WO2010054022A1 - Analyse de membrane chorio-allantoïque améliorée - Google Patents

Analyse de membrane chorio-allantoïque améliorée Download PDF

Info

Publication number
WO2010054022A1
WO2010054022A1 PCT/US2009/063316 US2009063316W WO2010054022A1 WO 2010054022 A1 WO2010054022 A1 WO 2010054022A1 US 2009063316 W US2009063316 W US 2009063316W WO 2010054022 A1 WO2010054022 A1 WO 2010054022A1
Authority
WO
WIPO (PCT)
Prior art keywords
embryonic
vascularization
skin tissue
cam
avian
Prior art date
Application number
PCT/US2009/063316
Other languages
English (en)
Inventor
Haiming Chen
Original Assignee
Institute Of Multiple Myeloma & Bone Cancer Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute Of Multiple Myeloma & Bone Cancer Research filed Critical Institute Of Multiple Myeloma & Bone Cancer Research
Publication of WO2010054022A1 publication Critical patent/WO2010054022A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates

Definitions

  • the field of the present invention generally pertains to methods of assaying vascularization. More specifically, the present invention is directed toward methods of assaying angiogenesis and/or vasculogenesis using an improved chorioallantoic membrane (CAM) assay to determine the anti- angiogenic or angiogenic activity of a test compound.
  • CAM chorioallantoic membrane
  • Angiogenesis defined as the growth or sprouting of new blood vessels from existing vessels, is a complex process that primarily occurs during embryonic development. Angiogenesis is typically limited in a normal adult to the placenta, ovary, endometrium and sites of hair growth or wound healing (Auerbach, W. and Auerbach, R., 1994, Pharmacol Ther 63(3):265-3 11 ; Ribatti et al., 1991 , Haematologica 76(4):3 11-20; Risau, 1997, Nature 386(6626):67 1-
  • Vasculogenesis is the process of blood vessel formation occurring by a de novo production of endothelial cells. Though similar to angiogenesis, the two are different in one aspect: the term angiogenesis denotes the formation of new blood vessels from pre-existing ones, while vasculogenesis is the term used for the formation of new blood vessels when there are no preexisting ones. For example, if a monolayer of endothelial cells begins sprouting to form capillaries, angiogenesis is occurring. Vasculogenesis, in contrast, first believed to occur only during embryologic development, is the term for when endothelial precursor cells (angioblasts) migrate and differentiate in response to local cues (such as growth factors and extracellular matrix) to form new blood vessels.
  • endothelial precursor cells angioblasts
  • vasculogenesis can also occur in the adult organism. Circulating endothelial progenitor cells (derivatives of stem cells) were identified and reportedly able to contribute, albeit to varying degrees, to neovascularization, such as during tumor growth, or to the revascularization process following trauma, e.g., after cardiac ischemia.
  • angiogenesis and/or vasculogenesis plays an important role in the maintenance of a variety of pathological states.
  • Some of these states are characterized by neovascularization, e.g., cancer, diabetic retinopathy, glaucoma, and age related macular degeneration.
  • pathological states characterized by neovascularization include lymphoma, hematologic cancers, melanoma, breast cancer, lung cancer, prostate cancer, colon cancer, ovarian cancer, liver cancer, Kaposi's sarcoma, metastatic disease, rheumatoid arthritis, psoriasis, and benign proliferative disorders including hemangiomas.
  • pathological states include, e.g., stroke, infertility, heart disease, ulcers, delayed wound healing, and scleroderma, are diseases of angiogenic insufficiency. Therefore, there is a need in the art to identify modulators of angiogenesis.
  • angiogenesis and vasculogenesis are highly complex biological processes that involve a number of different molecules and signaling pathways, the development of various strategies to study these processes have been an area of concentration for many research groups.
  • in vitro and in vivo strategies have been established to identify putative compounds which either stimulate or inhibit angiogenesis and/or vasculogenesis.
  • some in vitro strategies include the MatrigelTM tube-forming assay (see, e.g., Grant et al., Cell, 58:933-943 (1989) and Davis et al., J. Cell. Biochem., 51 :206-218 (1993)); the fibrin and collagen gel-cord-forming assays (see, e.g., Dvorak et al., Lab.
  • in vivo strategies have been developed to more thoroughly analyze the process of angiogenesis and/or vasculogenesis.
  • Some of these in vivo strategies include the rat, mouse and rabbit corneal pocket assays (see, e.g., Sholley et al., Lab. Invest., 51 :624-634 (1994), Friedlander ef a/., Science, 270:1500-1502 (1995), and Chen et al., Cancer Res., 55:4230-4233 (1995)); the primate iris neovascularization model (see, e.g., Miller et al., Am. J.
  • these strategies are limited by the length of time required for the assay and the complexity and expense required to carry them out.
  • most of the physiological effects of these molecules can generally only be monitored locally, with little information available as to their systemic effects.
  • the CAM is the major respiratory structure for the exchange of gases and nutrients during embryonic development; thus, the CAM becomes highly vascularized.
  • the CAM provides an ideal microenvironment in which to study angiogenesis and/or vasculogenesis.
  • the modified CAM assay developed by Brooks et al. (see Brooks et al., "Use of the 10-Day-Old Chick Embryo Model for Studying Angiogenesis," in Methods in Molecular Biology, Vol.129: lntegrin Protocols, ed. A. R.
  • Angiogenesis and/or vasculogenesis have been shown to play an important role in human diseases such as cancer. Therefore, the level of angiogenic and/or vasculogenic stimulation in a patient has become important diagnostically and the control of angiogenic and/or vasculogenic activity has become a promising therapeutic goal.
  • Compounds have been identified and contemplated for use in the treatment of human diseases involving unregulated angiogenesis and/or vasculogenesis.
  • anti- angiogenic agents examples include pigment epithelium derived factor (PEDF), angiostatin, thrombospondin, protamine, vasculostatin, endostatin, platelet factor 4, heparinase, interferons (e.g., IFN ⁇ ), and the like.
  • PEDF pigment epithelium derived factor
  • angiostatin thrombospondin
  • protamine vasculostatin
  • vasculostatin endostatin
  • platelet factor 4 heparinase
  • interferons e.g., IFN ⁇
  • active fragments of anti-angiogenic agents i.e., those fragments having biological activity sufficient to inhibit angiogenesis
  • active fragments of anti-angiogenic agents i.e., those fragments having biological activity sufficient to inhibit angiogenesis
  • the present invention provides a novel assay to test the anti-angiogenic and/or angiogenic activity of compounds in a rapid, accurate and inexpensive way.
  • the present invention provides a method of determining the ability of a test compound to modulate vascularization comprising the steps of: i) windowing an embyronated shelled egg; ii) culturing the embyronated shelled egg; iii) contacting an embryonic avian skin tissue in a culture with at least one test compound; iv) contacting the CAM of the embyronated shelled egg of step (ii), with the embryonic avian skin tissue of step (iii) and culturing the CAM and embryonic avian skin tissue composition; and v) determining the ability of the at least one test compound to modulate vascularization.
  • the embryonated shelled egg is from an avian species.
  • the avian species is selected from the group consisting of: chicken, turkey, duck, goose, quail, pheasant, grouse, ostrich, emu, cassowary and kiwi.
  • the avian species is selected from the group consisting of: chicken and quail.
  • the avian species of embryonated shelled egg is different from the species of embryonic avian skin tissue.
  • the avian species is chicken.
  • the avian embryonated shelled egg is obtained at about embryonic day 7 (E7) to about embryonic day 9 (E9) of gestation. In other particular embodiments, the avian embryonated shelled egg is cultured for about 0 to about 48 hours.
  • the embryonic avian skin tissue of step (c) is isolated from about an about embryonic day 7 (E7) to about embryonic day 9 (E9) embryo.
  • the embryonic avian skin tissue in a culture is contacted with the at least one test compound for about 0 to about 48 hours.
  • the CAM and embryonic avian skin tissue composition are cultured for about 1 to 4 days.
  • the present invention provides a method of determining the ability of a test compound to modulate vascularization that further comprises a step of measuring or monitoring a level of an indicator of vascularization of the at least one test compound. In certain related embodiments, the present invention provides a method of determining the ability of a test compound to modulate vascularization that further comprises a step of measuring or monitoring a level of an indicator of vascularization of the at least one test compound and comparing said level to a level of an indicator of vascularization of a control compound.
  • the control compound is a known angiogenic or anti-angiogenic modulator of vascularization.
  • the indicator of vascularization is selected from the group consisting of: new blood vessel growth, feather bud growth, feather bud vascularization, feather bud weight, and endothelial and/or Wnt pathway gene expression. In other related embodiments, the indicator of vascularization is selected from the group consisting of: new blood vessel growth, feather bud growth, feather bud vascularization, and feather bud weight. In yet other related embodiments, the indicator of vascularization is feather bud growth. In yet other related embodiments, the indicator of vascularization is feather bud weight.
  • the present invention provides a method of determining the anti-angiogenic activity of a test compound comprising the steps of: i) windowing an avian embyronated shelled egg; ii) culturing the avian embyronated shelled egg; iii) contacting an embryonic avian skin tissue in a culture with at least one test compound; iv) contacting the CAM of the embyronated shelled egg of step (ii), with the embryonic avian skin tissue of step (iii) and culturing the CAM and embryonic avian skin tissue composition; and v) determining the anti-angiogenic activity of the at least one test compound by measuring or monitoring a level of an indicator of vascularization of the at least one test compound.
  • a method of determining the anti- angiogenic activity of a test compound further comprises a step of contacting the embryonic avian skin tissue with the at least one test compound and an angiogenic compound.
  • a decrease in the level of the indicator of vascularization of the at least one test compound compared to a level of an indicator of vascularization of a non-angiogenic control compound or vehicle indicates that the at least one test compound has anti-angiogenic activity.
  • the present invention provides a method of determining the angiogenic activity of a test compound comprising the steps of: i) windowing an avian embyronated shelled egg; ii) culturing the avian embyronated shelled egg; iii) contacting an embryonic avian skin tissue in a culture with at least one test compound; iv) contacting the CAM of the embyronated shelled egg of step (ii), with the embryonic avian skin tissue of step (iii) and culturing the CAM and embryonic avian skin tissue composition; and v) determining the angiogenic activity of the at least one test compound by measuring or monitoring a level of an indicator of vascularization of the at least one test compound,
  • a method of determining the angiogenic activity of a test compound further comprises a step of contacting the embryonic avian skin tissue with the at least one test compound and an anti-angiogenic compound.
  • an increase in the level of the indicator of vascularization of the at least one test compound compared to a level of an indicator of vascularization of the anti-angiogenic compound indicates that the at least one test compound has angiogenic activity.
  • the present invention provides an assay for screening for a modulator of vascularization comprising: i) windowing an avian embryonated shelled egg; ii) culturing the avian embyronated shelled egg; iii) contacting an embryonic avian skin tissue in a culture with at least one test compound; iv) contacting the CAM of the embyronated shelled egg of step (ii), with the embryonic avian skin tissue of step (iii) and culturing the CAM and embryonic avian skin tissue composition; and v) determining the ability of the at least one test compound to modulate vascularization by measuring or monitoring a level of an indicator of vascularization of the at least one test compound and comparing said level to a level of an indicator of vascularization of a control compound.
  • FIG. 1 shows the results of a classical CAM angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E9 chicken chorioallantoic membranes were cultured with1.5 ⁇ M or 15 ⁇ M zoledronic acid or without zoledronic acid (positive control) for 24 and 48 hours at 37.5 0 C in a humidified incubator.
  • FIG. 2 shows a flowchart for a chorioallantoic membrane - feather bud (CAM-FB) angiogenesis assay.
  • Fertilized chick eggs E1
  • E8 On day 8 (E8), a window was opened in the shell and the embryo placed in a Petri dish.
  • the embryonic skin was detached from the body and cut into 5 mm 2 portions.
  • the embryonic skin/FB was cultured in culture medium with or without anti- angiogenesis compounds for 24 - 48 hours.
  • the treated FB was loaded onto E9 CAM and window was sealed.
  • Angiogenesis and feather bud development were examined after 4 days of co-culture.
  • FIG 3 shows the results of an early blood vessel (vascularization) assay conducted in a CAM/feather buds (CAM-FB) model angiogenesis assay.
  • Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8.
  • E8 chicken embryonic skin was cultured in insert culture dishes with 5 ⁇ M or 15 ⁇ M zoledronic acid or without zoledronic acid (positive control) for 24 hours.
  • the cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator.
  • Angiogenesis was determined by blood vessel growth under a microscope. The results show that zoledronic acid blocks new blood vessel formation and feather bud development through inhibition of endothelial proliferation and migration.
  • FIG. 4 shows the results of an early blood vessel
  • Figure 5 shows the results of an analysis for new blood vessel formation in a CAM-FB model assay using hematoxylin and eosin Y (H & E) staining and Tie2 protein immunohistochemistry.
  • A New blood vessel formation in feather buds was assayed using vehicle control (i.e., PBS) and zoledronic acid at 15 ⁇ M; 200X magnification.
  • B New blood vessel formation in feather buds was assayed using vehicle control (i.e., PBS) and zoledronic acid at 15 ⁇ M; 400X magnification.
  • C The leftmost two panels show H & E staining of feather buds in a model CAM-FB assay at 100X and 400X magnification. The rightmost two panels show antibody staining for the endothelial marker gene Tie2 in feather buds in a model CAM-FB assay at 200X using vehicle control (i.e., PBS) and zoledronic acid at 5 ⁇ M.
  • Figure 6 shows the results of a control feather bud development in vitro culture assay.
  • Three groups of E8 chicken embryonic skin were cultured in insert culture dishes and subjected to the following treatments for 24, 48, and 72 hours: i) no zoledronic acid, no vitamin-C; ii) 8 ⁇ g/mL zoledronic acid, 20 ⁇ M vitamin-C; and iii) 8 ⁇ g/mL zoledronic acid, no vitamin-C.
  • the results show that zoledronic acid does not affect normal feather bud (epithelial cells) growth in vitro.
  • FIG. 7 shows the results of an early blood vessel
  • E8 chicken embryonic skin were cultured in insert culture dishes and subjected to the following treatments for 24 hours: i) no zoledronic acid, no vitamin-C; ii) 100 ⁇ M vitamin-C; iii)15 ⁇ M zoledronic acid, 100 ⁇ M vitamin-C; and iv) 15 ⁇ M zoledronic acid.
  • the cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator.
  • Angiogenesis was determined by blood vessel growth and by degree of feather bud development. The results show that zoledronic acid inhibits feather bud development; vitamin- C does not affect feather bud development, and vitamin-C does not reverse zoledronic acid mediated inhibition of feather bud development.
  • Figure 8 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. Four groups of E8 chicken embryonic skin were cultured in insert culture dishes and subjected to the following treatments for 24 hours: i) no zoledronic acid, no vitamin-C; ii) 100 ⁇ M vitamin-C; iii)15 ⁇ M zoledronic acid, 100 ⁇ M vitamin-C; and iv) 15 ⁇ M zoledronic acid.
  • the cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator.
  • Angiogenesis was determined by blood vessel growth and by degree of feather bud development. The results show that zoledronic acid inhibits feather bud development; vitamin- C does not affect feather bud development, and vitamin-C slightly reverses zoledronic acid mediated inhibition of feather bud development.
  • Figure 9 shows the results of a control feather bud development in vitro culture assay.
  • Four groups of E8 chicken embryonic skin were cultured in insert culture dishes and subjected to the following treatments for 24, 48, and 72 hours: i) no zoledronic acid, no vitamin-C; ii) 100 ⁇ M vitamin-C; iii) 15 ⁇ M zoledronic acid, 100 ⁇ M vitamin-C; and iv) 15 ⁇ M zoledronic acid.
  • the cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for four days at 37.5 0 C in a humidified incubator. The results show that neither zoledronic acid nor vitamin-C alone or in combination affect normal feather bud (epithelial cells) growth in vitro.
  • FIG 10 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 24 hours: i) control (PBS); ii) 10 nM fumagillin; iii) 100 nM fumagillin; and iv) 1 ⁇ M fumagillin. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator. Angiogenesis was determined by blood vessel growth under a microscope. Results are shown for co-cultures at day one and day four. The results at day 4 show that the 100 nM fumagillin and 1 ⁇ M fumagillin inhibit new blood vessel formation and feather bud development.
  • PBS control
  • FIG 11 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 24 hours: i) control (PBS); ii) 100 nM fumagillin; and iii) 1 ⁇ M fumagillin. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator. Angiogenesis was determined by blood vessel growth under a microscope. The results show that at day four, 1 ⁇ M fumagillin (lower right panel), and 100 nM fumagillin (lower left panel) inhibit new blood vessel formation and feather bud development.
  • Figure 12 shows the results of a control feather bud development in vitro culture assay.
  • Five groups of E8 chicken embryonic skin were cultured in insert culture dishes and subjected to the following treatments for 72 hours: i) vehicle control (i.e., PBS; uppermost panel); ii) 100 nM fumagillin (left panel, center row); iii) 1 ⁇ M fumagillin (right panel, center row); iv) 500 nM minocycline (left panel, bottom row); and v) 5 ⁇ M minocycline (right panel, bottom row).
  • vehicle control i.e., PBS; uppermost panel
  • ii) 100 nM fumagillin left panel, center row
  • iii) 1 ⁇ M fumagillin right panel, center row
  • iv 500 nM minocycline
  • v 5 ⁇ M minocycline
  • Figure 13 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 24 hours: i) control (PBS); ii) 500 nM minocycline; and iii) 5 ⁇ M minocycline. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator. Angiogenesis was determined by blood vessel growth under a microscope. Results are shown for co-cultures at day one and day four.
  • Figure 14 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 24 hours: i) control (PBS); ii) 25 ⁇ g/mL Celebrex®; iii) 100 ⁇ g/mL Celebrex®; and iv) 250 ⁇ g/mL Celebrex®. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator.
  • PBS control
  • Angiogenesis was determined by blood vessel growth under a microscope. The results show the effect at day four of 25 ⁇ g/mL Celebrex® (celecoxib), 100 ⁇ g/mL Celebrex®, and 250 ⁇ g/mL Celebrex® on new blood vessel formation and feather bud development compared to the control (i.e., PBS).
  • FIG. 15 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 24 hours: i) control (PBS); ii) 0.2 ⁇ g/mL Velcade®; and iii) 0.2 ⁇ g/mL Velcade®. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator. Angiogenesis was determined by blood vessel growth under a microscope. The results show that at day four, 0.2 ⁇ g/mL Velcade® (bortezomib) inhibits new blood vessel formation and feather bud development.
  • PBS control
  • ii) 0.2 ⁇ g/mL Velcade® 0.2 ⁇ g/mL
  • Figure 16 shows a Western blot analysis of Tie2 protein in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes for 24 hours. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator. At day four, the embryonic skins/feather buds were removed and proteins samples prepared for Western blotting.
  • the results show that the expression of Tie2 protein is reduced in CAM-FB assays wherein the E8 chicken embryonic skins were exposed, for 24 hours, to increasing concentrations of fumagillin, compared to the CAM-FB assay using a control (i.e., PBS).
  • the results further show the expression of Tie2 protein in CAM-FB assays wherein the E8 chicken embryonic skins were exposed, for 24 hours, to increasing concentrations of Celebrex®. GAPDH was used as a loading control.
  • FIG 17 shows a RT-PCR analysis of Tie2 and FLK-1 gene expression in a CAM-FB model angiogenesis assay.
  • Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8.
  • E8 chicken embryonic skin was cultured in insert culture dishes for 24 hours.
  • the cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator.
  • the embryonic skins/feather buds were removed and samples prepared for RT/PCR.
  • Tie2 gene expression is reduced in CAM- FB assays wherein the E8 chicken embryonic skins were exposed, for 24 hours, to increasing concentrations of fumagillin, minocycline, or zoledronic acid, compared to the CAM-FB assay using a control (i.e., PBS).
  • FLK-1 gene expression is reduced in CAM-FB assays wherein the E8 chicken embryonic skins were exposed, for 24 hours, to increasing concentrations of fumagillin, minocycline, or zoledronic acid.
  • Tie2 gene expression is reduced in CAM-FB assays wherein the E8 chicken embryonic skins were exposed, for 48 hours, to increasing concentrations of fumagillin, minocycline, zoledronic acid, or melphalan compared to the CAM-FB assay using a control (i.e., PBS).
  • FLK-1 gene expression is reduced in CAM-FB assays wherein the E8 chicken embryonic skins were exposed, for 24 hours, to increasing concentrations of fumagillin, minocycline, zoledronic acid, or melpahlan.
  • GAPDH was used as a standardization control.
  • GAPDH was used as a standardization control.
  • Figure 18 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 24 hours: i) control (PBS); ii) 1.5 ⁇ g/mL anti-VEGF antibody; iii) 3.0 ⁇ g/mL anti-VEGF antibody; and iv) 6.0 ⁇ g/mL anti-VEGF antibody.
  • PBS control
  • the cultured embryonic skins were subsequently cultured in vitro and cultured for an additional day at 37.5 0 C in a humidified incubator or placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator.
  • Angiogenesis was determined by blood vessel growth under a microscope.
  • Figure 19 shows the results from a control mouse wound healing assay.
  • CD1 mice were injured with experimental gel containing15 ⁇ M zoledronic acid and gel only control. Two groups of ⁇ 6 mm incisions were made on the dorsal pelts of the mice for each experimental gel and control gel sample tested. Additionally a ⁇ 6 mm wound was made on the tails of mice 1 and 2. Experimental gel was applied to the tail would of mouse 1 , while control gel was applied to the tail wound of mouse 2. Wound healing was monitored at time 0, 2, 5, and 8 days post injury. The results show that determining the wound healing and angiogenesis by using this mouse wound healing model is difficult and inconclusive.
  • Figure 20 shows feather volume measurement and FLK-1 protein expression in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 48 hours: (Top panel) i) control (PBS), ii) 100 nM fumagillin, and iii) 1 ⁇ M fumagillin; (Middle panel) i) control (PBS), ii) 0.5 ⁇ M minocycline, and iii) 5 ⁇ M minocycline; and (Bottom panel) i) control (PBS), ii) 2 ⁇ M zoledronic acid, and iii) 8 ⁇ M zoledronic acid;.
  • PBS control
  • the cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional one, two, or four days at 37.5 0 C in a humidified incubator. At days, one, two, and four, the embryonic skins/feather buds were removed and feather bud (FB) weight was measured using a fine balance. FLK-1 protein expression was quantified using ELISA. (Top panel) Left-most panel shows reduced FB weight in CAM-FB assays treated with increasing concentrations of fumagillin; right-most panel shows reduced FLK1 protein expression in CAM-FB assays treated with increasing concentrations of fumagillin.
  • Figure 21 shows the comparative development of feather buds (FB) in embryonic skins cultured in vitro and on CAM. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. 8mm portions of E8 chicken embryonic skin were dissected and the epithelial layer of the FB was placed on an insert and exposed to specific drugs mixed with culture medium for 48 hours. One group of cultured embryonic skins was subsequently placed on E9 chicken CAM and the other group was cultured in the absence of E9 chicken CAM. Cultures were maintained for 4 days at 37.5 0 C in a humidified incubator. The bottom right panel show FB cultured on CAM.
  • FB feather buds
  • the co-cultures were not exposed to drugs or compounds that were able to prevent the development of blood vessels, placode and dermal condensation of the FB.
  • FB weight increased 5 - fold.
  • the top right panel shows FB cultured in the absence of CAM. In the absence of CAM, the FB does not develop feathers.
  • Figure 22 shows the results of a cytotoxicity assay for E8 chicken embryonic skins cultured in the presence of fumagillin, minocycline, or melphalan. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8.
  • Four groups of E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 48 hours: treatment group I) vehicle (e.g., PBS); treatment group II) 1 ⁇ M fumagillin; treatment group III) 5 ⁇ M minocycline; and treatment group IV) 30 ⁇ M melphalan.
  • Drugs showing anti-angiogenic activity, including fumagillin and minocycline did not show direct cytotoxic effects on cells in the FB. However, the chemotherapeutic drug without anti-angiogenic effects, the alkylating agent, melphalan, was toxic to the cells within the FB; causing the bud to diminish or disappear.
  • Figure 23 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 48 hours: i) control (PBS); ii) 0.5 ⁇ M doxorubicin; and iii) 5 ⁇ M doxorubicin. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator. Angiogenesis was determined by blood vessel growth under a microscope.
  • results are shown for co-cultures at day one and day four. The results show that at day four, both 0.5 ⁇ M and 5 ⁇ M doxorubicin inhibit new blood vessel formation and feather bud development.
  • B At days, one, two, and four, the embryonic skins/feather buds were removed and feather bud (FB) weight was measured using a fine balance. FLK-1 protein expression was quantified using ELISA. The left-most panel shows reduced FB weight in CAM-FB assays treated with increasing concentrations of doxorubicin. The right-most panel shows reduced FLK1 protein expression in CAM-FB assays treated with increasing concentrations of doxorubicin.
  • Figure 24 shows the results of an early blood vessel (vascularization) assay conducted in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 48 hours: i) control (PBS); ii) 1 ⁇ M melphalan; and iii) 10 ⁇ M melphalan. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator. Angiogenesis was determined by blood vessel growth under a microscope. Results are shown for co-cultures at day one and day four. The results show that at day four, both 1 ⁇ M and 10 ⁇ M melphalan fail to inhibit new blood vessel formation and feather bud development.
  • Figure 25 shows the results of a control feather bud development in vitro culture assay.
  • Six groups of E8 chicken embryonic skin were cultured in insert culture dishes and subjected to the following treatments for 48 hours: i) vehicle control (PBS); ii) 1 ⁇ M melphalan; iii) 10 ⁇ M melphalan; iv) 30 ⁇ M melphalan; v) 50 ⁇ M melphalan; and vi) 100 ⁇ M melphalan.
  • PBS vehicle control
  • ii) 1 ⁇ M melphalan iii) 10 ⁇ M melphalan
  • iv 30 ⁇ M melphalan
  • vi) 100 ⁇ M melphalan The results show that melphalan at low concentrations (1-10 ⁇ M) did not inhibit endothelial migration and proliferation in the CAM/FB model.
  • Figure 26 shows a Western blot analysis of Tie2 and FLK-1 protein expression in a CAM-FB model angiogenesis assay. Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8. E8 chicken embryonic skin was cultured in insert culture dishes for 48 hours. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator. At day four, the embryonic skins/feather buds were removed and protein samples prepared for Western blot analysis.
  • Figure 27 shows the results of an analysis for new blood vessel formation in a CAM-FB model assay using hematoxylin and eosin Y (H & E) staining and Tie2 and FLK-1 protein immunohistochemistry.
  • Fertilized chicken eggs were incubated horizontally at 37.5 0 C and windowed by day 8.
  • E8 chicken embryonic skin was cultured in insert culture dishes and subjected to the following treatments for 48 hours: i) vehicle control (PBS) and ii) 8 ⁇ M zoledronic acid. The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator.
  • PBS vehicle control
  • 8 ⁇ M zoledronic acid The cultured embryonic skins were subsequently placed on E9 chicken chorioallantoic membranes and cultured for an additional four days at 37.5 0 C in a humidified incubator.
  • CAM chorioallantoic membrane
  • CAM The chorioallantoic membrane
  • CAM provides an ideal microenvironment in which to study angiogenesis and/or vasculogenesis.
  • existing versions of the avian CAM assay suffer from several disadvantages. For example, to assess angiogenesis in a chicken CAM assay, methods in the art typically employ the use of a stereomicroscope to count the number of blood vessel branch points within the CAM directly beneath a filter disc impregnated with a test compound. Such a quantification method is very time-consuming, requires expensive equipment, and may not provide accurate results.
  • the major disadvantage of this assay is that the CAM already contains a well-developed vascular network, which makes it difficult to discriminate between new capillaries and previously existing ones.
  • the methods of the present invention provide an unambiguous measure for determining the ability of a compound to modulate angiogenesis and/or vasculogenesis in a modified CAM assay.
  • Methods of the invention are based, in part, on a finding that a modified CAM assay provides unique advantages in characterizing vascularization and in identifying compounds capable of modulating vascularization.
  • the present invention provides assay methods for determining the ability of a compound or mixture of compounds to modulate vascularization in ovo.
  • modulation refers to a positive or negative change in extent, duration, levels, or properties of a physiologic process.
  • modulation of vascularization can comprise an increase in the formation of new blood vessels or a decrease in the formation of new blood vessels.
  • vascularization refers to the formation and maintenance of blood vessels. Stimulation or enhancement of vascularization is defined as increasing blood vessel formation and resulting blood circulation beyond that which would occur naturally. Vascularization includes, but is not limited to, the processes of angiogenesis and vasculogenesis.
  • angiogenesis broadly refers to the process of developing new blood vessels. Angiogenesis involves proliferation, migration and tissue infiltration of capillary endothelial cells from pre-existing blood vessels. Angiogenesis is important in normal physiological processes, including for example, follicular growth, embryonic development and wound healing and in pathological processes such as tumor growth and metastasis.
  • vasculogenesis broadly refers to endothelial precursor cell (angioblast) migration and differentiation in response to local cues (such as growth factors and extracellular matrix) in order to form new blood vessels.
  • the in ovo assay methods of the present invention provide a simpler, more rapid, more reliable, and less expensive chorioallantoic membrane (CAM) assay, which can be used to determine the ability of one or more test compound to modulate vascularization.
  • CAM chorioallantoic membrane
  • modulatory test compounds may possess biochemical activities sufficient to render them useful as anti-angiogenic or angiogenic therapeutics.
  • the present invention provides methods for assessing the anti-angiogenic or angiogenic activity of a wide range of test compounds.
  • a candidate anti-angiogenic or angiogenic compound can be any chemical compound, for example, a macromolecule (e.g., a polypeptide, a protein complex, glycoprotein, or a nucleic acid) or a small molecule (e.g., an amino acid, a nucleotide, an organic or inorganic compound).
  • a macromolecule e.g., a polypeptide, a protein complex, glycoprotein, or a nucleic acid
  • a small molecule e.g., an amino acid, a nucleotide, an organic or inorganic compound.
  • anti-angiogenic compound or agent or “angiogenesis inhibitor” refers to a compound or agent having anti-angiogenic activity that inhibits, angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly.
  • inhibitor means to reduce, to suppress, to block, to prevent, to lessen and/or to down- regulate. Inhibition is meant to encompass both complete and partial inhibition.
  • the inhibition of angiogenesis can be relative to the angiogenesis measured in a control treatment (i.e., vehicle).
  • angiogenic compound or agent refers to a compound or agent having angiogenic activity as and stimulates the development of blood vessels, e.g., promotes angiogenesis, endothelial cell growth, stability of blood vessels, and/or vasculogenesis, etc.
  • angiogenesis induced by an angiogenic compound or agent can be relative to the angiogenesis measured in a control treatment (i.e., vehicle).
  • control compound or agent refers to a compound or agent as described herein throughout that has a known angiogenic or anti-antigenic effect, or no angiogenic effect, such as an inert compound (e.g., vehicle).
  • the methods of the present invention contemplate, in part, to modify a CAM assay by co-culturing an embryonic avian skin tissue with a CAM in order to monitor the process of vascularization.
  • an embryonic skin tissue cultured in contact with a CAM will normally result in the vascularization of the embryonic skin tissue.
  • the vascularization of the embryonic skin tissue can occur by endothelial cell proliferation and migration into the embryonic skin tissue.
  • the vascularization of the embryonic skin tissue can occur by endothelial precursor cell migration and differentiation. In other related particular embodiments, vascularization of the embryonic skin tissue can occur by any combination of endothelial cell proliferation and migration into the embryonic skin tissue and endothelial precursor cell migration and differentiation.
  • the present invention provides a method for determining the ability of a test compound to modulate vascularization, said method comprising the steps of: i) windowing an embyronated shelled egg; ii) culturing the embyronated shelled egg; iii) contacting an embryonic avian skin tissue in a culture with at least one test compound; iv) contacting the CAM of the embyronated shelled egg of step (ii), with the embryonic avian skin tissue of step (iii) and culturing the CAM and embryonic avian skin tissue composition; and v) determining the ability of the at least one test compound to modulate vascularization.
  • the present invention provides a method for determining the ability of a test compound to modulate vascularization, said method comprising the steps of: i) windowing an embyronated shelled egg; ii) culturing the embyronated shelled egg; iii) contacting an embryonic avian skin tissue in a culture with at least one test compound; iv) contacting the CAM of the embyronated shelled egg of step (ii), with the embryonic avian skin tissue of step (iii) and culturing the CAM and embryonic avian skin tissue composition; and v) determining the ability of the at least one test compound to modulate vascularization; and vi) determining the modulatory activity of the test compound by measuring an indicator of vascularization.
  • the level of the indicator of vascularization indicates the modulation of angiogenesis and/or vasculogenesis in an assay or method of the present invention.
  • determining if a test compound can modulate the vascularization of an embryonic avian skin tissue cultured in combination with a CAM can be accomplished by monitoring and/or measuring the level of an indicator of vascularization.
  • an indicator of vascularization in the assays of the present invention includes, but is not limited to, markers of endothelial cells, endothelial cell proliferation, and endothelial cell migration. Such markers also include by way of non-limiting example, particular members of the Wnt signal transduction pathway.
  • markers of proliferation or migration need not be specific to endothelial cells, but can also be used in combination with endothelial cell specific markers to assay endothelial cell proliferation and migration.
  • Exemplary endothelial cell markers suitable for use with the present invention are: 7B4 antigen, ACE, BNH9/BNF13, CD31 (PECAM-1 ), CD31 , CD34, CD54 (ICAM-1 ), CD62P (p-Selectin GMP140), CD105 (Endoglin), CD146 (P1 H12), D2-40, E-selectin, EN4, Endocan, ESM-1 , Endoglin (CD105), Endoglyx-1 , Endomucin, Endosialin (tumor endothelial marker 1 , TEM-1 , FB5), Eotaxin-3, EPAS1 (Endothelial PAS domain protein 1 ), Factor VIII related antigen, FB21 , Flk-1 (VEGFR-2), Flt-1 (VEGFR-1 ), GBP-1 (guanylate-binding protein-1 ), GRO-alpha, Hex, ICAM-2 (intercellular adhesion molecule 2), LYVE- 1
  • Exemplary markers of cell proliferation are: AgNOR, Alpha-smooth muscle actin (alpha SMA), ANT2, B23, Cdk1 , Choline, Claspin, Cyclin A, CYR61 , Fen1 , Geminin, Histone H3, HsMCM2, IL-2, IL-6, lnterleukin-2 (IL-2), Ki-67 (MIB-1), Ki-SI , Ki- S2, Ligl (5H5), Mitosin, p120, PCNA, PDPK, PLK (polo-like kinase), PTTG (Pituitary tumor transforming gene), Stathmin, STK1 , TK-1 (Thymidine Kinase 1 ), Topoisomerase Il alpha (topo Il alpha), TPS (Tissue polypeptide specific antigen), Transcobalamin II, WT1 , and the like.
  • an indicator of vascularization is selected from the group consisting of endothelial cell markers, endothelial cell proliferation markers, endothelial cell migration markers, and Wnt signal transduction pathway genes.
  • an indicator of vascularization is selected from the group consisting of Flk-1 , CD144, Tie-2, vWF, CD31 , and CD34.
  • the gene expression of endothelial cell markers, endothelial cell proliferation markers, endothelial cell migration markers, and Wnt signal transduction pathway genes can be measured by polymerase chain reaction (PCR), semi-quantitative PCR, realtime PCR, Taqman PCR, reverse transcriptase PCR, ligase-mediated PCR, primer extension, S1 nuclease assay, RNase protection assay, Northern blotting, nuclear run-on assay, nuclear run-off assay, slot blots, dot blots, in situ hybridization, and in situ hybridization with tyramide signal amplification, among others.
  • PCR polymerase chain reaction
  • semi-quantitative PCR realtime PCR
  • Taqman PCR reverse transcriptase PCR
  • ligase-mediated PCR primer extension
  • S1 nuclease assay RNase protection assay
  • Northern blotting nuclear run-on assay
  • nuclear run-off assay nuclear run-off assay
  • the protein levels of endothelial cell markers, endothelial cell proliferation markers, endothelial cell migration markers, and Wnt signal transduction pathway genes can be measured by methods known in the art, including, but not limited to immunohistochemistry, western blotting, mass spectrometry, 2D-gel electrophoresis, and chromatography, among others.
  • methods of the present invention assess vascularization using light microscopy to count the number of blood vessel branch points that infiltrate the embryonic skin tissue in co-culture with the CAM.
  • methods of the present invention assess vascularization using a fine balance to measure the mass or weight of the feather buds in the embryonic skin tissue in co-culture with the CAM.
  • the present methods determine the vascularization of an embryonic skin tissue by monitoring feather bud growth of the embryonic skin tissue in co-culture with a CAM (i.e., chorioallantoic membrane feather bud assay; CAM-FB).
  • a CAM i.e., chorioallantoic membrane feather bud assay
  • a CAM-FB assay conducted with a test compound will be compared to an equivalent CAM-FB assay conducted with vehicle or a control compound, so that the modulation of vascularization by a test compound can be compared to a reference.
  • a level of an indicator of vascularization for a test compound is compared to a level of an indicator of a control compound or vehicle.
  • the control compound can be any known anti-angiogenic, angiogenic, non- angiogenic compound or vehicle that has been previously assayed in a CAM- FB assay of the present invention or any compound known in the art to have an anti-angiogenic, angiogenic or non-angiogenic activity.
  • a decrease in the level of the indicator of vascularization of one or more test compounds compared to a level of an indicator of vascularization of a non-angiogenic control compound or vehicle indicates that the one or more test compounds have anti-angiogenic activity.
  • an indicator of vascularization for an anti-angiogenic compound indicates that a test compound reduces angiogenesis and/or vascularization and/or negatively affects the feather bud growth of an embryonic skin co-cultured with a CAM compared to normal feather bud growth in a non-treated or vehicle treated embryonic skin co-cultured with a CAM.
  • an increase in the level of the indicator of vascularization of one or more test compounds compared to a level of an indicator of vascularization of an anti-angiogenic compound indicates that the one or more test compounds have angiogenic activity.
  • an indicator of vascularization for an angiogenic compound indicates that a test compound increases angiogenesis and/or vascularization and positively affects feather bud growth of an embryonic skin co-cultured with a CAM compared to normal feather bud growth in a non- treated, vehicle treated, or anti-angiogenic compound treated embryonic skin co-cultured with a CAM.
  • the angiogenic activity of a test compound can be monitored or measured for an indicator of vascularization in the presence of an amount of known anti- angiogenic compound or the anti-antigenic activity of a test compound can be monitored or measured for an indicator of vascularization in the presence of an amount of known angiogenic compound.
  • embryonic skin tissue is cultured in the presence of an amount of a known compound with anti- angiogenic activity (e.g., zoledronic acid, fumagillin, minocycline, Celebrex®, doxrubicin), which is preferably the least amount of anti-angiogenic compound necessary to provide an anti-angiogenic effect.
  • a known compound with anti- angiogenic activity e.g., zoledronic acid, fumagillin, minocycline, Celebrex®, doxrubicin
  • the embryonic skin tissue and anti-angiogenic compound culture is then further contacted with a test compound or mixture of test compounds. If the test angiogenic compound lessens, or inhibits the anti-angiogenic effect of the known anti-angiogenic compound; the test compound can be considered angiogenic or to have angiogenic properties.
  • an embryonic skin tissue is contacted with an amount of a test anti-angiogenic compound. If the test compound lessens, or inhibits the normal angiogenesis and/or vasculogenesis (e.g., vascularization) in an embryonic skin cultured in contact with a CAM, then the test compound can be considered anti-angiogenic or to have anti- angiogenic properties.
  • a test anti-angiogenic compound e.g., vascularization
  • an embryonic skin tissue is uuiiiciuieu with an amount of a known angiogenic compound (e.g., vitamin-C), which is preferably the least amount of angiogenic compound necessary to provide an angiogenic effect.
  • a known angiogenic compound e.g., vitamin-C
  • the embryonic skin tissue and angiogenic compound culture is then further contacted with a test compound or mixture of compounds. If the test anti-angiogenic compound can lessen, or inhibit the angiogenic effect of the known angiogenic compound, then the test compound can be considered anti-angiogenic or to have anti-angiogenic properties.
  • the amount of known anti-angiogenic or angiogenic compound can be any amount, including, but not limited to a concentration that does not produce an effect; a concentration that produces a low, moderate, or strong effect; a concentration that produces a minimal effect, half-maximal, or maximal effect; or a saturating concentration, wherein no additional effect is produced beyond the concentration of compound that elicits a maximal effect.
  • the present invention provides a method for determining the anti-angiogenic activity of a test compound or combination of test compounds, said method comprising the steps of: i) obtaining, windowing, and culturing an embryonated shelled egg; ii) contacting an embryonic avian skin tissue in a culture with a test compound or combination of test compounds; iii) contacting the CAM of the cultured egg of step (i) with the cultured embryonic avian skin tissue of step (ii), and culturing the CAM and the embryonic avian skin tissue; and iv) determining the anti-angiogenic activity of the test compound or mixture of compounds.
  • the present invention provides a method for determining the anti-angiogenic activity of one or more test compounds, said method comprising the steps of: i) obtaining, windowing, and culturing an embryonated shelled egg; ii) contacting an embryonic avian skin tissue in a culture with a known angiogenic compound and one or more test compounds; iii) contacting the cultured CAM of step (i) with the cultured embryonic avian skin tissue of step (ii), and culturing the CAM and embryonic avian skin tissue composition; and iv) determining the anti-angiogenic activity of the test compound or mixture of compounds.
  • a method for determining the anti- angiogenic activity of a test compound or combination of test compounds comprises the additional step of comparing an indicator of vascularization of a test compound and an indicator of vascularization of a control anti-angiogenic or angiogenic compound or vehicle.
  • the present invention provides a method for determining the angiogenic activity of a test compound or mixture of test compounds, said method comprising the steps of: i) obtaining, windowing, and culturing an embryonated shelled egg; ii) contacting an embryonic avian skin tissue in a culture with a test compound or mixture of test compounds; iii) contacting the cultured CAM of step (i) with the cultured embryonic avian skin tissue of step (ii), and culturing the CAM and the embryonic avian skin tissue; and iv) determining the angiogenic activity of the test compound or mixture of compounds.
  • the present invention provides a method for determining the angiogenic activity of a test compound or mixture of test compounds, said method comprising the steps of: i) obtaining, windowing, and culturing an embryonated shelled egg; ii) contacting an embryonic avian skin tissue in a culture with a known anti-angiogenic compound and a test compound or mixture of test compounds; iii) contacting the cultured CAM of step (i) with the cultured embryonic avian skin tissue of step (ii), and culturing the CAM and embryonic avian skin tissue composition for a period of time sufficient to determine the angiogenic activity of the test compound or mixture of compounds; and iv) determining the angiogenic activity of the test compound or mixture of compounds.
  • a method for determining the angiogenic activity of a test compound comprises the additional step of comparing an indicator of vascularization of a test compound and an indicator of vascularization of a control anti-angiogenic or angiogenic compound or vehicle.
  • a CAM-FB assay of the present invention comprises the steps of: i) obtaining an embryonated shelled egg; windowing the embryonated shelled egg, and culturing the embryonated shelled egg at an early stage of development; ii) contacting an early embryonic avian skin tissue with at least one test compound in a culture; iii) contacting the cultured CAM of step (i) with the cultured embryonic avian skin tissue of step (ii), and culturing the CAM and the embryonic avian skin tissue for a period of time sufficient to determine the modulatory, angiogenic, or anti-angiogenic activity of the test compound; and iv) determining the modulatory, angiogenic, or anti-angiogenic activity of the test compound by measuring an indicator of vascularization.
  • methods of the present invention provide for obtaining an embryonated egg (i.e., having an embryo).
  • the egg is obtained from an avian species.
  • the avian egg is obtained from a fowl.
  • the term "fowl" means a bird belonging to one of two orders, namely the gamefowl or landfowl (Galliformes) and the waterfowl (Anseriformes), which together form the fowl clade scientifically known as Galloanserae.
  • a fowl egg used in the context of the invention can be any suitable fowl egg.
  • Suitable fowl eggs typically include those deriving from a fowl belonging to the order Galliformes.
  • the fowl egg is derived from a fowl belonging to the family Phasianidae, and, more preferably, from a fowl belonging to the genus Gallus.
  • the fowl egg is derived from the fowl Gallus gallus (i.e., the chicken).
  • an egg or embryonic skin tissue obtained from any species of fowl, including, but not limited to those of the chicken, turkey, duck, goose, quail, pheasant, grouse, ostrich, emu, cassowary, and kiwi.
  • an egg and an embryonic skin tissue suitable for use in a single assay may be from different species.
  • the CAM of a chick egg is contacted with embryonic skin tissue from quail or vice versa.
  • non-avian shelled eggs including, but not limited to, reptilian eggs, such as a turtle egg, a crocodile egg, an alligator egg or the like can be used in combination with embryonic skin tissue from any species of fowl as described herein throughout. It would further be understood by the skilled artisan that such xenogenic assays require optimization. For example, developmental timing of the CAM and embryonic skin tissue from different species should be synchronized in order to produce comparable results to a non-xenogenic assay.
  • eggs and/or skin tissue of the present invention are obtained from the avian species selected from the group consisting of: chicken, turkey, duck, goose, quail, pheasant, grouse, ostrich, emu, cassowary, and kiwi.
  • the avian species of eggs and/or skin tissue are selected from the group consisting of: chicken, turkey, duck, goose, and quail.
  • the avian species of eggs and/or skin tissue are selected from the group consisting of: chicken and quail.
  • the egg and skin tissue are from different avian species.
  • the egg and skin tissue are from the same avian species.
  • Methods of preparing an embryonated avian egg for use in the present invention are well-known in the art and are described for species of fowl in, for example, Brooks et al. Cell, 79, 1157- 1164 (1994) and Brooks et al. Methods in Molecular Biology, 129, 257-269 (1999).
  • the age of the fertilized avian egg e.g., an embyronated fowl egg used in the present inventive methods described herein throughout may be of any gestational age.
  • An egg of the present invention may be obtained at any time following fertilization and then cultured until any particular gestational age suitable for use in the methods of the present invention.
  • fertilized avian eggs are obtained from about embryonic day 6- 12 (E6-E12), about E7-E10, or about E8-E9 of gestation.
  • an embryonic avian egg suitable for use in the methods of the present invention is obtained at about E7, about E8, about E9, about E10, about E11 , or about E12.
  • the terminology to describe the embryonic age of a fertilized or embryonated egg begins at time 0, which is the time of fertilization.
  • time of fertilization can not always be precisely measured.
  • an embryonated egg that is about embryonic day 7 is typically the developmental age of an E6.5-E7.5 day old embryo.
  • an embryonated egg that has an age of E7 means the stage of embryonic development is likely to be between 6.5-7.5 days of gestation.
  • the term “about” or “approximately” refers to a time period, quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference time period, quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1 %.
  • the phrase "about embryonic day 8" means from embryonic day 7.5 to embryonic day 8.5 of development. In other embodiments, about embryonic day 7, 8, 9, 10, 11 , or 12 would be similarly understood to refer to the embryonic day stated plus or minus a half day.
  • the fertilized eggs are collected at an early developmental time period (e.g., E7-E9), windowed, and subsequently cultured in a humidified incubator (e.g., 70% relative humidity) at a temperature of about 37.5 0 C and 5% CO 2 .
  • a windowed fertilized or embryonated shelled egg of the present invention is cultured for about 0 to about 96 hours, about 0 to about 72 hours, about 0 to about 48 hours, about 12 to about 36 hours, about 18 to about 30 hours, or about 24 hours.
  • the fertilized or embryonated shelled egg is subsequently cultured for about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25 , about 26, about 27, about 28, about 29, or about 30 hours.
  • incubation conditions may be optimized for particular models of incubators or methods of incubation.
  • eggs may be incubated at about 0%-100%, about 50%-90%, about 60%-80%, about 65% to 75% or any percent value of relative humidity in between.
  • eggs of the present invention can be incubated at about 36.5 0 C, 37.O 0 C, 37.5 0 C, 38.O 0 C, 38.5 0 C, 39.O 0 C, 39.5 0 C, or 40.0 0 C or any temperature value in between.
  • the temperature may need to be varied according to the humidity used in the experiment.
  • the growth conditions for proper embryonic development of a particular avian species are not necessarily the same growth conditions used for a different species.
  • one of skill in the art may optimize the incubation conditions for maximum survivability of the eggs of the present invention. Such methods are routinely performed in the art.
  • the term "windowing” refers to cutting, grinding or otherwise removing an area of an egg so as to allow access to the contents of the egg.
  • the egg Prior to contacting the CAM of the egg with an embryonic skin tissue, the egg is windowed. Windowing can be performed at any point during the culture of an embyronated egg prior to contacting the CAM of the egg with an embryonic skin tissue.
  • the embryonated egg is obtained, cultured to a particular developmental age, windowed, and then cultured to another particular developmental age.
  • the embryonated egg is obtained, windowed, and then cultured to a particular developmental age.
  • the embryonated egg is obtained, cultured to a particular developmental age, and then windowed.
  • a skilled artisan can obtain E6-E12 fertilized eggs and candle them using a hand held egg candler at the blunt end of the egg to identify the air sac and prominent blood vessels.
  • the CAM can be separated from the shell by making a shallow burr hole at the blunt end on the egg and another burr hole made perpendicular to the previously identified blood vessels in the center of the egg. Mild suction is applied to the blunt end burr hole to displace the air sac and drop the CAM away from the shell. Fine forceps are then used to pick away the shell over the false air sac, so that a window can be made and the CAM identified and be made accessible.
  • the skilled artisan is aware of many such techniques as they are routine in the art. Also included within the general term "windowing" are methods by which the window is sealed to allow further development of the embryo.
  • Sealing can be accomplished using tape or any heat melted or heat softened composition.
  • heat melted composition or “heat softened composition” refer to any composition of natural or artificial origin that is solid or gel-like at one temperature and liquefies on the application of a heat source. When the heat source is removed, the liquefied composition will cool and revert to the original solid or gel form.
  • Heat softened compositions within the scope of the present invention include, but are not limited to, hot melt glues, gelatin gels, gelatin-glycerol compositions, and waxes.
  • Waxes may include, but are not limited to: paraffin waxes comprising mixtures of high molecular weight solid hydrocarbons; natural waxes such as, but not limited to beeswax, lanolin, shellac wax; vegetable waxes including carnauba, candelilla; and synthetic waxes including ethylenic polymers, polyol ether-esters, and chlorinated napthalenes.
  • the heat softened compositions of the present invention are non-toxic and therefore physiologically inert to a developing embryo. It is further preferred that the melting point of the heat softened or heat melted compositions be below a temperature that would harm the viability of the embryo or the contents of the egg.
  • a CAM-FB assay of the present invention comprises the steps of: i) obtaining an embryonated shelled egg (e.g., an avian egg) at embryonic day E6-E12 of gestation; creating a window in the shell of the egg, thereby exposing the CAM; and culturing the windowed egg; ii) contacting an early embryonic avian skin tissue in a culture with at least one test compound; iii) contacting the cultured CAM of step (i) with the cultured embryonic avian skin tissue of step (ii), and culturing the CAM and the embryonic avian skin tissue for a period of time sufficient to determine the modulatory, angiogenic, or anti-angiogenic activity of the test compound; and iv) determining the modulatory, angiogenic, or anti-angiogenic activity of the test compound by measuring an indicator of vascularization.
  • an embryonated shelled egg e.g., an avian egg
  • the embryonated egg is obtained at a developmental time of about: E7-E10.
  • the embryonated egg is obtained at a developmental time selected from the group consisting of: E6, E7, E8, E9, E10, E11 , and E12.
  • the embryonated egg is obtained at a developmental time selected from the group consisting of: E7, E8, E9, and E10.
  • the embryonated egg is obtained at a developmental time selected from the group consisting of: E7, E8, and E9.
  • the embryonated egg is obtained at a developmental time of E8 of gestation.
  • a CAM-FB assay of the present invention comprises the steps of: i) obtaining an embryonated shelled egg (e.g., an avian egg) at an early gestational age; creating a window in the shell of the egg, thereby exposing the CAM; and culturing the windowed egg; ii) contacting an E6-E9 embryonic avian skin tissue with at least one test compound in a culture; iii) contacting the cultured CAM of step (i) with the cultured embryonic avian skin tissue of step (ii), and culturing the CAM and the embryonic avian skin tissue for a period of time sufficient to determine the modulatory, angiogenic, or anti-angiogenic activity of the test compound; and iv) determining the modulatory, angiogenic, or anti-angiogenic activity of the test compound by measuring an indicator of vascularization.
  • an embryonated shelled egg e.g., an avian egg
  • One having ordinary skill in the art is familiar with the standard tissue culture protocols required to accomplish early embryonic avian skin tissue explant cultures.
  • the skilled artisan obtains eggs of the appropriate developmental age, as described herein throughout and removes the embyro from the egg and washes the embryo in Hanks Buffered Salt Solution or any other suitable physiological buffer such as Ringer's Physiological Saline Solution, .9M NaCI, or Phosphate Buffered Saline pH 7.4 (PBS).
  • the embryo is then dissected in the physiological buffer with fine point forceps or a tungsten needle in combination with a scalpel to remove dorsal skin from E5-E9 age embryos.
  • the dissected embryonic skin tissue can then be placed in a tissue culture insert (Falcon), which is subsequently transferred into a 60mm tissue culture dish.
  • the embryonic avian skin tissue explant is then cultured at about 30-42 0 C or any temperature in between, for example, about 36 0 C, about 36.5 0 C, about 37 0 C, about 37.5 0 C, about 38 0 C or about 38.5 0 C in about 5% CO 2 /95% air.
  • Suitable growth medium for culture of embryonic skin tissue explants include, but are not limited to, such media as Dulbecco Modified Eagle's Medium (DMEM), McCoys 5A medium (Gibco), Eagle's basal medium, CMRL medium, Glasgow minimum essential medium, Ham's F-12 medium, Iscove's modified Dulbecco's medium, Liebovitz' L-15 medium, and RPMI 1640, among others.
  • DMEM Dulbecco Modified Eagle's Medium
  • McCoys 5A medium Gibco
  • Eagle's basal medium fetal medium
  • CMRL medium Glasgow minimum essential medium
  • Ham's F-12 medium Ham's F-12 medium
  • Iscove's modified Dulbecco's medium Liebovitz' L-15 medium
  • RPMI 1640 RPMI 1640
  • the culture medium may be supplemented with 0%-20% serum, including, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% serum.
  • Suitable types of serum for use in the present invention are fetal bovine serum (FBS), fetal calf serum (FCS), equine serum (ES), and human serum (HS).
  • Culture media of the present invention may further comprise one or more antibiotics and/or antimycotics to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination, among others. Additionally, the culture medium may be changed about every 12, 24, 36, or 48 hours or any interval of time in between or not changed.
  • antibiotics and/or antimycotics to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination, among others.
  • the culture medium may be changed about every 12, 24, 36, or 48 hours or any interval of time in between or not changed.
  • embryonic avian skin tissue is isolated from an E6-E9 embryo.
  • embryonic skin from different avian species may be used at different gestational ages of the embryo depending on the species of avian used, the indicator of vascularization being measured, and/or the particular goals of the experiment.
  • particular embodiments of the present invention isolate embryonic chicken skin from embryonic day 7.5-8.5 chicken embryos, the developmental age at which the skin tissue from chick embryos is ready for feather bud development.
  • the embryonic skin tissue is cultured from 0-3 days in culture.
  • embryonic avian skin tissue dissected from an E6 chick embryo may be cultured for up to about 3 days in culture
  • embryonic avian skin tissue dissected from an E7 chick embryo may be cultured for up to about 2 days in culture
  • embryonic avian skin tissue dissected from an E8 chick embryo may be cultured for up to about 1 day in culture
  • embryonic avian skin tissue dissected from an E9 chick embryo may be placed in contact with a cultured CAM of the appropriate age without the need for further culturing.
  • a CAM-FB assay of the present invention comprises the steps of: i) obtaining a fertilized avian egg at an early stage of development; creating a window in the shell of the egg, thereby exposing the CAM; and culturing the windowed egg; ii) contacting an E6-E9 embryonic avian skin tissue with at least one test compound and at least one known anti- angiogenic compound or at least one angiogenic compound in a culture; iii) contacting the cultured CAM of step (i) with the cultured embryonic avian skin tissue of step (ii), and culturing the CAM and the embryonic avian skin tissue for a period of time sufficient to determine the modulatory, angiogenic, or anti- angiogenic activity of the test compound; and iv) determining the modulatory, angiogenic, or anti-angiogenic activity of the test compound by comparing the level of an indicator of vascularization of at least one test compound to the level of an indicator
  • the embryonic avian skin tissue used in the methods and assays of the present invention is at a developmental age of E6-E9.
  • the embryonic avian skin tissue used is at a developmental age selected from the group consisting of E6, E7, E8, and E9.
  • the embryonic avian skin tissue used is at a developmental age selected from the group consisting of E7, E8, and E9.
  • the embryonic avian skin tissue used is at a developmental age of E8 of gestation.
  • the present invention provides for methods and assays that use embryonic avian skin dissected from an early stage embryo.
  • the embryonic avian skin tissue is isolated (e.g., by dissection) from an embryo at an age in a range of about E6-E9, about E6-E8, about E6- E7, or about E7-E8 of gestation.
  • the embryonic avian skin tissue is dissected from an embryo of about E6, about E7, about E8, or about E9 days of gestation.
  • an embryonic avian skin tissue is cultured in combination with at least one compound or a mixture of compounds for a sufficient period of time, prior to contacting the cultured embryonic avian skin tissue with a CAM.
  • the compounds can be any number of test compounds, compounds with known angiogenic, anti-angiogenic or non- angiogenic activity or any number of control compound(s) or vehicle, or any combination thereof.
  • an embryonic avian skin tissue is contacted with at least one compound or a mixture of compounds and cultured for a period of about 0-72 hours, about 6-48 hours, about 12-36 hours, about 18-30 hours, about 22-26 hours.
  • an embryonic avian skin tissue that is contacted with at least one compound or a mixture of compounds can be cultured for a period of about 1 hour, about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 48 hours, about 72 hours, or any interval of time in between.
  • an embryonic avian skin tissue that is contacted with at least one compound or a mixture of compounds can be cultured for a period of about 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, or 36 hours.
  • the at least one compound or mixture of compounds that contacts an embryonic skin tissue in a culture of the present invention can be any number of angiogenic, anti- angiogenic, or unknown compounds in any combination and in any concentration.
  • the embyronic avian skin tissue is cultured for a period of time before at least one compound (e.g., a test compound) or combination of compounds is added to the culture.
  • an embryonic avian skin tissue can be cultured for a period of about 0 hours, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours or any number of hours in between before the addition of at least one compound (e.g., a test compound) or mixture of compounds to the embryonic avian skin tissue explant culture.
  • an embryonic avian skin tissue can be cultured for a period of about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours before the addition of at least one compound or mixture of compounds to the embryonic avian skin tissue explant culture.
  • an embryonic avian skin tissue explant is contacted with a combination of compounds, each compound of said mixture having known or unknown anti-angiogenic, angiogenic, or non-angiogenic properties.
  • a combination of compounds can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more compounds, any number of which are test compounds.
  • embryonic avian skin tissue explants are contacted with a particular concentration of at least one test compound, selected from a plurality of test compound concentrations.
  • the degree of vascularization of the embryonic avian skin tissue explants directly and/or linearly correlates to the concentration of test compound used to contact the embryonic avian skin tissue in culture. It would be readily understood to those of skill in the art that once a dose response curve is known for a particular test compound, that test compound is capable of being utilized as a control angiogenic or anti-angiogenic compound. The skilled artisan would also recognize that the control angiogenic or anti-angiogenic compounds and test compounds should not be the same compound at the same concentration in any given assay.
  • a test compound may elicit a dose-dependent response in modulating vascularization of an embryonic avian skin tissue in co-culture with a CAM.
  • a greater concentration of an anti-angiogenic compound would prevent vascularization to a greater extent than a relatively smaller concentration of said compound; thus, the indicator of vascularization would be less in the case of the greater concentration of said compound relative to the lesser concentration of said compound.
  • the concentration of a known compound is tested with different concentrations of the test compound in order to ascertain the activity of the test compound.
  • the embryonic skin tissue is contacted with a particular concentration of a known anti-angiogenic compound and different concentrations of a test compound.
  • a known anti-angiogenic compound concentrations of a test compound.
  • the present invention is suitable to establish the relative angiogenic activities among different angiogenic compounds, which may further aid in the selection of an appropriate angiogenic compound in a particular therapeutic setting.
  • test compounds can be assayed at any concentration.
  • test compounds assayed at several concentrations within the range of about 1 pM to about 1 M is commonly useful to determine the ability of a compound to modulate vascularization. It will be possible or even desirable to conduct certain of these assays at concentrations of about 1 nM to about 1 mM, or of about 0.5 nM to about 0.5 mM.
  • the range of concentrations to be tested consists of a plurality of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different concentrations within a range of about 1 pM to about 1 M, or alternatively, within a concentration range of about 1 nM to about 1 mM.
  • the range of test compound concentrations to be tested is about 10 nM to about 100 ⁇ M, about 100 nM to about 10 ⁇ M, and about 250 nM to about 1 ⁇ M or any intervening ranges of values.
  • the plurality of different test compounds or combinations of compounds or substances are 2-fold serial dilutions, 3-fold serial dilutions, 4-fold serial dilutions, 5-fold serial dilutions, 6- fold serial dilutions, 7-fold serial dilutions, 8-fold serial dilutions, 9-fold serial dilutions, or 10-fold serial dilutions in a range of concentrations of about 1 pM to about 1 M, or alternatively, within a concentration range of about 1 nM to about 1 mM.
  • a series of ten, 10-fold serial dilutions of a test compound starting at a concentration of about 1 M would include test compound concentrations of about 100 mM, about 10 mM, about 1 mM, about 100 ⁇ M, about 10 ⁇ M, about 1 ⁇ M, about 100 nM, about 10 nM, about 1 nM, and about 10O pM.
  • concentrations assayed in an in ovo CAM-FB assay of the present invention may vary from one test compound to another.
  • concentrations assayed for a mixture of compounds varies depending on the compounds in the mixture, and thus, mixtures of compounds may contain lower, the same as, or greater concentrations of compounds than would be present in an individual CAM-FB assay of a compound in the mixture.
  • test compound that is determined to modulate, inhibit, down-regulate, reduce, potentiate, promote or increase angiogenesis according to the present invention, should do so by failing to affect normal development of the cultured embryonic avian skin tissue in the absence of a CAM co-culture.
  • Techniques for assessing these compound "off-target" effects are well known in the art, and can be conducted according to art known methods or those described in the Examples section herein.
  • the embryonic avian skin tissue is contacted with a compound in order to identify the anti-angiogenic activity of said compound.
  • compound used interchangeably herein, with the interchangeable terms “test compound,” and “test agent” referring to compounds used in the CAM assays as described herein throughout to identify those having a desired activity in modulating, inhibiting, down-regulating, reducing, potentiating, promoting or increasing angiogenesis according to the present invention.
  • Compound encompasses numerous biological and chemical classes, including synthetic, semi-synthetic, or naturally-occurring inorganic or organic molecules, including synthetic, recombinant or naturally-occurring polypeptides (e.g., peptidomimetics, peptoids, antibodies, and the like) and nucleic acids (e.g., nucleic acids encoding a gene product, antisense RNA, siRNA, and the like).
  • Test compounds or “test agents” include those found in large libraries of synthetic or natural compounds.
  • assays of the present invention are suitable for determining the modulatory, angiogenic, or anti-angiogenic effects of a compound or mixture of compounds, wherein the mixture of compounds is any combination of unknown or known anti-angiogenic and/or angiogenic compounds in any concentration.
  • assays and methods of the present invention involve testing a compound from a combinatorial small organic molecule or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds).
  • potential therapeutic compounds potential modulator or ligand compounds
  • Such "combinatorial chemical libraries” or “ligand libraries” can be screened separately or screened in pools, to identify those library members particular chemical species or subclasses) that display a desired characteristic activity.
  • screening libraries with pools of compounds may reduce the ultimate number of screens for any given library. For example, pools containing the activity of interest can be iterively subdivided until the activity is restricted to a particular compound or mixture of compounds.
  • the identified compounds can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No.
  • chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091 ), benzodiazepines (e.g., U.S. Pat. No.
  • test compounds of the present invention include small organic or inorganic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Test compounds may comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups.
  • the agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Agents, particularly candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • the compound can be purified or can be contained in a complex substance.
  • a complex substance is comprised of a plurality of components and/or compounds.
  • a complex substance can, for example, be an animal's body fluid. Suitable animal body fluids include, for example, blood, plasma, serum, bone marrow, urine, cerebrospinal fluid, saliva, synovial fluid, ocular fluid, amniotic fluid, bile, seminal fluid, or secretions. Suitable secretions include pancreatic secretions, gastric secretions, nasal secretions, pulmonary secretions, vaginal secretions, and perspiration. Accordingly, the substances identified herein are in no way limiting. The animal providing the compound can be a human patient. Furthermore, there is no need in the context of the invention to identify the nature or any characteristics of the compound. Accordingly, the invention encompasses embodiments in which the nature and characteristics of the test compound is unknown.
  • test compounds of the present invention include, but are not limited to, mixtures of compounds including both known and unknown compounds.
  • Unknown test compounds in said mixture of compounds can be any of the foregoing classes of compounds or substances.
  • an assay of the present invention determines the anti-angiogenic activity of a compound or mixture of compounds; known compounds can include, for example, an antibody or other antagonist to an angiogenic agent as described herein throughout, e.g., antibodies to VEGF, antibodies to VEGF receptors, small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, SUTENT®/SU 11248 (sunitinib malate), AMG706, or those described in, e.g., international patent application WO 2004/113304).
  • Anti-angiogenesis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti- angiogenic therapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003) (e.g., Table 2 listing antiangiogenic factors); and, Sato Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 lists Anti-angiogenic agents used in clinical trials).
  • native angiogenesis inhibitors e.g., angiostatin, endostatin, etc. See, e.g., Kla
  • anti-angiogenic factors include antibiotics, NSAIDs, fumagillin, minocycline, Celebrex®, zoledronic acid, doxorubicin, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res.
  • SP-PG Sulphated Polysaccharide Peptidoglycan Complex
  • the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha.alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4- pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChlMP-3 (Pavloff et ai, J.
  • an assay of the present invention determines the angiogenic activity of a compound or mixture of compounds
  • known test compounds which can be tested alone or in combination with other known or unknown test compounds, can include, but are not limited to, e.g., VEGF and members of the VEGF family, PIGF, PDGF family, fibroblast growth factor family (FGFs), TIE ligands (Angiopoietins), ephrins, ANGPTL3, DLL4, etc.
  • IGF-I insulin-like growth factor-l
  • VIGF insulin-like growth factor
  • EGF epidermal growth factor
  • CTGF CTGF and members of its family
  • TGF- ⁇ and TGF- ⁇ TGF- ⁇ .
  • the compound or mixture of compounds to be tested can be an unknown, known, or suspected anti- angiogenic and or angiogenic compound or a mixture of any of the foregoing types of compounds in any combination and concentration.
  • a method of determining the ability of a test compound to modulate vascularization comprises the steps of: i) windowing an embyronated shelled egg; ii) culturing the embyronated shelled egg; iii) contacting an embryonic avian skin tissue in a culture with at least one test compound; iv) contacting the CAM of the embyronated shelled egg of step (b), with the embryonic avian skin tissue of step (c) and culturing the CAM and embryonic avian skin tissue composition; and iv) determining the ability of the at least one test compound to modulate vascularization by measuring a level of an indicator of vascularization of at the least one test compound.
  • determining the ability of the at least one test compound to modulate vascularization is accomplished by measuring a level of an indicator of vascularization of at the least one test compound and comparing said level to a level of an indicator of vascularization of a control compound (e.g., known compound or vehicle).
  • a control compound e.g., known compound or vehicle
  • an indicator of vascularization may be measured at any time after the onset of contacting a CAM with an embryonic avian skin tissue.
  • the CAM and embryonic avian skin tissue are co-cultured for a period of about 0 days to about 7 days, about 1 day to about 5 days, about 1 day to about 4 days, about 2 days to about 4 days, about 3 days to about 5 days, or about 3 days to about 4 days, or about any number of days in between.
  • the CAM and embryonic avian skin tissue are co-cultured for a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.
  • the CAM and embryonic avian skin tissue are co-cultured for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In certain particular embodiments, the CAM and embryonic avian skin tissue are co-cultured for a period of about 1 or more days, about 2 or more days, about 3 or more days, about 4 or more days, about 5 or more days, about 6 or more days, or about 7 or more days.
  • an indicator of vascularization and be monitored or measured at any time during the CAM and embryonic avian skin tissue co-culture, and/or at multiple times during the CAM and embryonic avian skin tissue co-culture.
  • Indicators of vascularization in methods and assays of the present invention include, but are not limited to, new blood vessels in the embryonic avian skin tissue co-cultured with a CAM, increases in feather bud growth and/or the vascularization of feather buds, feather bud weight, and an increased expression of endothelial genes and/or Wnt pathway genes.
  • One having ordinary skill in the art is familiar with the routine practices used to monitor and/or measure indicators of vascularization of the present invention.
  • the skilled artisan can monitor and/or measure new blood vessels in the embryonic avian skin tissue co-cultured with a CAM, increases in feather bud growth and/or the vascularization of feather buds, using a stereomicroscope.
  • increases in feather bud growth in an avian embryonic skin tissue co-cultured with a CAM can be monitored by the naked human eye.
  • increased feather bud growth can be qualitatively assessed by the presence or absence of feather bud growth or by the relative amount of feather bud growth compared to a control assay, wherein the amount of feather bud growth to a particular compound is already known and/or has been quantified in an assay of the present invention. It is well- established in the art that changes in the gene expression of particular endothelial genes, and/or genes involved in the Wnt signaling pathway are regulated during the process of vascularization.
  • the present invention quantifies the expression of a gene that indicates vascularization such as Flk-1 , CD144, Tie-2, vWF, CD31 , and CD34, and the like.
  • a gene that indicates vascularization such as Flk-1 , CD144, Tie-2, vWF, CD31 , and CD34, and the like.
  • PCR polymerase chain reaction
  • semi-quantitative PCR real-time PCR
  • Taqman PCR reverse transcriptase PCR
  • ligase-mediated PCR primer extension
  • S1 nuclease assay RNase protection assay
  • Northern blotting nuclear run-on assay
  • nuclear run-off assay slot blots
  • dot blots dot blots
  • in situ hybridization and in situ hybridization with tyramide signal amplification
  • immunohistochemistry western blotting
  • mass spectrometry 2D-gel electrophoresis
  • 2D-gel electrophoresis 2D-gel
  • an indicator of vascularization in an embryonic avian skin tissue co-cultured with a CAM is selected from the group consisting of: new blood vessel growth, feather bud growth, feather bud vascularization, feather bud weight, and endothelial and/or Wnt pathway gene expression.
  • the indicator of vascularization is feather bud growth or feather bud weight,.
  • the present invention provides for assays and methods that determine the ability of a compound to modulate vascularization by monitoring an indicator of vascularization in an embryonic avian skin tissue co-cultured with a CAM.
  • an indicator of vascularization may be monitored or measured at any period during the co-culture of embryonic avian skin tissue with a CAM.
  • the indicator of vascularization may be measured at about 12-144 hours, about 12-120 hours, about 24-96 hours, 48- 96 hours, or about 72-96 hours or any number of hours in between after the onset of the co-culture of embryonic avian skin tissue with a CAM.
  • the indicator of vascularization may be measured at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 96 hours, about 120 hours, about 144 hours or any number of intervening hours.
  • the indicator of vascularization is monitored and/or measured at multiple times at regular intervals, including, but not limited to every hour, every 6 hours, every 12 hours, or every 24 hours.
  • an indicator of vascularization is measured continuously.
  • the indicator of vascularization is measured at about 24 hours and 96 hours after the onset of embryonic avian skin tissue and CAM co-culture.
  • the present invention provides an assay to determine a test compound's ability to modulate vascularization.
  • the modulatory activity of the test compound is determined by comparing an indicator of vascularization for the test compound to an indicator of vascularization for a control compound (e.g., treatment with a known angiogenic, anti-antigenic compound or vehicle).
  • a test compound that is determined to be a modulator of vascularization has a measure of the indicator of vascularization that is greater than, equal to, or less than the measure of the indicator of vascularization of a control compound.
  • the present invention provides an assay to determine the anti-angiogenic activity of a test compound, optionally in the presence of a known angiogenic compound.
  • the anti-angiogenic activity of the test compound is determined by comparing an indicator of vascularization for the test compound to an indicator of vascularization for a control compound (e.g., treatment with a known angiogenic, anti-antigenic compound or vehicle).
  • a test compound that is determined to be an anti-angiogenic compound has a level of the indicator of vascularization that is less than or equal to the level of the indicator of vascularization of a control compound.
  • an anti- angiogenic compound results in less, reduced, or no feather bud growth or feather bud weight compared to the feather bud growth or feather bud weight measured in an assay that uses a control compound.
  • the feather bud growth or feather bud weight measured in an assay using at least one anti-angiogenic compound can be about 0% less, 1% less, 5% less, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, 100% less, or any percentage of feather bud growth or feather bud weight less than feather bud growth or feather bud weight in a control experiment.
  • the feather bud growth or feather bud weight measured in an assay using at least one anti-angiogenic compound can be about 1-fold less, about 1.5-fold less, about 2-fold less, about 2.5-fold less, about 3-fold less, about 3.5-fold less, about 4-fold less, about 4.5-fold less, about 5-fold less, about 7.5-fold less, about 10-fold less, or any fold of feather bud growth or feather bud weight less than feather bud growth or feather bud weight in a control experiment.
  • the present invention provides an assay to determine the angiogenic activity of a test compound, optionally in the presence of a known anti-angiogenic compound.
  • the angiogenic activity of the test compound is determined by comparing an indicator of vascularization for the test compound to an indicator of vascularization for a control compound (e.g., treatment with vehicle).
  • a test compound that is determined to be an angiogenic compound has a measure of the indicator of vascularization that is greater than or equal to the measure of the indicator of vascularization of a control compound.
  • an angiogenic compound results in more, or a greater amount of feather bud growth or feather bud weight compared to the feather bud growth or feather bud weight measured in an assay that uses a control compound.
  • the feather bud growth or feather bud weight measured in an assay using at least one anti-angiogenic compound can be about 0% more, 1 % more, 5% morel 0% more, 20% more, 30% more, 40% more, 50% more, 60% more, 70% more, 80% more, 90% more, 100% more, or any percentage of feather bud growth or feather bud weight more than feather bud growth or feather bud weight in a control experiment.
  • the feather bud growth or feather bud weight measured in an assay using at least one angiogenic compound can be about 1-fold more, about 1.5-fold more, about 2-fold more, about 2.5-fold more, about 3-fold more, about 3.5-fold more, about 4-fold more, about 4.5-fold more, about 5-fold more, about 7.5-fold more, about 10-fold more, or any fold of feather bud growth or feather bud weight more than feather bud growth or feather bud weight in a control experiment.
  • the present invention provides an assay to determine the angiogenic activity of a test compound.
  • the angiogenic activity of the test compound is determined by contacting the embryonic avian skin tissue in combination with a known anti-angiogenic compound.
  • an angiogenic compound will promote or increase feather bud growth or feather bud weight in an embryonic avian skin tissue cultured in the presence of the angiogenic compound and the anti-angiogenic compound.
  • a test compound that is determined to be an angiogenic compound has a measure of the indicator of vascularization that is greater than or equal to the measure of the indicator of vascularization of the anti-angiogenic compound performed in a separate assay as a control.
  • an angiogenic compound results in more, or an increased amount of feather bud growth or feather bud weight compared to the feather bud growth or feather bud weight measured in an assay that uses an anti-angiogenic compound performed in a separate assay as a control.
  • the feather bud growth or feather bud weight measured in an assay using at least one angiogenic test compound can be about 0% more, 10% more, 20% more, 30% more, 40% more, 50% more, 60% more, 70% more, 80% more, 90% more, 100% more, or any percentage of feather bud growth or feather bud weight more than feather bud growth or feather bud weight in a control that uses an anti-angiogenic compound performed in a separate assay as a control.
  • the feather bud growth or feather bud weight measured in an assay using at least one angiogenic test compound can be about 1-fold more, about 1.5-fold more, about 2-fold more, about 2.5-fold more, about 3-fold more, about 3.5-fold more, about 4-fold more, about 4.5-fold more, about 5-fold more, about 7.5-fold more, about 10-fold more, or any fold of feather bud growth or feather bud weight more than feather bud growth or feather bud weight in a control that uses an anti-angiogenic compound performed in a separate assay as a control.
  • One having ordinary skill in the art would understand that methods are known in the art to qualitatively and/or quantitatively measure the level of the indicator of vascularization.
  • the methods and assays of the present invention provide for the identification of a test compound or mixture of compounds suitable in the treatment and/or prevention of a condition or disorder resulting from pathological angiogenesis, or a lack thereof.
  • Conditions and disorders amenable to treatment include, but are not limited to, cancer; atherosclerosis; proliferative retinopathies such as diabetic retinopathy, age-related maculopathy, retrolental fibroplasia; excessive fibrovascular proliferation as seen with chronic arthritis; psoriasis; and vascular malformations such as hemangiomas; or a disorder that is associated with or that results from pathological angiogenesis, or that is facilitated by neovascularization (e.g., a tumor that is dependent upon neovascularization), is amenable to treatment with an anti-angiogenic or angiogenic compound or mixture of compounds identified with the methods and assays of the present invention.
  • the instant methods also provide for the identification of compounds useful in the treatment of both primary and metastatic solid tumors, including carcinomas, sarcomas, leukemias, and lymphomas. Of particular interest is the treatment of tumors occurring at a site of angiogenesis.
  • the anti-angiogenic compounds identified herein are useful in the treatment of any neoplasm, including, but not limited to, carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract, (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma) and tumors of the brain, nerves, eyes,
  • the anti-angiogenic compounds identified by the methods and assays of the present invention are also useful for treating solid tumors arising from hematopoietic malignancies such as leukemias (i.e. chloromas, plasmacytomas and the plaques and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia) as well as in the treatment of lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).
  • leukemias i.e. chloromas, plasmacytomas and the plaques and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia
  • lymphomas both Hodgkin's and non-Hodgkin's lymphomas
  • the anti- angiogenic compounds identified herein are useful for reducing metastases from the tumors described above either when used alone or in combination with radiotherapy and/or other chemotherapeutic agents.
  • autoimmune diseases such as rheumatoid, immune and degenerative arthritis
  • various ocular diseases such as diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization due to macular degeneration, hypoxia, angiogenesis in the eye associated with infection or surgical intervention, and other abnormal neovascularization conditions of the eye
  • skin diseases such as psoriasis
  • blood vessel diseases such as hemangiomas, and capillary proliferation within atherosclerotic plaques
  • Osier-Webber Syndrome plaque neovascularization
  • telangiectasia hemophiliac joints
  • excessive wound granulation keloids
  • an anti-angiogenic or angiogenic compound identified by the methods of the present invention can be administered as the sole active ingredient for preventing or treating pathological angiogenesis or the lack of angiogenesis
  • more than one anti-angiogenic or angiogenic compound disclosed herein can be administered either in combination, concurrently or sequentially.
  • one or more of the anti-angiogenic compounds disclosed herein can be administered with other agents such as other anti-angiogenic compounds that are effective for preventing or treating the disease.
  • one or more anti-angiogenic compounds disclosed herein can be administered with another anti-cancer drug such as a chemotherapeutic agent (e.g., alkylating agents, anti-metabolites, natural products and their derivatives, hormones and steroids, and synthetics).
  • chemotherapeutic agent e.g., alkylating agents, anti-metabolites, natural products and their derivatives, hormones and steroids, and synthetics.
  • chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (
  • calicheamicin especially calicheamicin gammai l and calicheamicin omegali
  • dynemicin including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCI N®, morpholino-doxorubicin,
  • Patent No. 6,344,321 which is herein incorporated by reference in its entirety; anti HGF monoclonal antibodies (e.g., AV299 from Aveo, AMG102, from Amgen); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTI N® vaccine, LEUVECTI N® vaccine, and VAXI D® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); COX-2 inhibitors such as celecoxib (CELEBREX®; 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1 -yl) benzenesulfonamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • compositions and methods of the present invention are described, for example, in the "Physicians Desk Reference, 62 nd edition. Oradell, NJ: Medical Economics Co., 2008 ", Goodman & Gilman's "The Pharmacological Basis of Therapeutics, Eleventh Edition. McGraw-Hill, 2005", “Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.”, and "The Merck Index, Fourteenth Edition. Whitehouse Station, NJ: Merck Research Laboratories, 2006", incorporated herein by reference in relevant parts. H. Pharmaceutical compositions
  • a "pharmaceutical composition” means therapeutically or prophylactically effective amount of an anti-angiogenic or angiogenic compound together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers can be found in Remington: The Science and Practice of Pharmacy, supra.
  • a pharmaceutical composition according to the present invention can be administered orally, parenterally (e.g., subcutaneously, intradermal ⁇ , intramuscularly, intraperitoneally, and intravenously), topically, or transdermally.
  • a pharmaceutical composition is administered orally, topically, or intravenously.
  • the pharmaceutical composition may be administered to a tumor or hyperplastic tissue directly (e.g., intratumorally) or to a region in proximity to a tumor of hyperplastic tissue (e.g., paracancerally).
  • compositions may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, oxidation state, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as tert-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents [such as ethylenediamine tetra-acetic acid (EDTA)]; complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins
  • compositions will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See, for example, Remington: The Science and Practice of Pharmacy, supra. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the anti-angiogenic or angiogenic compounds identified herein.
  • the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier may be water or physiological saline solution, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof.
  • the pharmaceutical compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington: The Science and Practice of Pharmacy, supra) in the form of a lyophilized cake or an aqueous solution. Further, the pharmaceutical composition may be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • the anti-androgenic compounds can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the anti-androgenic compounds. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • compositions for oral administration can also be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by a patient.
  • compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
  • auxiliaries can be added, if desired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used orally also include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • Another pharmaceutical composition may involve an effective quantity of angiogenic or anti-angiogenic compound disclosed herein in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • the pharmaceutical compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising one or more anti-androgenic compounds disclosed herein in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which an anti-angiogenic or angiogenic compound is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of an anti-angiogenic or angiogenic compound with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes that provides for the controlled or sustained release of the product which may then be delivered via a depot injection.
  • an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes that provides for the controlled or sustained release of the product which may then be delivered via a depot injection.
  • Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of an anti-angiogenic or angiogenic compound includes implantable drug delivery devices.
  • pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • citric acid and its salts and sodium EDTA are suitable stabilizing agents.
  • parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.
  • the anti-angiogenic or angiogenic compounds disclosed herein may be prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent. Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving anti-angiogenic or angiogenic compounds disclosed herein in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly (2-hydroxyethyl- methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.
  • Sustained-release compositions also include liposomes, which can be prepared by any of several methods known in the art.
  • the pharmaceutical composition to be used for in vivo administration may need to be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution.
  • the composition for parenteral administration may be stored in lyophilized form or in solution.
  • parenteral compositions generally are 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.
  • the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
  • Fertilized chicken eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development. A sterile filter disc was exposed to either vehicle (control), 1.5 ⁇ M zoledronic acid (zoledronic acid), or 15 ⁇ M zoledronic acid.
  • E9 chicken CAMs were cultured with one of the three types of treated discs for 24 and 48 hours at 37.5 0 C in a humidified incubator. Typical results from a classical CAM assay are shown in Figure 1.
  • E8 chicken embryonic skins were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Three groups of E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) with vehicle, 3.75 ⁇ M zoledronic acid, or 15 ⁇ M zoledronic acid for 24 hours.
  • the embryonic chicken skin tissue explants now age E9, were transferred to the cultured CAMs, now age E9 and cultured at 37.5 0 C in a humidified incubator.
  • endothelial cells of CAM normally started to proliferate and migrate into feather buds of the embryonic skin tissue after 2 days of co- culture.
  • the blood vessel growth was determined after 4 days culturing under the microscope.
  • the results from a representative CAM-FB assay conducted as described in this example are shown in Figure 3.
  • the results showed that zoledronic acid blocks new blood vessel formation and feather bud development through inhibition of endothelial proliferation and migration.
  • Example 2 One having ordinary skill in the art would have no trouble discerning that zoledronic acid blocks new blood vessel formation and feather bud development through inhibition of endothelial proliferation and migration in the assay of Example 2, whereas the results of the assay conducted in Example 1 are subject to much more uncertainty. Thus, the skilled artisan would interpret the modified CAM assay of Example 2 (e.g., CAM-FB assay) to provide significant benefits over the CAM assays conducted in the art.
  • CAM-FB assay modified CAM assay
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) with: treatment group A) vehicle (e.g., no zoledronic acid, no vitamin-C); treatment group B) 15 ⁇ M zoledronic acid and 100 ⁇ M vitamin-C; or treatment group C) 15 ⁇ M zoledronic acid with no vitamin-C for 24 hours.
  • treatment group A vehicle (e.g., no zoledronic acid, no vitamin-C)
  • treatment group B 15 ⁇ M zoledronic acid and 100 ⁇ M vitamin-C
  • treatment group C 15 ⁇ M zoledronic acid with no vitamin-C for 24 hours.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • Embryonated chicken eggs were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • the three or four groups of E8 embyronic chicken skin tissue were cultured in vitro in order to examine the effects of zoledronic acid and vitamin C on feather bud development.
  • the embryonic skin tissue explants were examined at day 0, day1 and day 4 of the treatments.
  • treatment group A vehicle (e.g., control group); treatment group B) 8 ⁇ g/ml (20 ⁇ M) zoledronic acid; and treatment group C) 8 ⁇ g/ml (20 ⁇ M) zoledronic acid and 20 ⁇ M vitamin-C (see results, Figure 6).
  • vehicle e.g., control group
  • treatment group B 8 ⁇ g/ml (20 ⁇ M) zoledronic acid
  • treatment group C 8 ⁇ g/ml (20 ⁇ M) zoledronic acid and 20 ⁇ M vitamin-C
  • treatment group A vehicle (e.g., control group); treatment group B) 100 ⁇ M vitamin-C; treatment group C) 8 15 ⁇ M zoledronic acid; and treatment group D) 100 ⁇ M vitamin-C and 15 ⁇ M zoledronic acid (see results, Figure 9).
  • vehicle e.g., control group
  • treatment group B 100 ⁇ M vitamin-C
  • treatment group C 8 15 ⁇ M zoledronic acid
  • treatment group D 100 ⁇ M vitamin-C and 15 ⁇ M zoledronic acid
  • results of these experiment showed that there was no alteration of normal feather bud development (e.g., epithelial cell development) in vitro using the above compounds in response to treatment with vehicle or with any of the drug combinations tested.
  • the results of the assays and methods of the present invention depend on the interaction of the feather bud epithelial cells layer with the CAM in order to establish angiogenesis are not due to treatment effect with drugs alone.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 24 hours: treatment group A) vehicle (e.g., no zoledronic acid, no vitamin-C); treatment group B) 100 ⁇ M vitamin-C; treatment group C) 15 ⁇ M zoledronic acid and 100 ⁇ M vitamin-C; or treatment group D) 15 ⁇ M zoledronic acid with no vitamin-C.
  • treatment group A vehicle (e.g., no zoledronic acid, no vitamin-C)
  • treatment group B 100 ⁇ M vitamin-C
  • treatment group C 15 ⁇ M zoledronic acid and 100 ⁇ M vitamin-C
  • treatment group D 15 ⁇ M zoledronic acid with no vitamin-C.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • Angiogenesis was determined by blood vessel growth and by degree of feather bud development. The results showed that zoledronic acid inhibited feather bud development; vitamin-C did not affect feather bud development, and vitamin-C slightly reversed zoledronic acid mediated inhibition of feather bud development.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development. In parallel, another set embryonated chicken eggs were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • treatment group A vehicle (e.g., no zoledronic acid, no vitamin-C); treatment group B) 100 ⁇ M vitamin-C; treatment group C) 15 ⁇ M zoledronic acid and 100 ⁇ M vitamin-C; or treatment group D) 15 ⁇ M zoledronic acid with no vitamin-C.
  • vehicle e.g., no zoledronic acid, no vitamin-C
  • treatment group B 100 ⁇ M vitamin-C
  • treatment group C 15 ⁇ M zoledronic acid and 100 ⁇ M vitamin-C
  • treatment group D 15 ⁇ M zoledronic acid with no vitamin-C.
  • E8 chicken embryonic skins were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Three groups of E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) with vehicle, 5 ⁇ M zoledronic acid, or 15 ⁇ M zoledronic acid for 24 hours.
  • the embryonic chicken skin tissue explants now age E9, were transferred to the cultured CAMs, now age E9 and cultured at 37.5 0 C in a humidified incubator.
  • H & E staining was used to determine new blood vessel formation in feather buds treated with vehicle control (i.e., PBS) and zoledronic acid at 15 ⁇ M; 200X magnification (see Figure 5A).
  • H & E staining was used to determine new blood vessel formation in feather buds treated with vehicle control (i.e., PBS) and zoledronic acid at 15 ⁇ M; 400X magnification (see Figure 5B).
  • H & E staining was used to determine new blood vessel formation in feather buds treated with vehicle control (i.e., PBS) in a model CAM-FB assay at 100X and 400X magnification (see Figure 5C, leftmost two panels).
  • Immunohistochemistry for Tie2 protein expression was used to determine new blood vessel formation in feather buds treated with vehicle control (i.e., PBS) and zoledronic acid at 5 ⁇ M using standard protocols.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 24 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 10 nM fumagillin; treatment group C) 100 nM fumagillin; and treatment group D)1 ⁇ M fumagillin.
  • treatment group A vehicle (e.g., PBS)
  • treatment group B 10 nM fumagillin
  • treatment group C 100 nM fumagillin
  • treatment group D 1 ⁇ M fumagillin.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development. In parallel, another set embryonated chicken eggs were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Three groups of E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 24 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 100 nM fumagillin; and treatment group C) 1 ⁇ M fumagillin. At the end of the 24 hour culture period, the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • vehicle e.g., PBS
  • treatment group B 100 nM fumagillin
  • treatment group C 1
  • Embryonated chicken eggs were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Five groups of E8 embyronic chicken skin tissue were cultured in vitro in order to examine the effects of fumagillin and minocycline on feather bud development.
  • the embryonic skin tissue explants were examined after 72 hours of treatment.
  • the five groups of embryonic skin tissue explants were treated with the following compounds: treatment group A) vehicle (e.g., control group); treatment group B) 100 nM fumagillin; treatment group C) 1 ⁇ M fumagillin; treatment group D) 500 nM minocycline; and treatment group E) 5 ⁇ M minocycline.
  • vehicle e.g., control group
  • treatment group B 100 nM fumagillin
  • treatment group C 1 ⁇ M fumagillin
  • treatment group D 500 nM minocycline
  • treatment group E 5 ⁇ M minocycline.
  • the results of these experiment showed that there was no alteration of normal feather bud development (e.g., epithelial cell development) in vitro using the above compounds in response to treatment with vehicle or with any of the drug combinations tested.
  • the results of the assays and methods of the present invention depend on the interaction of the feather bud epithelial cells layer with the CAM in order to establish angiogenesis are not due to treatment effect with drugs alone
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 chicken embryonic skins were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Three groups of E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 24 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 500 nM minocycline; and treatment group C) 5 ⁇ M minocycline.
  • treatment group A vehicle (e.g., PBS); treatment group B) 500 nM minocycline; and treatment group C) 5 ⁇ M minocycline.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 24 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 25 ⁇ g/mL Celebrex®; treatment group C) 100 ⁇ g/mL Celebrex®; and treatment group D) 250 ⁇ g/mL Celebrex®.
  • treatment group A vehicle (e.g., PBS); treatment group B) 25 ⁇ g/mL Celebrex®; treatment group C) 100 ⁇ g/mL Celebrex®; and treatment group D) 250 ⁇ g/mL Celebrex®.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development. In parallel, another set embryonated chicken eggs were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Three groups of E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 24 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 0.2 ⁇ g/mL Velcade®; and treatment group C) 0.2 ⁇ g/mL Velcade®. At the end of the 24 hour culture period, the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • vehicle e.g., PBS
  • treatment group B 0.2 ⁇ g/mL
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 24 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 0.1 ⁇ M fumagillin; treatment group C) 0.5 ⁇ M fumagillin; treatment group D) 1 ⁇ M fumagillin; treatment group E) 100 ⁇ g/mL Celebrex®; and treatment group F) 250 ⁇ g/mL Celebrex®.
  • vehicle e.g., PBS
  • treatment group B 0.1 ⁇ M fumagillin
  • treatment group C 0.5 ⁇ M fumagillin
  • treatment group D 1 ⁇ M fumagillin
  • treatment group E 100 ⁇ g/mL Celebrex®
  • treatment group F 250 ⁇ g/mL Celebrex®.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to four groups of treatments, each with three conditions. E8 embryonic skin tissue explants were exposed to the following treatments for 24 hours: treatment group A) condition i) vehicle (e.g., PBS), condition ii) 0.1 ⁇ M fumagillin, and condition iii) 1 ⁇ M fumagillin; treatment group B) condition i) vehicle (e.g., PBS), condition ii) 50 ⁇ g/mL minocycline, and condition iii) 250 ⁇ g/mL minocycline; and treatment group C) condition i) vehicle (e.g., PBS), condition ii) 7.5 ⁇ M zoledronic acid, and condition iii) 15 ⁇ M zoledronic acid.
  • condition i) vehicle e.g., PBS
  • condition ii) 7.5 ⁇ M zoledronic acid e.g., 7.5 ⁇ M
  • E8 embryonic skin tissue explants were exposed to the following treatments for 48 hours: treatment group A) condition i) vehicle (e.g., PBS), condition ii) 0.1 ⁇ M fumagillin, and condition iii) 1 ⁇ M fumagillin; treatment group B) condition i) vehicle (e.g., PBS), condition ii) 50 ⁇ g/mL minocycline, and condition iii) 250 ⁇ g/mL minocycline; treatment group C) condition i) vehicle (e.g., PBS), condition ii) 2 ⁇ M zoledronic acid, and condition iii) 8 ⁇ M zoledronic acid; and D) condition i) vehicle (e.g., PBS), condition ii) 1 ⁇ M melphalan and condition iii) 10 ⁇ M melphalan.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAM
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 24 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 1.5 ⁇ g/mL mouse anti-VEGF antibody; treatment group C) 3.0 ⁇ g/mL mouse anti-VEGF antibody; and treatment group D) 6.0 ⁇ g/mL mouse anti-VEGF antibody.
  • vehicle e.g., PBS
  • treatment group B 1.5 ⁇ g/mL mouse anti-VEGF antibody
  • treatment group C 3.0 ⁇ g/mL mouse anti-VEGF antibody
  • treatment group D 6.0 ⁇ g/mL mouse anti-VEGF antibody.
  • the cultured embryonic chicken skin tissue explants were subsequently cultured in vitro and cultured for an additional day at 37.5 0 C in a humidified incubator or were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator for four days.
  • the CAM-FB cultures showed an effect among experimental treatment groups compared to the control treatment.
  • Angiogenesis was determined by blood vessel growth and by degree of feather bud development.
  • the results showed that mouse anti-human VEGF (6.0 ug/ml) reduced new blood vessel formation and feather bud development (see, Figure 18A) and also reduced feather bud formation (see, Figure 18B).
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 48 hours: treatment group I) vehicle (e.g., PBS); treatment group II) 100 nM or 1 ⁇ M fumagillin; treatment group III) 0.5 ⁇ M or 5 ⁇ M minocycline; and treatment group IV) 2 ⁇ M or 8 ⁇ M zoledronic acid.
  • treatment group I vehicle (e.g., PBS); treatment group II) 100 nM or 1 ⁇ M fumagillin; treatment group III) 0.5 ⁇ M or 5 ⁇ M minocycline; and treatment group IV) 2 ⁇ M or 8 ⁇ M zoledronic acid.
  • treatment group I vehicle
  • treatment group II 100 nM or 1 ⁇ M fumagillin
  • treatment group III 0.5 ⁇ M or 5 ⁇ M minocycline
  • treatment group IV 2 ⁇ M or 8 ⁇ M zoledronic acid.
  • CAM-FB assays showed marked inhibition of FB development occurred in the presence of fumagillin, minocycline, and zoledronic acid in a concentration-dependent manner within 2 days, compared to control treatment groups. Maximum effects were observed after 4 days of culture as determined by feather weight and Flk-1 expression (see, Figure 20, for example).
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 chicken embryonic skins 8mm portions of E8 chicken embryonic skin were dissected and the epithelial layer of the FB was placed on an insert and exposed to specific drugs mixed with culture medium for 48 hours.
  • One group of cultured embryonic skins was subsequently placed on E9 chicken CAM and the other group was cultured in the absence of E9 chicken CAM. Cultures were maintained for 4 days at 37.5 0 C in a humidified incubator. The co-cultures were not exposed to drugs or compounds that were able to prevent the development of blood vessels, placode and dermal condensation of the FB.
  • FB weight increased 5 - fold relative to embryonic skins cultured in the absence of CAM. In the absence of CAM, the FB does not develop feathers.
  • Embryonated chicken eggs were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Four groups of E8 embyronic chicken skin tissue were cultured in vitro in order to examine the effects of fumagillin and minocycline on feather bud development.
  • the embryonic skin tissue explants were examined after 48 hours of treatment.
  • treatment group A vehicle (e.g., PBS); treatment group B) 1 ⁇ M fumagillin; treatment group C) 5 ⁇ M minocycline; and treatment group D) 30 ⁇ M melphalan.
  • vehicle e.g., PBS
  • treatment group B 1 ⁇ M fumagillin
  • treatment group C 5 ⁇ M minocycline
  • treatment group D 30 ⁇ M melphalan.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 48 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 0.5 ⁇ M doxorubicin; and treatment group C) 5 ⁇ M doxorubicin.
  • treatment group A vehicle (e.g., PBS); treatment group B) 0.5 ⁇ M doxorubicin; and treatment group C) 5 ⁇ M doxorubicin.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • day 1 there was no visible difference among the different treatment groups.
  • At day 4 there was an effect among experimental treatment groups compared to the control treatment.
  • Angiogenesis was determined by blood vessel growth and by degree of feather bud development. The results showed that both concentrations of doxorubicin had an inhibitory effect on angiogenesis and feather bud development (see, Figure 23A, for example
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 48 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 0.5 ⁇ M doxorubicin; and treatment group C) 5.0 ⁇ M doxorubicin.
  • treatment group A vehicle (e.g., PBS); treatment group B) 0.5 ⁇ M doxorubicin; and treatment group C) 5.0 ⁇ M doxorubicin.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • the embryonic skins/feather buds were removed and feather bud (FB) weight was measured using a fine balance.
  • FLK-1 protein expression was quantified using ELISA.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) and exposed to the following treatments for 48 hours: treatment group A) vehicle (e.g., PBS); treatment group B) 1 ⁇ M melphalan; and treatment group C) 10 ⁇ M melphalan.
  • treatment group A vehicle (e.g., PBS)
  • treatment group B 1 ⁇ M melphalan
  • treatment group C 10 ⁇ M melphalan.
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator.
  • Embryonated chicken eggs were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Six groups of E8 embyronic chicken skin tissue were cultured in vitro in order to examine the effects of fumagillin and minocycline on feather bud development.
  • the embryonic skin tissue explants were examined after 48 hours of treatment.
  • treatment group A vehicle (e.g., PBS); treatment group B) 1 ⁇ M melphalan; treatment group C) 10 ⁇ M melphalan; treatment group D) 30 ⁇ M melphalan; treatment group E) 50 ⁇ M melphalan; and treatment group F) 100 ⁇ M melphalan.
  • vehicle e.g., PBS
  • treatment group B 1 ⁇ M melphalan
  • treatment group C 10 ⁇ M melphalan
  • treatment group D 30 ⁇ M melphalan
  • treatment group E 50 ⁇ M melphalan
  • treatment group F 100 ⁇ M melphalan.
  • Embryonated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • condition i) vehicle e.g., PBS
  • condition ii) 0.1 ⁇ M fumagillin e.g., 0.1 ⁇ M fumagillin
  • condition iii) 1 ⁇ M fumagillin treatment group B
  • condition i) vehicle e.g., PBS
  • condition ii) 50 ⁇ g/mL minocycline treatment group B
  • treatment group C condition i) vehicle (e.g., PBS), condition ii) 2 ⁇ M zoledronic acid, and condition iii) 8 ⁇ M zoledronic acid
  • condition i) vehicle e.g., PBS
  • condition ii) 1 ⁇ M melphalan condition iii) 10
  • the cultured embryonic chicken skin tissue explants were transferred to the cultured CAMs and incubated at 37.5 0 C in a humidified incubator for four days.
  • Embronated chick eggs were incubated horizontally at 37.5 0 C in a humidified incubator and windowed by E8 or day 8 of embryonic development. Subsequent to windowing, fertilized chick eggs were cultured until a developmental age of E9 or day 9 of embryonic development.
  • E8 chicken embryonic skins were obtained and dissected in order to isolate E8 chicken embryonic skins.
  • Two groups of E8 embryonic skin tissue explants were cultured in insert dishes (Falcon) with vehicle, or 8 ⁇ M zoledronic acid for 48 hours. At the end of the 48 hour culture period, the embryonic chicken skin tissue explants were transferred to the cultured CAMs, and cultured at 37.5 0 C in a humidified incubator.
  • the blood vessel growth was determined after 4 days culturing using hematoxylin and eosin Y (H & E) staining and Tie2 and FLK-1 protein immunohistochemistry.
  • H & E staining was used to determine new blood vessel formation in feather buds treated with vehicle control (i.e., PBS) and zoledronic acid at 8 ⁇ M; (see Figure 27A).
  • Immunohistochemistry for Tie2 and FLK-1 protein expression was used to determine new blood vessel formation in feather buds treated with vehicle control (i.e., PBS) and zoledronic acid at 8 ⁇ M using standard protocols.
  • zoledronic acid Compared to new blood vessel formation in feather buds treated with PBS, zoledronic acid inhibited organized feather formation and blood vessel formation in feather buds and reduced the number of the cells expressing the endothelial marker proteins Tie2 and FLK-1.
  • FB feather bud Fertilized chick eggs (Charles River) were incubated horizontally at 37.5°C in a humidified incubator for 8 days and staged according to Hamburger and Hamilton (1951). Stage 33 chicken embryonic dorsal skin with FB was collected under a dissecting microscope (Olympus) in Hank's buffered saline solution (GIBCO/lnvitrogen). The FB skin was cut into 2 x 2 mm pieces and placed on culture inserts in 6 well culture dishes (Falcon).
  • the FBs were cultured with DMEM containing 2% fetal calf serum, gentamicin (1 :1000), and with or without drugs at 37 0 C at an atmosphere of 5% carbon dioxide air for 24 - 48 hours.
  • FB images were analyzed by dissection microscope to determine size, area, shape factor, orientation of each feather bud.
  • HRP Horseradish peroxidase
  • KPL Gaithersburg, MD
  • AEC 3-amino-9-ethylcarbazole
  • the slides were blocked with 0.05% Tween-20 (TBST) and 3% BSA for 1 hour at RT and were incubated overnight with anti-mouse IgG antibody conjugated to phycoerythhn (PE) (1 :100; BD Biosciences, San Jose, CA) at 4 0 C.
  • PE phycoerythhn
  • the slides were washed three times with PBS for 15 minutes at RT and incubated with FITC-conjugated swine anti-goat or anti-mouse antibody (Biosource, Camarillo, CA) for 2 hours at RT.
  • Anti-DAPI antibody was added to slides as a nuclear marker.
  • the slides were washed as before and mounted with aqueous mounting media (Biomeda, Foster City, CA).
  • Endothelial markers were identified under the microscope (Olympus BX51 , San Diego, CA) and merged cells were analyzed by Microsuite Biological Suite program (Olympus BX51 , San Diego, CA).
  • RNA (1 ⁇ g) was reverse-transcribed to cDNA and amplified using the ThermoScript RT-PCR System (Invitrogen, Carlsbad, CA). PCR was performed again using the ThermoScript RT-PCR System (Invitrogen, Carlsbad, CA) and a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) for one cycle at 94°C

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention a pour objet des procédés et des analyses permettant de déterminer la capacité d’un composé d’essai à moduler la vascularisation au moyen d’une nouvelle analyse de membrane chorio-allantoïque (CAM) améliorée. La présente invention concerne en outre des procédés et des analyses permettant de déterminer l’activité anti-angiogénique et/ou l’activité angiogénique d’un composé d’essai au moyen de la nouvelle analyse de CAM.
PCT/US2009/063316 2008-11-05 2009-11-04 Analyse de membrane chorio-allantoïque améliorée WO2010054022A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11158008P 2008-11-05 2008-11-05
US61/111,580 2008-11-05

Publications (1)

Publication Number Publication Date
WO2010054022A1 true WO2010054022A1 (fr) 2010-05-14

Family

ID=41503760

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/063316 WO2010054022A1 (fr) 2008-11-05 2009-11-04 Analyse de membrane chorio-allantoïque améliorée

Country Status (1)

Country Link
WO (1) WO2010054022A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111685077A (zh) * 2020-06-05 2020-09-22 广东贞山鳄鱼养殖有限公司 一种鳄鱼的人工孵化方法
WO2023059904A2 (fr) 2021-10-07 2023-04-13 Children's Cancer Therapy Development Institute Dispositif d'ouverture d'oeuf, méthode de xénogreffe de tumeur ex ovo, dosage de composé thérapeutique, méthode de test de composé thérapeutique par dosage de xénogreffe d'oeuf de caille ex ovo
RU2808738C1 (ru) * 2023-05-26 2023-12-04 Федеральное государственное автономное образовательное учреждение высшего образования "Белгородский государственный национальный исследовательский университет" (НИУ "БелГУ") Способ оценки процесса формирования сосудистой сети на хориоаллантоисной мембране куриного эмбриона

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005033300A1 (fr) * 2003-10-01 2005-04-14 Cedars-Sinai Medical Center Systeme et methode de culture tissulaire et cellulaire xenobiotique favorisee par circulation vasculaire aviaire de substitution
WO2006001021A2 (fr) * 2004-06-28 2006-01-05 Bar-Ilan University Systeme de criblage fonctionnant sur la base d'elements aviaires chimeres contenant des greffes mammiferes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005033300A1 (fr) * 2003-10-01 2005-04-14 Cedars-Sinai Medical Center Systeme et methode de culture tissulaire et cellulaire xenobiotique favorisee par circulation vasculaire aviaire de substitution
WO2006001021A2 (fr) * 2004-06-28 2006-01-05 Bar-Ilan University Systeme de criblage fonctionnant sur la base d'elements aviaires chimeres contenant des greffes mammiferes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BORGES J ET AL: "Chorioallantoic membrane angiogenesis model for tissue engineering: A new twist on a classic model", TISSUE ENGINEERING, LARCHMONT, NY, US, vol. 9, no. 3, 1 June 2003 (2003-06-01), pages 441 - 450, XP002997977, ISSN: 1076-3279 *
RIBATTI D: "The chick embryo chorioallantoic membrane in the study of tumor angiogenesis.", October 2008, ROMANIAN JOURNAL OF MORPHOLOGY AND EMBRYOLOGY = REVUE ROUMAINE DE MORPHOLOGIE ET EMBRYOLOGIE 2008, VOL. 49, NR. 2, PAGE(S) 131 - 135, ISSN: 1220-0522, XP002563935 *
SALVATORE PONTICELLI ET AL: "Modulation of angiogenesis by a tetrameric tripeptide that antagonizes vascular endothelial growth factor receptor 1", THE JOURNAL OF BIOLOGICAL CHEMISTRY,, vol. 283, no. 49, 15 October 2008 (2008-10-15), pages 34250 - 34259, XP002556278 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111685077A (zh) * 2020-06-05 2020-09-22 广东贞山鳄鱼养殖有限公司 一种鳄鱼的人工孵化方法
WO2023059904A2 (fr) 2021-10-07 2023-04-13 Children's Cancer Therapy Development Institute Dispositif d'ouverture d'oeuf, méthode de xénogreffe de tumeur ex ovo, dosage de composé thérapeutique, méthode de test de composé thérapeutique par dosage de xénogreffe d'oeuf de caille ex ovo
RU2808738C1 (ru) * 2023-05-26 2023-12-04 Федеральное государственное автономное образовательное учреждение высшего образования "Белгородский государственный национальный исследовательский университет" (НИУ "БелГУ") Способ оценки процесса формирования сосудистой сети на хориоаллантоисной мембране куриного эмбриона

Similar Documents

Publication Publication Date Title
US20230034473A1 (en) Methods of treating fibroblast growth factor 19-mediated cancers and tumors
ES2541925T3 (es) Métodos y composiciones para el uso diagnóstico en pacientes de cáncer
US8835473B2 (en) Methods and compositions for treating cancer
US20110076271A1 (en) Diagnostic methods and compositions for treatment of cancer
KR101557545B1 (ko) 암 환자에서 진단 용도를 위한 방법 및 조성물
US20230113705A1 (en) Methods for diagnosing, prognosing and managing treatment of breast cancer
Abrass et al. Abnormal development of glomerular endothelial and mesangial cells in mice with targeted disruption of the lama3 gene
Sobol et al. Anticancer activity of repurposed hemostatic agent desmopressin on AVPR2‑expressing human osteosarcoma
Sun et al. Preconditioning of mesenchymal stem cells with ghrelin exerts superior cardioprotection in aged heart through boosting mitochondrial function and autophagy flux
WO2010054022A1 (fr) Analyse de membrane chorio-allantoïque améliorée
WO2021063970A1 (fr) BIOMARQUEURS PRÉDICTIFS POUR LE TRAITEMENT D'UN PATIENT ATTEINT D'UN CANCER AVEC DES INHIBITEURS DE LA VOIE DE SIGNALISATION DU TGFβ
US20210293769A1 (en) Method for culturing cancer tissue or tissue analogous to cancer tissue
US10001485B2 (en) Methods for sensitizing a quiescent cancer stem cell to a BCR-ABL inhibitor
US20220305001A1 (en) Methods of treating acute myeloid leukemia with farnesyltransferase inhibitors
US20220142983A1 (en) Methods of treating cancer with farnesyltransferase inhibitors
US10806730B2 (en) Methods of treating cancer with farnesyltransferase inhibitors
WO2021063972A1 (fr) Cthrc1 en tant que biomarqueur pour un micro-environnement tumoral activé par tgf-bêta
Avolio et al. Contractile reprogramming of cardiac pericytes by MEK inhibition promotes arteriologenesis of the ischemic heart
BR112020002591A2 (pt) método para o tratamento de câncer
KR101146597B1 (ko) 백반증 진단용 조성물
Garofano Differences in the African American and European American Platelet Transcriptomes Alter Platelet Function and May Contribute to Racial Disparities in Cardiovascular Disease and Cancer
US9682090B2 (en) Methods for treating and preventing prostate cancer bone metastases
WO2023083863A1 (fr) Procédé de surveillance de la réponse à une thérapie néoadjuvante pour le cancer du sein
Åleskog et al. Evaluation of purine and pyrimidine analogues in human tumor cells from patients with low‐grade lymphoproliferative disorders using the FMCA
WO2018028369A1 (fr) Utilisation de tnfr2

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09756879

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09756879

Country of ref document: EP

Kind code of ref document: A1