Eisenberg et al. vivo animal models for the particular disease being targeted, needed to exist, in order to ensure that any lead compounds identified in initial screening campaigns could be furthe developed pharmacologically to a point where drug development value could be firmly established. Fifth, assays needed to offer the possibility of eventual patent-protected licensable opportunities(compound, target, or both), since it was recognized that eventual development of lead compounds would depend on interest by the pharmaceutical industry, which would likely require appropriate intellectual property protections allowing for a return n investment Guided by the above five principles, the first several thousand fractions were screened across a range of assays representing a variety of therapeutic areas. Although beyond the scope of this manuscript, active screens included targets in the following areas: oncology (angiogenesis, tumor-specific differential cytotoxicity, and cell survival pathways), anti infectives(bacterial, anthrax), anti-virals(HIv-1), diabetes and neurodegeneration (Alzheimer's disease, Parkinsons disease). Other therapeutic areas under consideration included assays for novel targets in arthritis, cancer(and non-cancer) stem cells, cardiovascular disease, anti-infectives (Methicillin-resistant Staphylococcus aureus, tuberculosis, influenza A/HINI), hepatitis C, immuno-oncology approaches, inflammation, multiple sclerosis, and pain 5.9. Description of selected (initial)screens involving cell based and whole organism(e.g Zebra fish)assays Human fibroblasts and cancer cell lines [LNCaP, PC3 (Prostate), MCF7, MDA-231 ( Breast, A549 (Lung), BJ Fibroblasts] were propagated using standard conditions as suggested by ATCC. Cell proliferation and/or survival were determined by using Cell Titer lo(Promega)in a 384-well format assay as previously described [40] For zebra fish screening, fractions were delivered robotically to 96 well plates. Four embryos were added per well by pipette at roughly 6 h post fertilization to allow early development to occur before treatment. More than 1000 extract fractions were initially screened at nominal concentrations(based on the assumption of a pure compound of Mw from 6 h post fertilization to 72 h post fertilization. All wells were examined by lighr s 500)on embryos in 10% Hanks Saline with 0.003% phenyl thiourea in water, obse microscope to look for signs of altered development and impaired angiogenesis Fractions that showed grossly defective development were not further analyzed 5.9.1. LC/Ms and NMR techniques for compound characterization-LC/MS was performed on an Agilent 6130 Single Quadrupole system. NMR spectra of active compounds were obtained on a Varian 600 MHz spectrometer. Compounds were identified either using authentic samples or comparing their chemical shifts with those reported in the 9 5.9.2. International collaboration agreements-Agreements covering the scope of work, ownership of materials and intellectual property, sharing of potential financial revenues and co-authorship of academic publications were negotiated by representatives of the participating US and Chinese Academic Institutions. These were also reviewed by representatives of the Chinese Ministry of Health, Chinese Ministry of Education, State Administration of TCM, Chinese Fda and the chinese ministry of Science and Authovivo animal models for the particular disease being targeted, needed to exist, in order to ensure that any lead compounds identified in initial screening campaigns could be further developed pharmacologically to a point where drug development value could be firmly established. Fifth, assays needed to offer the possibility of eventual patent-protected licensable opportunities (compound, target, or both), since it was recognized that eventual development of lead compounds would depend on interest by the pharmaceutical industry, which would likely require appropriate intellectual property protections allowing for a return on investment. Guided by the above five principles, the first several thousand fractions were screened across a range of assays representing a variety of therapeutic areas. Although beyond the scope of this manuscript, active screens included targets in the following areas: oncology (angiogenesis, tumor-specific differential cytotoxicity, and cell survival pathways), anti￾infectives (bacterial, anthrax), anti-virals (HIV-1), diabetes and neurodegeneration (Alzheimer’s disease, Parkinson’s disease). Other therapeutic areas under consideration included assays for novel targets in arthritis, cancer (and non-cancer) stem cells, cardiovascular disease, anti-infectives (Methicillin-resistant Staphylococcus aureus, tuberculosis, influenza A/H1N1), hepatitis C, immuno-oncology approaches, inflammation, multiple sclerosis, and pain. 5.9. Description of selected (initial) screens involving cell based and whole organism (e.g. Zebra fish) assays Human fibroblasts and cancer cell lines [LNCaP, PC3 (Prostate), MCF7, MDA-231 (Breast), A549 (Lung), BJ Fibroblasts] were propagated using standard conditions as suggested by ATCC. Cell proliferation and/or survival were determined by using Cell Titer Glo (Promega) in a 384-well format assay as previously described [40]. For zebra fish screening, fractions were delivered robotically to 96 well plates. Four embryos were added per well by pipette at roughly 6 h post fertilization to allow early development to occur before treatment. More than 1000 extract fractions were initially screened at nominal concentrations (based on the assumption of a pure compound of MW 500) on embryos in 10% Hank’s Saline with 0.003% phenyl thiourea in water, observing from 6 h post fertilization to 72 h post fertilization. All wells were examined by light microscope to look for signs of altered development and impaired angiogenesis. Fractions that showed grossly defective development were not further analyzed. 5.9.1. LC/MS and NMR techniques for compound characterization—LC/MS was performed on an Agilent 6130 Single Quadrupole system. NMR spectra of active compounds were obtained on a Varian 600 MHz spectrometer. Compounds were identified either using authentic samples or comparing their chemical shifts with those reported in the literature. 5.9.2. International collaboration agreements—Agreements covering the scope of work, ownership of materials and intellectual property, sharing of potential financial revenues and co-authorship of academic publications were negotiated by representatives of the participating US and Chinese Academic Institutions. These were also reviewed by representatives of the Chinese Ministry of Health, Chinese Ministry of Education, State Administration of TCM, Chinese FDA and the Chinese Ministry of Science and Technology. Eisenberg et al. Page 11 Fitoterapia. Author manuscript; available in PMC 2012 January 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript