linked immunosorbent assay (ELISA) for each of several key T cell- and macrophage-derived cytokines and effector molecules thought to mediate the destructive damage by infiltrating immune cells (e.g., TNF-a, IL-2, TGF-b, and IFN-g).
• ELISA enzyme-linked immunosorbent assay
• the functional inhibitory activity of EY301-EY305 will then be determined by calculation of the IC50 of each compound for each cytokine of interest.
• the compounds will then be ranked ordered by IC50 concentration for each cytokine of interest to assess the relative efficacy and potency of each SMIC for immune cell effector function.
• OGVHD Ocular graft versus host disease
• GOVHD ocular graft versus host disease
• 2-3-month old mice of C57BL/6 and BALB/c strains are used for this model.
• C57BL/6 mice are sacrificed, following which RPMI-1640 medium is flushed in the diaphyseal channel of both recovered tibias and femurs. Bone marrow is then homogenized and filtered, and unpurified bone marrow (BM) cells, devoid of red blood cells, are recovered from donor mice.
• BM bone marrow
• donor spleen is harvested aseptically, cut into small pieces, mashed with a spatula, and filtered and isolated to a single-cell suspension of isolated spleen-derived T cells.
• Recipient BALB/c mice undergo total body lethal irradiation at a dose of irradiated 950 cGy (with Cesium- 137 source), following which donor C57BL/6 BM cells and spleen-derived T cells are transplanted to irradiated recipient mice via tail vein injection.
• GVHD BMT severity score at -90 days, compared to safe BMT Keratopathy is observed to be severe, as evidenced by intense fluorescein staining of the ocular surface (FIG. 9B), as compared to control safe BMT with no comeal staining (FIG. 9A) (Fluorescein staining quantified by a clinical grading system, described in (33). Quantitative scoring of these parameters, using a validated clinical grading system (34), demonstrated a mean clinical score of 24 ⁇ 2 in the BMT + T cell recipient group with experimental OGVHD (FIG. 9C), as compared to 4 ⁇ 2 in the control safe BMT recipient group without signs of OGVHD (FIG. 9C).
• the OGVHD model described in Example 4 was used to test whether local ocular administration of tool compound CaMKK2 inhibitor STO-609 (1.5 mg/mL) might prevent or ameliorate severity of experimental OGVHD.
• CaMKK2 inhibitor STO-609 1.5 mg/mL
• recipient BALB/c mice were allowed to convalesce for 14 days prior to initiation of experimental procedures.
• STO-609 or vehicle was administered daily for 2 weeks, with regular examination occurring throughout the treatment period.
• eyes were then graded (in masked fashion) for signs of OGVHD and scored according to a modified clinical grading system (33, 34).
• mice were then sacrificed and eyes recovered for histologic analysis of ocular adnexal tissue (lids, conjunctiva) and anterior segment (conjunctiva, sclera, cornea), with pathologic assessment of OGVHD (i.e. immune cell infiltration, conjunctival scarring, goblet cell loss, Meibomian gland scarring, and lacrimal gland disruption/infiltration/scarring) .
• OGVHD i.e. immune cell infiltration, conjunctival scarring, goblet cell loss, Meibomian gland scarring, and lacrimal gland disruption/infiltration/scarring
• STO-609 treated mice had fewer signs of OGVHD (FIG. 11B).
• cells will be cultured in serum reduced conditions prior to treatment with 0.5 pg/mL ionomycin and 10 ng/mL PMA for 15 minutes, in the presence of vehicle, STO-609, candidate SMICs, or vehicle control. Cells will then be recovered and lysed for total protein, and Western blot analysis performed for pCaMKK2, pAMPK, pCaMKIV, with densitometry analysis normalized to actin, with SMICs inhibitory capacity compared to STO-609 and vehicle.
• MLR mixed lymphocyte reaction
• irradiated B ALB/c splenocytes will be added to C57BL/6 splenocytes in triplicates for each condition: STO-609 and candidate SMICs (all at the following concentrations: 10 pM, 3 pM, 1 pM, 0.3 pM, and 0.1 pM) as well as vehicle control.
• ELISA will then be performed for inflammatory cytokines IL-2, TNF-a, IL- 6, TGF-b, and IFN-g with calculation of drug IC50 for each cytokine. Measures of viability and apoptosis will also be performed for drug concentrations to ascertain potential dose-limiting toxicities in vitro.
• a kinome scan will be performed to assess for off-target activity of candidate SMICs for >450 common kinases, using a commercially available fee-for- service technology available through eurofins / DiscoverX (Fremont, CA). The top 5 SMICs will be ranked according to least off-target activity. The goal of this collective screening strategy (Western blot analysis, MLR reaction, kinome scan) will be to nominate at least five (5)_SMICs, ranked by potency and lack of off-target activity, to advance to in vivo screening and testing.
• mice will be sacrificed and eyes recovered for histology.
• Standard H&E sections of ocular tissue will be examined in a masked fashion for evidence of inflammation or tissue morphology change suggestive of toxicity.
• Superficial assessment for systemic toxicity will be performed, assessing failure to thrive and mortality. Any finding greater than “trace abnormal” will disqualify that compound concentration.
• the goal of these in vivo tolerability studies will be to identify at least two (2) SMICs with lack of ocular toxicity with topical application.
• C57BL/6 mice are sacrificed, following which RPMI-1640 medium is flushed in the diaphyseal channel of both recovered tibias and femurs.
• Recovered bone marrow is then homogenized and filtered, and unpurified bone marrow (BM) cells, devoid of red blood cells, are recovered from donor mice.
• donor spleen is harvested aseptically, cut into small pieces, mashed with a spatula, and filtered to a single-cell suspension of isolated splenocytes, containing T cells.
• Recipient BALB/c mice undergo total body lethal irradiation at a dose of 950 cGy with Cesium- 137 source, following which donor C57BL/6 BM cells and spleen- derived T cells are transplanted to irradiated recipient mice via tail vein injection. Recipient BALB/c mice will then be allowed to convalesce for approximately 14 days before initiation of further experimental procedures. At that time, twice-daily, bilateral topical treatment with selected drug will be initiated, with regular examination throughout a four-week treatment period. Clinical scoring will be performed after two weeks and four weeks of treatment. OGVHD clinical findings include lid margin edema, lid crusting, tear film disruption, conjunctival chemosis, and keratopathy.
• mice After the treatment period (six weeks post-BMT), mice will be sacrificed and eyes recovered for histologic analysis of ocular adnexal tissue (lids, conjunctiva) and anterior segment (conjunctiva, sclera, cornea), with pathologic assessment of OGVHD (i.e. immune cell infiltration, conjunctival scarring, goblet cell loss, Meibomian gland scarring, and lacrimal gland disruption/infiltration/scarring) .
• OGVHD i.e. immune cell infiltration, conjunctival scarring, goblet cell loss, Meibomian gland scarring, and lacrimal gland disruption/infiltration/scarring
• Treatment groups will be as follows:
• nonparametric statistics e.g. Mann- Whitney test
• Parametric statistics will be used to compare median scores among treatment groups, since clinical scoring reflects categorical grades.
• Parametric statistics will be used for comparison of continuous variables (e.g. quantitative immune cell infiltration) on pathologic assessment.
• the initial set of synthesized SMICs are referenced in Table 1. From among this set, six candidate SMICs (EY1003.A001.B001, EY1005.A001.B001, EY1006.A001.B001,
• EY1007.A001.B001, EY1008.A001.B001, EY1001.A002.B001) were selected for a cell-based biochemical screening assay in HEK293 cells.
• HEK-293 cells were treated with one of the following: STO-609, EY1003.A001.B001, EY1005.A001.B001, EY 1006.
• NonGLP toxicology studies of locally administered candidate SMICs EY1006.A001.B001 and EY1001.A002.B001 was assessed in 2-3-month old wild-type BALB/c mice assessing local toxicity by clinical assessment and by histopathology. Both compounds were topically administered at 1.5 mg/mL twice daily for 14 days. No signs of local toxicity were observed for either drug by clinical biomicro scopic eye examination or postmortem histologic assessment. Additionally, neither compound was associated with any apparent systemic toxicity (i.e., no evidence of failure to thrive or mortality).
• STO-609 acetate 1.5 mg/mL
• lead SMIC compounds EY1006.A001.B001 and EY1001.A002.B001 both at 1.5 mg/mL
• prednisolone acetate beginning at day 14 post-BMT and continuing for two weeks.
• OGVHD clinical findings include lid margin edema, lid crusting, tear film disruption, conjunctival chemosis, and keratopathy. These findings were clinically graded following one week and two weeks of treatment using a quantitative scoring system adapted from previously published studies (33, 34). Both lead SMICs EY1006.A001.B001 and EY1001.A002.B001 reduced severity of clinical OGVHD findings with efficacy similar to STO-609 (FIG. 13). By contrast, vehicle control- treated eyes had lid margin swelling and scarring with lash and periocular fur loss, eyelid crusting, chemosis, abnormal tear film, and keratopathy.
• STO-609 and lead SMICs were superior to vehicle and prednisolone, in preventing signs of OGVHD (p ⁇ 0.05 for STO-609, EY1006.A001.B001, EY1001.A002.B001 vs vehicle or prednisolone).
• Mild disease was score 0-5, moderate disease was score 6-10 and severe disease was score > 10.
• Step 1 As shown in the procedure for Compounds 6-9, Stepl
• Ri is selected from oxygen (O) or sulfur (S);
• R 2 is selected from hydrogen (H), an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.
• R is selected from an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.
• X oxygen (O) or sulfur (S), and n is 1 or 2.
• R is an ester or ester prodrug comprised of but not limited to an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.
• X is oxygen (O) or sulfur (S); wherein Ri is selected from hydrogen (H), an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.;
• R 2 is selected from hydrogen (H), an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.
• R, Ri, and R 2 are selected from an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group; and wherein n is 1, 2, or 3.
• A denotes an atom in a carbocyclic or heterocyclic aromatic ring or a pharmaceutically acceptable salt, solvate, hydrate, or derivative thereof.
• Ri is hydrogen (H) or COOCH3, and wherein R 2 is CFhCN, CH2COOC2H5, or CFhCOPh.
• Statement 10 A method of modulating CaMKK2 in a subject, the method comprising administering an effective amount of the composition of Formula (I) to a subject:
• Ri is selected from oxygen (O) or sulfur (S); and wherein R2 is selected from hydrogen (H), an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.
• a method of modulating CaMKK2 in a subject comprising administering an effective amount of the composition of Formula (I) to a subject:
• a method of modulating CaMKK2 in a subject comprising administering an effective amount of the composition of Formula (I), Formula (II), or both to a subject:
• X is oxygen (O) or sulfur (S), and n is 1 or 2.
• X is oxygen (O) or sulfur (S); wherein Ri is selected from hydrogen (H), an, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.; and wherein R 2 is selected from hydrogen (H), cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.
• a method of modulating CaMKK2 in a subject comprising administering an effective amount of the composition of Formula (I), Formula (II), Formula (III), Formula (IV), or combinations thereof to a subject:
• R, Ri, and R 2 are selected from an, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.; and wherein n is 1, 2, or
• a method of modulating CaMKK2 in a subject comprising administering an effective amount of the composition of Formula (I) to a subject: (Formula I) wherein A denotes an atom in a carbocyclic or heterocyclic aromatic ring or a pharmaceutically acceptable salt, solvate, hydrate, or derivative thereof.
• a method of modulating CaMKK2 in a subject comprising administering an effective amount of the composition of Formula (I) to a subject:
• a method of modulating CaMKK2 in a subject comprising administering an effective amount of the composition of Formula (I), Formula (II), or both to a subject: I) or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof,
• Ri is hydrogen (H) or COOCH3, and wherein R 2 is CFhCN, CH2COOC2H5, or CFhCOPh.
• a method of treating any ophthalmic disease which includes but is not limited to: 1) ocular surface inflammatory diseases (OSIDs), including but not limited to ocular graft versus host disease, ocular cicatricial pemphigoid, vernal keratoconjunctivitis, allergic eye disease, meibomian gland dysfunction, aqueous tear deficiency (common dry eye disease), corneal scarring, and conjunctival scarring and fibrosis; 2) uveitis and other inflammatory diseases of the eye, including but not limited to keratitis, scleritis, ulceris, iridocyclitis, intermediate uveitis, pars planitis, posterior uveitis, choroiditis, chorioretinitis, retinitis, or panuveitis of noninfectious, infectious, or idiopathic etiologies; and 3)“back of the eye” retinal diseases, which
• OSIDs
• retinal vein occlusion retinal vein occlusion
• retinal artery occlusion retinal degenerations and dystrophies
• the method comprising administering an effective amount of the compound of any of Statements 1-9 to the subject such that the ophthalmic disease is treated.
• Statement 20 A method of treating a frontal or distal eye indication in a subject, the method comprising administering an effective amount of the compound of any of Statements 1-9 to the subject such that the indication is treated.
• Statement 21 A method of treating cancer in a subject, the method comprising administering an effective amount of the compound of any of Statements 1-9 to the subject such that the cancer is treated.
• Statement 22 A method of treating an appetite disease in a subject, the method comprising administering an effective amount of the compound of any of Statements 1-9 to the subject such that the appetite disease is treated.
• Statement 23 A method of treating systemic inflammatory or autoimmune diseases, such as graft versus host disease, sarcoidosis, systemic lupus erythematosus, others, in a subject, the method comprising administering an effective amount of the compound of any of Statements 1-9 to the subject such that the systemic inflammatory disease is treated.
• systemic inflammatory or autoimmune diseases such as graft versus host disease, sarcoidosis, systemic lupus erythematosus, others.
• XX or a pharmaceutically acceptable salt, solvate, hydrate, or derivative thereof, wherein X is oxygen (O) or sulfur (S); wherein Y is NR 4 , or CR 5 R 6 ; wherein Ri is -CHiCOOH, -COOH, -CH 2 COOCR 7 , - COOR7; -CH2CONH2, -CONH2, -CH2CONR5R6, or -CONR 5 R 6 ; wherein each R 2 and R 3 are independently hydrogen (H), C1-C10 alkyl, -OR 7 , -OCH2CH2OR7, -OCH2CH2NR5R6, -OCH2CH2COOR7 or -OCH2CH2PO3H;
• R 4 hydrogen (H), C1-C10 alkyl, -CH 2 CN, -CH 2 C(0)NH 2 , -CH 2 COOH,
• n is an integer from 1 to 5
• m is an integer from 0 to 5
• Z is oxygen (O) or sulfur (S);
• each R 5 and R 6 are independently hydrogen (H), C1-C5 alkyl, or R 5 and R 6 together may a 3 to 7-member cycloalkyl ring; and wherein R 7 is C1-C5 alkyl.
• Statement 25 The compound of Statement 24, wherein X is oxygen.
• Statement 26 The compound of any of Statement 24 or 25, wherein Y is NH, NCH3,
• Statement 27 The compound of any of Statement 24-26, wherein Ri is COOH.
• Statement 28 The compound of any of Statement 24-27, wherein R2 or R3 is -OCH3.
• Statement 29 The compound of any of Statement 24-28, wherein R2 and R3 are -OCH3.
• X oxygen (O) or sulfur (S); wherein Ri is -CH2COOH, -COOH, -CH2COOCR7, -COOR7; - CH2CONH2, -CONH2, -CH2CONR5R6, or -CONR5R6; wherein each R2 and R3 are independently hydrogen (H), C1-C10 alkyl, -OR 7 , -OCH2CH2OR7, -OCH2CH2NR5R6, -OCH2CH2COOR7 or - OCH2CH2PO3H;
• each R5 and R 6 are independently hydrogen (H), C1-C5 alkyl, or R5 and R 6 together may a 3 to 7-member cycloalkyl ring; wherein R 7 is C1-C5 alkyl; and wherein w is 1 or 2.
• Statement 31 The compound of Statement 30, wherein X is oxygen.
• Statement 32 The compound of any of Statement 30-31, wherein Ri is COOH.
• Statement 33 The compound of any of Statement 30-32, wherein R2 or R3 is -OCH3.
• Statement 34 The compound of any of Statement 30-33, wherein R2 and R3 are -OCH3.
• Statement 35 A method of modulating CaMKK2 in a subject, the method comprising administering to the subject an effective amount of the compound of any of Statements 24-29.
• Statement 36 A method of modulating CaMKK2 in a subject, the method comprising administering to the subject an effective amount of the compound of any of Statements 30-34.
• Statement 37 A method of treating a frontal or distal eye indication in a subject, the method comprising administering an effective amount of the compound of any of Statements 24-29 to the subject.
• Statement 38 A method of treating a frontal or distal eye indication in a subject, the method comprising administering an effective amount of the compound of any of Statements 30-34 to the subject.
• Statement 39 A method of treating cancer in a subject, the method comprising administering an effective amount of the compound of any of Statements 24-29 to the subject.
• Statement 40 A method of treating cancer in a subject, the method comprising administering an effective amount of the compound of any of Statements 30-34 to the subject.
• Statement 41 A method of treating an appetite disease in a subject, the method comprising administering an effective amount of the compound of any of Statements 24-29 to the subject.
• Statement 42 A method of treating an appetite disease in a subject, the method comprising administering an effective amount of the compound of any of Statements 30-34 to the subject.
• Statement 43 A method of treating systemic inflammatory or autoimmune diseases, such as graft versus host disease, sarcoidosis, systemic lupus erythematosus, others, in a subject, the method comprising administering an effective amount of the compound of any of Statements 24-29 to the subject.
• systemic inflammatory or autoimmune diseases such as graft versus host disease, sarcoidosis, systemic lupus erythematosus, others.
• Statement 44 A method of treating systemic inflammatory or autoimmune diseases, such as graft versus host disease, sarcoidosis, systemic lupus erythematosus, others, in a subject, the method comprising administering an effective amount of the compound of any of Statements 30-34 to the subject.
• systemic inflammatory or autoimmune diseases such as graft versus host disease, sarcoidosis, systemic lupus erythematosus, others.

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