WO2008107912A2 - Procédés de dosage in vitro pour classifier l'embryotoxicité de composés - Google Patents

Procédés de dosage in vitro pour classifier l'embryotoxicité de composés Download PDF

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Publication number
WO2008107912A2
WO2008107912A2 PCT/IN2008/000126 IN2008000126W WO2008107912A2 WO 2008107912 A2 WO2008107912 A2 WO 2008107912A2 IN 2008000126 W IN2008000126 W IN 2008000126W WO 2008107912 A2 WO2008107912 A2 WO 2008107912A2
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WIPO (PCT)
Prior art keywords
human
compound
embryotoxic
embryonic stem
stem cells
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PCT/IN2008/000126
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English (en)
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WO2008107912A3 (fr
Inventor
Apama Khanna
Ashish Mehta
Vijay Bhaskar Reddy
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Reliance Life Sciences Pvt. Ltd.
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Priority to US12/043,899 priority Critical patent/US20090220996A1/en
Publication of WO2008107912A2 publication Critical patent/WO2008107912A2/fr
Publication of WO2008107912A3 publication Critical patent/WO2008107912A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/04—Pretreatment of vegetable raw material
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B13/00—Recovery of fats, fatty oils or fatty acids from waste materials
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/12—Refining fats or fatty oils by distillation
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/08—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils with fatty acids
    • 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
    • G01N33/5014—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 for testing toxicity
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/74—Recovery of fats, fatty oils, fatty acids or other fatty substances, e.g. lanolin or waxes

Definitions

  • the present invention provides in vitro assay techniques for determining the teratogenic or embryotoxic potential of compounds, as wel ⁇ as compositions comprising human embryonic stem cells.
  • the disclosed techniques are useful, for example, for high throughput screening of potential drug candidates to determine their effects on embryonic stem cells at the molecular and/or cellular level.
  • a leading cause of drug candidate attrition is reproductive toxicity.
  • Mutagenic, embryotoxic, or teratogenic substances may exert direct cytotoxic effects and/or induce alterations of embryonic development as a result of mutations at the DNA level. Additionally, developmental defects may be generated by interference of mutagenic or embryotoxic substances with regulatory processes of proliferation and differentiation at the levels of gene and protein expression, respectively. Medical drugs and xenobiotics, when administered during pregnancy, may interfere with embryonic development and as a consequence induce embryonic lethality or teratogenic effects.
  • Salicylates e.g., aspirin
  • aspirin are well- known therapeutic drugs, which have been taken by pregnant women for years with no signs of being responsible for birth defects
  • aspirin causes birth defects in rats, mice, monkeys, guinea-pigs, cats and dogs.
  • test chemicals are analyzed by segment studies, which cover pre-conceptional exposure and postnatal development including the lactation period (Schmidt, et al, Int. J. Dev. Biol. 45:421-429, 2001). Therefore there is a need for alternatives to living animals to test the potential reproductive toxicity of chemical substances by in vitro systems.
  • ES cells are derived from the inner cell mass (“ICM”) of the mammalian blastocyst (Evans and Kaufman, Nature 292:154-156, 1981; Martin, Proc. Natl. Acad. Sci. USA 78:7634-7638, 1981.). These cells are pluripotent, and thus capable of developing into any organ or tissue type. ES cells are capable of indefinite proliferation in vitro in an undifferentiated state, maintaining a normal karyotype through prolonged culture.
  • Embryonic stem cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation. Embryonic stem cells have been isolated from the ICM of blastocyst stage embryos in multiple species (Bhattacharya, et al, BMC Dev. Biol. 5:22, 2005), including mice (Solter and Knowles, Proc. Natl. Acad.
  • ES cells Appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells.
  • Differentiated cells from stem cells offer considerable advantages compared with primary or immortalized cells in that these cells are genetically normal, demonstrate uniform physiological responses, are maintained in culture for long periods of time, and are grown at scale, all of which enhance their usefulness in screening processes (McNeish, Nat. Rev. Drug Discov. 3:70-80, 2004).
  • a unique advantage of ES cells is their ability to undergo homologous recombination at a relatively high frequency, which enables the selection of reproducible and precise genetic modifications of the endogenous genome.
  • ES cells Under certain culture conditions, e.g., in the absence of leukemia inhibitory factor, ES cells can differentiate in vitro into embryo-like aggregates termed embryoid bodies ("EB"), which can also differentiate into derivatives of all three germ layers (i.e., mesoderm, ectoderm and endoderm), e.g., cardiogenic cells (Doetschman, et al. , J. Embryol Exp. Morphol. 87:27-45, 1985), myogenic cells (Rohwedel, et al , Dev. Biol 164:87-101, 1994), and neuronal and haematopoietic cells (Wiles and Keller, Development 111:259-267, 1991).
  • EB embryoid bodies
  • retinoic acid causes changes in tissue specific genes at specific times of embryoid body differentiation, and activation, repression, or modulation of the expression of myocardial-specific or somatic-specific genes, thus demonstrating retinoic acid to be teratogenic (Wobus, et al, 1994, supra). Therefore embryoid bodies can be used as a toll for checking the embryotoxicity of known compounds.
  • the use of blastocyst-derived pluripotent ES cells has been used to develop in vitro methods for testing various medical drugs and xenobiotics.
  • the EST has been validated in a study coordinated by European Centre for the Validation of Alternative Methods ("ECVAM”; Genschow, et al, In Vitr. MoI Toxicol 13:51-65, 2000; Spielmann, et al, Altern. Lab Anim. 29:301-303, 2001).
  • EVAM European Centre for the Validation of Alternative Methods
  • the validation study included in vitro cultivation of rat whole embryos (WEC test), the micromass test employing primary cultures of dissociated limb-bud cells of rat embryos, and the differentiation analysis of a pluripotent mouse embryonic stem cell line (the EST) (Genschow, et al, 2000, supra).
  • the EST involves 1) cytotoxic effects of test substances on differentiated 3T3 fibroblasts, 2) cytotoxic effects on the undifferentiated ES cells and 3) the influence of test compounds on ES cell derived cardiac differentiation.
  • a prediction model for the embryotoxic potential of a given substance was established (Scholz, et al, 1999, supra).
  • 10 compounds were tested for their embryotoxic potential: 100, 88.9 and 91.7% of the prediction for non embryo toxic (Class 1), weekly embryo toxic (Class 2) and strongly embryotoxic (Class 3) substances, respectively, were in accordance with classification derived from in vivo data (Scholz, et al, 1999, supra, Genschow, et al, 2000, supra).
  • the mouse EST procedure is already used not only to test chemical compounds, but also physical factors, such as electromagnetic fields emitted by digital mobile communication systems (Rohwedel, et al, 2001, supra; Schonborn, et al, Bioelectromagnetics 21:372-384, 2000).
  • EST protocol Despite the recent improvements of embryotoxicity testing using the EST protocol, there are some drawbacks of the mouse EST regarding the differentiation analysis and the parameters used.
  • One of the main concerns of the protocol is that it utilizes manual counting of beating cardiomyocytes, which is cumbersome, time consuming, and requires technical expertise.
  • mouse embryonic stem cells mES
  • human embryonic stem cells hES
  • the morphology, cell surface markers, and growth requirement of embryonic cells from other species are significantly different from mouse ES cells.
  • mouse and human embryos differ significantly in temporal expression of embryonic genes, such as in the formation of the egg cylinder versus the embryonic disc (Kaufman, The Atlas of Mouse Development, Academic Press, London, UK, 1992), in the proposed derivation of some early lineages (O'Rahilly and Muller; Developmental Stages in Human Embryos, Carnegie Institution of Washington, Publication 637, Washington, DC, 1987), in the structure and function of the extra-embryonic membranes and placenta (Mossman, Vertebrate Fetal Membranes, Rutgers University Press, New Brunswick, NJ, 1987), in growth factor requirement for development ⁇ e.g., the hematopoietic system (Lapidot, et al, Lab Anim. Sci. 43:147-150, 1993; Vormoor, et al, Blood Cells 20:316-322, 1994)), and in adult structure and function (e.g., central nervous system).
  • embryonic genes such as in the formation of the egg cylinder versus
  • LIF leukemia inhibitory factor
  • U.S. Patent No. 5,811,231 discloses a method of screening for toxic compounds by determining the level of transcription of genes linked to selected stress promoters in cells of various cell lines.
  • WO 97/01644 describes a method of creating a molecular profile of a chemical composition (toxicity screening) using transgenic embryoid bodies containing tissue specific promoters and reporter genes. The method involves stable transfection of embryonic stem cells and embryonic germ cells with a reporter gene/promoter construct, allowing the transfected cells to differentiate into embryoid bodies in the presence of test substances, and detecting the expression of the reporter gene.
  • WO 00/34525 discloses methods and systems for identifying and typing toxicity of chemical compositions as well as screening new compositions for toxicity.
  • the method involves detecting alteration in gene or protein expression in isolated mammalian embryoid bodies contacted with various chemical compositions of known and unknown toxicities, thus establishing molecular profiles, and correlating the molecular profiles with toxicities of the chemical compositions.
  • Alteration in the level of gene or protein expression can be detected by use of a label, such as a fluorescent, colorimetric, radioactive, enzyme, enzyme substrate, nucleoside analog, magnetic, glass, or latex bead, colloidal gold, or electronic transponder label.
  • a label such as a fluorescent, colorimetric, radioactive, enzyme, enzyme substrate, nucleoside analog, magnetic, glass, or latex bead, colloidal gold, or electronic transponder label.
  • a label such as a fluorescent, colorimetric, radioactive, enzyme, enzyme substrate, nucleoside analog, magnetic, glass, or latex bea
  • the present invention has focused in providing an assay method for determining the embryotoxicity in terms of qualitative and quantitative techniques.
  • the present invention provides an in vitro embryotoxicity testing method, which is a simple and effective method for assessing the toxicity of chemicals and /or compositions and provides a high throughput screening of the chemicals ands/or compositions.
  • the present invention provides the use of multiple markers, which focuses on improving the efficiency of the test and aims to provide an objective measurement instead of subjective assessment.
  • the techniques provided results, which are reliable, reproducible and highly sensitive than the conventional mouse embryonic screening models.
  • the present invention also focuses on the use of hES of INDIAN origin for the detection of embryotoxicity of chemicals and/or compositions.
  • the object of the present invention is to provide a routinely employable in vitro embryotoxicity test method for the detection of chemically induced embryotoxic / teratogenic effects.
  • IC50 inhibitory concentration
  • ID50 inhibition of differentiation concentration
  • the present disclosure provides in vitro embryotoxicity testing methods that are simple and effective for assessing the toxicity of chemicals and/or compositions, and allows for high throughput embryotoxicity screening of chemicals and/or compositions.
  • the present disclosure also provides in vitro embryotoxicity testing methods that use multiple markers, which improves the efficiency of the test and provides an objective measurement instead of a subjective assessment.
  • the presently disclosed techniques provide results that are reliable, reproducible, and highly sensitive compared to conventional mouse embryonic screening models.
  • the present disclosure also provides methods that use hES of Indian origin for the detection of embryotoxicity of chemicals and/or compositions.
  • the present disclosure provides routinely employable in vitro embryotoxicity test methods for the detection of chemically-induced embryotoxic/teratogenic effects.
  • the present disclosure also provides methods for pre-clinical in vitro embryotoxicity testing of chemicals and/or compositions using human embryoid bodies.
  • the present disclosure further provides in vitro embryotoxicity testing methods that classify compounds, chemicals, drugs, xenobiotics, compositions, and combinations thereof, as non-embryotoxic, weakly embryotoxic, or strongly embryotoxic.
  • the present disclosure provides in vitro embryotoxicity testing methods that are rapid, less expensive than animal pre-clinical toxicity studies, and provide information that is more relevant to human clinical trials.
  • the present disclosure also provides in vitro embryotoxicity testing models that yield results that are the same or similar to those generated by animal pre-clinical toxicity studies, and also yield results that are different from, but more relevant to human clinical trials than, those generated from animal studies, due at least in part to genetic differences (or genetically-related differences, such as expression patterns, expression levels, timing of expression, etc.) between animal and humans.
  • the present disclosure provides in vitro embryotoxicity testing methods for detection of embryotoxicity/teratogenic properties of chemicals, compounds, compositions, xenobiotics, drugs, etc., by giving suitable indications of possible developmental disturbances and differentiation disturbances during early development.
  • the present disclosure also provides in vitro embryotoxicity testing methods that can predict the effects of chemicals on the organs of ectoderm, mesoderm, and/or endoderm origin, as well as other cells types of ectoderm, mesoderm, and/or endoderm origin.
  • the present disclosure provides in vitro embryotoxicity testing methods that utilize controlled experimental conditions, which therefore provide results that are easily quantified.
  • the present disclosure also provides in vitro embryotoxicity testing methods that predict the cellular and molecular effects of a chemical or drug.
  • the present disclosure provides in vitro embryotoxicity testing methods that correctly convert the results into useful predictions of toxicity, so that appropriate safety assessments can be made.
  • the present disclosure provides in vitro embryotoxicity testing methods that are reliable and provide reproducible results following application of a clearly stated prediction model.
  • the present disclosure provides in vitro embryotoxicity testing methods that are relevant in establishing the scientific meaningfulness and usefulness of results for a particular purpose in terms of hazard prediction.
  • the present disclosure provides in vitro embryotoxicity testing methods to predict the mechanism(s) of toxic chemicals at the molecular and/or cellular level, thus providing data that allows for the chemical modification of appropriate substituents of the chemical core structure of the toxin.
  • the present disclosure provides in vitro embryotoxicity testing methods that evaluate the cytotoxic potential of test compounds on three different cell types: human foreskin fibroblasts, which represent mature adult cell types; embryonic stem cells, which resemble germ cells; and embryoid bodies, which represent the early developmental stages of pregnancy and/or fetal development.
  • the present disclosure provides in vitro embryotoxicity testing methods that determine the effects of compounds on the differentiation potential of human embryoid bodies into different cell types based on gene expression levels.
  • the methods involve the use of human embryoid bodies.
  • the embryoid bodies are formed from human embryonic stem cells, and in other embodiments the embryoid bodies are formed from RELICELL®hES human embryonic stem cells of Indian origin.
  • the present disclosure also provides in vitro embryotoxicity testing methods and assay techniques that comprise qualitative assessment of embryotoxicity using a 3-(4,5,-di-methylthiazol-2yl)-2,5-diphenyltetrazolium bromide (“MTT”) assay, fluorescent activated cell sorter (“FACS”) techniques, luminescent techniques, or quantitative assessment of embryotoxicity by molecular end points for detection of gene expression levels.
  • MTT can be used for qualitative assessment of embryotoxicity of chemicals and/or compositions by determining the viability of cells in the presence of MTT.
  • the test procedure is based on the capacity of mitochondrial dehydrogenase enzymes in living cells to convert the yellow substrate MTT into a dark blue formazan product, which is then detected quantitatively, for example using a microplate ELISA reader.
  • the present disclosure provides in vitro embryotoxicity testing methods that allow for the calculation of IC 5 0 (inhibitory concentration) and ID 5O (inhibition of differentiation concentration) values for known and/or unknown compounds, chemicals, xenobiotics, medicinal drugs, pesticides, metals, or any other compounds or compositions that are used by humans, or that come into contact with humans.
  • the present disclosure also provides new methods to calculate ID 50 values based on gene expression profiles of genes that are predominantly involved in early developmental stages of pregnancy or fetal developmental stages.
  • the present disclosure provides human embryonic stem cells as a model to test the effects of three reference compounds that are well-known through in vitro and in vivo studies as strongly embryotoxic, weakly embryotoxic, and non-embryotoxic.
  • the present disclosure provides in vitro embryotoxicity testing methods checked against three reference compounds, such as a strongly embryotoxic (5-fluorouracil; 5-FU), a weakly embryotoxic (caffeine), and a non-embryotoxic (penicillin G) compound.
  • Non-embryotoxic drugs include, but are not limited to, penicillin G, saccharin, ascorbic acid, and isoniazid.
  • Weakly embryotoxic drugs include, but are not limited to, caffeine, lithium chloride, diphenhydramine, indomethacin, aspirin, dexamethasone, methotrexate, and diphenylhydantoin.
  • Strongly embryotoxic drugs include, but are not limited to, 5-fluorouracil, hydroxyurea, busulfan, cytosinearabinoside, and retinoic acid.
  • the present disclosure provides in vitro embryotoxicity testing methods that compare the cytotoxic effects of one or more compounds on three different cell types: human foreskin fibroblasts ("HFF"), which represent an adult or mature cell type; human embryonic stem cells, which represent the early growth stages of development post-fertilization, or the germ lineages; and human embryoid bodies, which represent the stages of development during early pregnancy.
  • HFF human foreskin fibroblasts
  • human embryonic stem cells which represent the early growth stages of development post-fertilization, or the germ lineages
  • human embryoid bodies which represent the stages of development during early pregnancy.
  • molecular end points are used to increase the sensitivity and reproducibility of the assay.
  • the present disclosure utilizes a panel of genes representative of some of the major organs in the process of development.
  • the present disclosure also provides qualitative in vitro embryotoxicity testing methods for assessment of the effects of compounds on various lineages, comprising detection of gene expression by isolating total RNA, and using the isolated RNA to determine the expression of genes specific for ectoderm, mesoderm, and/or endoderm. The changes in gene expression in the presence or absence of the drug are then compared.
  • the in vitro embryotoxicity testing methods involve culturing human embryonic stem cells by the "hanging drop” method, for example by seeding human embryonic stem cells onto the lid of a culture dish and growing the cells for three days in the presence of a concentration range of a test chemical.
  • the embryoid bodies that are formed i.e., the aggregates of cells
  • the embryoid bodies can then be transferred to bacteriological petri dishes containing the appropriate concentration of test chemical for another two days.
  • the embryoid bodies can be seeded into a 96 well plate and incubated for another five days under controlled conditions. After 15 days, MTT and/or RT -PCR tests can be performed.
  • the test chemical composition has a predetermined toxicity.
  • a test chemical can be identified through one of the presently disclosed testing protocols as exhibiting an identical molecular profile as the known chemical composition.
  • the toxicity of a test chemical composition can be ranked according to a comparison of its molecular profile in EB cells to those of chemical compositions with predetermined toxicities.
  • the present disclosure provides an embryotoxicity prediction model that correctly converts the results into a prediction of toxicity. Such methods provide results that appropriately classify compounds as non-embryotoxic, weakly embryotoxic, or strongly embryotoxic.
  • the present disclosure also provides in vitro embryotoxicity testing methods that can predict the molecular or cellular mechanism of chemicals exhibiting toxicity. These methods provide data on the molecular and cellular mechanism of action, which allows for changes to the chemical, for example changes to the substitutions on the core structure, followed by further testing to determine the embryotoxicity of the new chemical variants.
  • the chemical compositions tested using the presently disclosed in vitro embryotoxicity testing methods can be therapeutic agents (or potential therapeutic agents), agents of known toxicities, such as neurotoxins, hepatic toxins, toxins of hematopoietic cells, myotoxins, carcinogens, teratogens, or toxins to one or more reproductive organs, agricultural chemicals, such as pesticides, fungicides, nematicides, and fertilizers, cosmetics, including so-called "cosmeceuticals," industrial wastes or by-products, or environmental contaminants, animal therapeutics or potential animal therapeutics, or biopharmaceutical products, where human testing is mandatory.
  • FIG. 1 Flow sheet depicting a process of making embryoid bodies and embryotoxicity testing.
  • FIG. 2A, FIG. 2B, and FIG. 2C Photomicrographs of the dose dependent effects of 5- fluorouracil (FIG. 2A), caffeine (FIG. 2B) and penicillin G (FIG. 2C) on day 5 human embryoid bodies (hEBs).
  • FIG. 2A Photomicrographs 1-6 represent different doses of 5-fluorouracil: 1 - Control (no treatment); 2 - 0.0001 ⁇ g/ml; 3 - 0.001 ⁇ g/ml; 4 - 0.01 ⁇ g/ml; 5 - 0.1 ⁇ g/ml; 6 - 1 ⁇ g/ml.
  • FIG. 2B Photomicrographs of the dose dependent effects of 5- fluorouracil (FIG. 2A), caffeine (FIG. 2B) and penicillin G (FIG. 2C) on day 5 human embryoid bodies (hEBs).
  • FIG. 2A Photomicrographs 1-6 represent different doses of 5-fluorouracil: 1 - Control (
  • Photomicrographs 1-6 represent different doses of caffeine: 1 - Control (no treatment); 2 - 0.05 ⁇ g/ml; 3 - 0.5 ⁇ g/ml; 4 - 5 ⁇ g/ml; 5 - 50 ⁇ g/ml; 6 - 500 ⁇ g/ml.
  • FIG. 2C Photomicrographs 1-6, represent different doses of penicillin G: 1 - Control (no treatment); 2 - 1 ⁇ g/ml; 3 - 10 ⁇ g/ml; 4 - 100 ⁇ g/ml; 5 - 1000 ⁇ g/ml; 6 - 5000 ⁇ g/ml. Photomicrographs are representative of 3 experiments.
  • Cytotoxicity analysis of three cell types using CyQUANT assay following drug treatment.
  • Dose dependent survival percentage curve of human ES, human EB, and HFF cells following treatment of penicillin G (FIG. 4A), saccharin (FIG. 4B), caffeine (FIG. 4C), indomethacin (FIG. 4D), busulfan (FIG. 4E), and hydroxyurea (FIG. 4F). All data are a mean of three independent experiments for each cell type. Data represented as mean ⁇ SE. Photomicrographs on day 7 of human EB formation following drug treatment.
  • FIG. 4A Penicillin G (a- control; b- 10 ⁇ g/ml; c- 5000 ⁇ g/ml).
  • FIG. 4B Saccharin (a- control; b- 10 ⁇ g/ml; c- 5000 ⁇ g/ml).
  • FIG. 4C Caffeine (a- control; b- 0.5 ⁇ g/ml; c- 500 ⁇ g/ml).
  • FIG. 4D Indomethacin (a-control; b- 1 ⁇ g/ml; c- 100 ⁇ g/ml).
  • FIG. 4F Hydroxyurea (a- control; b- 0.1 ⁇ g/ml; c- 10 ⁇ g/ml).
  • FIG. 5A and FIG. 5B Gene expression profile following treatment with different doses of 5- fluorouracil on hEBs on day 15.
  • FIG. 5A The gene expression pattern at different doses: lane 1 - Control (no treatment); lane 2 - 0.0001 ⁇ g/ml; lane 3 - 0.001 ⁇ g/ml; lane 4 - 0.01 ⁇ g/ml, lane 5 - 0.1 ⁇ g/ml.
  • FIG. 6A and FIG. 6B Gene expression profile following treatment with different doses of caffeine on hEBs on day 15.
  • FIG. 6A Gene expression pattern at different doses: lane 1 - Control (no treatment); lane 2 - 0.05 ⁇ g/ml; lane 3 - 0.5 ⁇ g/ml; lane 4 - 5 ⁇ g/ml; lane 5 - 50 ⁇ g/ml; lane 6 - 500 ⁇ g/ml.
  • FIG. 7A and FIG. 7B Gene expression profile following treatment with different doses of penicillin G on hEBs on day 15.
  • FIG. 7A Gene expression pattern at different doses: lane 1 - Control (no treatment); lane 2 - 1 ⁇ g/ml; lane 3 - 10 ⁇ g/ml; lane 4 - 100 ⁇ g/ml; lane 5 - 1000 ⁇ g/ml; lane 6 - 5000 ⁇ g/ml.
  • FIG. 8A Busulfan.
  • FIG. 8B Hydroxyurea.
  • i - undifferentiated markers ii - ectodermal markers
  • iii - mesodermal markers iii - mesodermal markers
  • FIG. 9A and FIG. 9B Gene expression profile of day 15 differentiated human EBs following treatment of weakly embryotoxic compounds.
  • FIG. 9A Caffeine.
  • FIG. 9B Indomethacin.
  • i - undifferentiated markers ii - ectodermal markers
  • iii - mesodermal markers iii - mesodermal markers
  • FIG. 1OA and FIG. 1OB Gene expression profile of day 15 differentiated human EBs following treatment of non embryotoxic compounds.
  • FIG. 1OA Penicillin G.
  • FIG. 1OB Saccharin.
  • i - undifferentiated markers ii - ectodermal markers; iii - mesodermal markers; and iv - endodermal markers.
  • embryonic body typically refers to a morphological structure comprised of a population of cells, the majority of which are derived from embryonic stem (“ES") cells that have undergone differentiation.
  • toxicity means any adverse effect of a chemical on a living organism or portion thereof. The toxicity can be to individual cells, to a tissue, to an organ, or to an organ system. A measurement of toxicity is therefore integral to determining the potential effects of the chemical on human or animal health, including the significance of chemical exposures in the environment.
  • Chemical composition refers to the inhibitory concentration at which 50% of the cells show cytotoxicity.
  • ID 50 refers to the inhibitory concentration at which 50% of the cells show inhibition of differentiation to a cell type.
  • the present disclosure provides assay methods for determining embryotoxicity in terms of qualitative and quantitative techniques. Since hES cells are believed to be the closest in vitro system to humans (McNeish, 2004, supra), the present disclosure uses hES cells to evaluate embryotoxicity, based on human fibroblast IC 5 0, undifferentiated hES cell IC 50 , and differentiated hES cell ID 5O , and classify compounds into non-teratogen, weak teratogen, and strong teratogen categories.
  • the present disclosure provides details of a study protocol wherein chemicals and/or compositions can be evaluated for their embryotoxicity potential.
  • the techniques provided herein involve the qualitative assessment of the toxicity by MTT assay, and the effect of the compounds and/or compositions on various lineages by using specific markers and the quantitative detection of IC 5O values.
  • the tests are compared against standard known toxic or safe compounds, for example a strongly embryotoxic (5-fluorouracil; 5-FU), a weakly embryotoxic (caffeine), and a non-embryotoxic (penicillin G) compound..
  • 5-FU strongly embryotoxic
  • caffeine a weakly embryotoxic
  • penicillin G penicillin G
  • the aim in selecting the three compounds relies on the fact that teratogenic effects of penicillin G are neither observed in mouse or human (Boucher and Delost, C. R. Seances Soc. Biol. FiI.
  • the present disclosure provides techniques or test procedures that determine the correlation of the toxicity effects of a compound and/or composition on various lineages, and thus enables determination of the toxicity and the efficacy potential of various groups or side chains (for example in new chemical entities (NCE's)), or the substitution of different groups or side chains, present on a core or main structure.
  • the present disclosure provides test procedures that are able to identify more than just embryotoxicity potential, in that the procedures provide toxicity data on various lineages. This data is very helpful to academic and industrial researchers, enabling them to potentially alter drug delivery techniques in order to avoid toxic effects on a particular lineage.
  • the present disclosure provides embryotoxicity assay techniques that characterize the toxicity of chemicals and/or compositions in a more elaborate manner.
  • the present disclosure provides results using human ES cells that can be compared with results from mouse ES cells, to highlight the sensitivity using human ES cells.
  • the present disclosure also provides testing of certain gene expression markers specific for the Asian and/or Indian population.
  • hES cells are believed to be the closest in vitro system to mimic humans, and the toxicity in hES cell could resemble post-fertilization stages.
  • present disclosure provides test systems that provide a comparative effect of the compounds on different lineages during development, and since these test systems are based on gene expression they are more sensitive, reproducible, and robust, and requires less dependency on visual expertise, which is an absolute requirement in conventional techniques based on visual observation of beating cardiomyocytes.
  • Gene expression can be analyzed by isolating total RNA using the TRIzol method (Invitrogen Corporation, Carlsbad, CA) or RNAeasy Spin Columns (QIAGEN, Incorporated, Valencia, CA), and using the isolated RNA to study the expression of genes specific for ectoderm, mesoderm, and endoderm by an appropriate method, such as RT-PCR, qPCR, microarray, or TaqMan Low Density Array (“TLDA").
  • TRIzol method Invitrogen Corporation, Carlsbad, CA
  • QIAGEN RNAeasy Spin Columns
  • TLDA TaqMan Low Density Array
  • beating cardiomyocytes were used as an indictor of developmental toxicity. More recently, literature supports the hypothesis to use hES cells utilizing PCR and FACS as a tool for developing better end points for testing developmental toxicity (Huuskonen, 2005, supra).
  • researchers have also suggested the use of automated in vitro screening methods for teratogens that are based on cytotoxicity and cell morphology (Walmod, et al, Toxicol. In Vitro 18:511- 525, 2004).
  • PCR of key genes involved in cardiomyocytes development alpha myosin heavy chain, Oct-4, Brachyury and Nkx2.5
  • mice embryonic stem cells Apart from the in vitro data available, similar results have been shown using mouse embryonic stem cells (zur Nieden, et al. , 2004, supra). The results disclosed herein are in accordance with published data about the non-toxic and toxic effects of penicillin G and 5 -FU, respectively. The results also indicate that the methods are sensitive and specific, as they do not cause gene down- regulation in a non-specific manner, as only endodermal markers are down-regulated by penicillin G, whereas all the three lineage markers are effected by caffeine and 5-FU.
  • the present disclosure provides evidence that the use of human embryonic stem cells for drug toxicity evaluations is a far better choice that the use of any other alternative method for toxicity testing, at least in part because: (1) hES cells are believed to be the closest in vitro system to mimic humans, and the toxicity in hES cells could resemble post-fertilization stages; (2) the test systems determine the effects of a compound and/or composition on different lineages during development; and (3) since, in certain embodiments, the test systems are gene expression based, they are more sensitive, reproducible, robust, and requires less expertise than visual observation of beating cardiomyocytes. The use of increased numbers of markers provides a clearer picture of developmental toxicity in humans.
  • the results of the presently disclosed methods demonstrate the potential of human ES cells as an in vitro model to study developmental toxicity.
  • the present disclosure provides results that human EBs are more sensitive to drug treatment as assessed by cytotoxicity assays. The down- regulation of early developmental markers were seen upon drug treatment.
  • the methods also provide a correlation between the therapeutic range of the tested compounds in serum and the toxicity dose as tested by human ES cells.
  • the present disclosure provides a simple, reliable, and robust model system, and a more clinically relevant assay than using mouse ES cells.
  • the presently disclosed embryotoxicity testing protocols comprise the following sequence of steps.
  • Studies using embryoid bodies generally comprise: a) preparation of various concentrations of test solutions in culture medium or a desired solvent; b) preparation of a cell suspension by enzymatic digestion of human embryonic stem cell colonies; c) culturing the cell suspension for formation of embryoid bodies in the presence and absence of one or more test solutions, and incubation for three days in hanging drops; d) cultivation of the embryoid bodies in bacteriological petri dishes for two days under controlled conditions; e) transfer of the embryoid bodies into 96 well plates and incubation for 10 days in the presence of one or more test solutions; and f) detection of the viability of the embryoid bodies after 15 days by MTT assay, or fluorescent or luminescent method, and isolation of RNA for studying gene expression.
  • HFF human foreskin fibroblasts
  • Studies using human embryonic stem cells generally comprise: a) preparation of various concentrations of test solutions in culture medium or a desired solvent; b) preparation of hES colonies by manual passage in the presence and absence of one or more test solutions, and incubation for 15 days on matrigel coated plates changing the media and test solutions every 2 days; c) disruption of the colonies after 15 days with enzymes to form single cells; d) labeling of single cells with a fluorescently-labeled dye, such as propidium iodide ("PI"); and e) detection of the percentage viability with FACS as a measure of cytotoxicity.
  • PI propidium iodide
  • test solution Various concentrations of test chemicals are prepared in culture medium or other appropriate solvent (termed a "test solution” herein).
  • the culture medium comprises 80% DMEM/F-12, 15% ES tested FBS, 5% serum replacement, 1% nonessential amino acid solution, 1 mM glutamine (GIBCO®, Invitrogen Corporation), 0.1% ⁇ -mercaptoethanol, 4 ng/ml human bFGF, and 20 ng/ml LIF.
  • Preparation of a cell suspension of human embryonic stem cells generally involves enzymatic digestion of 3-4 day old human embryonic stem cell colonies. The process involves washing the colonies with 2 ml DPBS, and then 2 ml of collagenase (2 mg/ml) was added for 15-20 minutes. After incubation, collagenase is removed and 1 ml of trypsin (0.05%) is added for 1 minute. The trypsin is then removed, the colonies are collected in the medium, and a cell suspension is prepared by trituration. A cell suspension of approximately 10,000 cells is prepared with an appropriate test solution.
  • the culturing of the cell suspension is done by dispensing about 20 ⁇ l of the cell suspension along with test chemicals on the inner side of a 100 mm bacteriological petri dish. Around 50-80 drops are dispensed for each concentration, the untreated control, and the solvent control. The lid is turned carefully into its regular position and is put on top of a petri dish containing 5 ml of phosphate buffered saline ("PBS"). The "hanging drops" were incubated for three days in a humidified atmosphere with 5% CO 2 at 37 0 C.
  • PBS phosphate buffered saline
  • the EBs are transferred into 96 well plates by placing 10 EBs in each well. About 150 ⁇ l of freshly prepared test solution is added to each well, and three wells are used for each concentration. The 96 well plates are incubated for 5 days in a humidified atmosphere with 5%CO 2 at 37 0 C. The remaining EBs were plated on a 24 well plate coated with 0.2% gelatin and were allowed to differentiate. Media was replaced every second day until day 15. On the 15th day, an MTT assay is performed. MTT solution (5 mg/ml) was prepared in culture medium containing test chemical.
  • the MTT solution is added to all wells and incubated at 37 0 C in a humidified atmosphere of 5% CO 2 for 4 hours, after which the MTT desorb solution (acidified isopropanol) was added to each well.
  • the plate is shaken on a micrometer plate for 15 minutes to dissolve the formazan.
  • the absorbance is measured at 550-570 nm in a microtiter plate reader using 630 nm as a reference wavelength.
  • 5-fluorouracil was treated at concentrations from 0.0001 — 1 ⁇ g/ml, caffeine at concentrations from 0.1 - 500 ⁇ g/ml, penicillin G at concentrations from 0.1 - 5000 ⁇ g/ml, busulfan at concentrations from 0.1 - 10 ⁇ g/ml, hydroxyurea at concentrations from 0.1 - 10 ⁇ g/ml, indomethacin at concentrations from 1 - 100 ⁇ g/ml, and saccharin at concentrations from 10 - 5000 ⁇ g/ml. These concentrations were selected based on previously published reports (Genschow, et al, 2000, supra). Caffeine and penicillin G were dissolved in media, while 5-FU was dissolved in DMSO and further diluted in media.
  • Undifferentiated ES cells RELICELL®hES 1
  • RELICELL®hES 1 Undifferentiated ES cells, RELICELL®hES 1
  • HFF Human foreskin fibroblasts
  • HFF Human Foreskin Fibroblasts
  • HFF HFF were trypsinized, and a cell suspension of 1 x 10 4 cells/ml in routine culture medium was prepared. Using a multi-channel pipette, 50 ⁇ l volumes of the cell suspension (500 cells/well) was dispensed. Viability of the cells can be checked by staining an aliquot of the cell suspension with trypan blue. A viability of 90% is acceptable. The cells were incubated for 2 hours in a humidified atmosphere with 5% CO 2 at 37°C, which allows for adherence of the cells.
  • the viability of the cells was determined using the MTT test, which was detected quantitatively using a microplate ELISA reader at 570 nm with a 630 nm reference filter. The percent viability at each test concentration was expressed based on the absorbance, where the absorbance of the control was considered as 100% viable cells, and 50% inhibitory concentration values were calculated from the concentration-response curve (IC 50 HFF).
  • hES cells were trypsinised and added last, after the preparation of test chemicals in medium, to avoid prolonged storage outside the incubator.
  • 20 ⁇ l of cell suspension containing the appropriate test chemicals (-5000 cells) was dispensed on the inner side of a 100 mm tissue culture petri dish lid. Approximately 50-80 drops were pipetted per lid.
  • 150 ⁇ l of assay medium containing the appropriate concentration of test chemical 150 ⁇ l volume contains 1.333x the final chemical concentration
  • test solution was removed with the care, so that the cell layer on the bottom of the wells was not disturbed.
  • 200 ⁇ l of freshly prepared test solution (final concentration/well as on day 0) was added, and the cell cultures were incubated at 5% CO 2 and 37°C for 3 days. This process was repeated on days 6, 9, and 12. Determination of cell growth inhibition was performed at day 15 of the assay using MTT reagent. Cytotoxicity for hES cells was calculated using flow cytometry. Briefly, cells were placed on matrigel coated plates along with different test compound concentrations. Media was replaced every 3 days along with the different test compound concentrations. At day 15, hES cells were collected by trypsinization, and after washing with PBS, the cells were incubated with PI for 10 minutes in the dark. Percent viability for various test compound concentrations was calculated using flow cytometry.
  • FIG. 2A shows the dose dependent effects of 5-FU on 5 day old EBs; there was a significant change in the morphology of the EBs at a dose of 1 ⁇ g/ml (FIG. 2A - 6) when compared to the control (FIG. 2A - 1). There was a loss of compactness and a decrease in the size of the EBs, suggesting that the effects at this dose were detrimental for growth.
  • the hEBs at the dose of 1 ⁇ g/ml of 5-FU did not survive until day 15 of the study.
  • FIG. 2B represents the effects of caffeine in a dose dependent manner.
  • the morphology did not show any major changes, as was the case for 5-FU, but there was a significant darkening of the cells as the dose increased (FIG. 2B - 5 and 6).
  • the effects were more prominent at a dose of 500 ⁇ g/ml when compared to the lower doses.
  • FIG. 2C represents the effects of penicillin G in a dose dependent manner. There was no significant change observed in the penicillin G treated EBs up to 1000 ⁇ g/ml (FIG. 2C - 5). However, there was some non-significant darkening of the EBs at the highest dose, suggesting that even penicillin G at a very high dose could cause some toxicity (5000 ⁇ g/ml, FIG. 2C - 6).
  • the data also suggest that morphology evaluations at an early stage (day 5) of the treatment could give some indications of the toxic effects of different compounds.
  • FIG. 3A represents the dose dependent effects of 5-FU on HFF, hES cells, and hEBs. The results suggest that there is a significant decrease in the cell viability of all the three cell types following treatment with 5-FU.
  • FIG. 3B shows the effects of caffeine in a log dose response. The highest dose of 500 ⁇ g/ml of caffeine caused a significant decrease in the survival of all the three cell types to about 10%. hEBs seem to be more vulnerable to toxic effects of caffeine than hES and HFF cells.
  • a dose of 50 ⁇ g/ml caused a significant decrease in cell survival percentage when compared to controls.
  • the effects of penicillin G on the survival patterns in a dose-dependent manner are presented in FIG. 3C. All three cell lines showed a statistically significant decrease in cell survival at a dose of 5000 ⁇ g/ml.
  • hEBs showed more cytotoxic effects at a dose of 1000 ⁇ g/ml when compared to hES cells and HFF, suggesting that differentiating cells are more susceptible to the toxic effects of penicillin G than hES and HFF cells.
  • Penicillin G (FIG. 4A) and saccharin (FIG. 4B) showed more than 95% cell death in hEBs, hES and HFF cells at a dose of 5000 ⁇ g/ml.
  • hEBs showed about 60% cell survival in both of the compounds at 1000 ⁇ g/ml.
  • 20% cell survival was observed after caffeine (500 ⁇ g/ml) treatment (FIG. 4C), whereas about 20-30% survived after indomethacin (100 ⁇ g/ml) treatment (FIG. 4D).
  • 2-5% cell survival in HFF and hES was observed following treatment of 10 ⁇ g/ml of the strongly embryotoxic compounds busulfan (FIG.
  • PCR was performed with the initial denaturation cycle at 94 0 C for 5 min, followed by 35 cycles of 94 0 C for 30 seconds, annealing temperature varying (Table 1) for 30 seconds, 72 0 C for 1 minute, followed final extension at 72 0 C for 5 min.
  • Electrophoresis was performed on 1.5% agarose gels. Paired comparisons were conducted using a paired t test, and all data are presented as mean values ⁇ S.E. Differences were considered significant at a 0.05 level of confidence.
  • FIG. 5A represents the effects of 5-FU on gene expression profiles of EBs.
  • the genes were selected to provide an indication of the organogenesis of major organs from each lineage (i.e., ectoderm, mesoderm, and endoderm).
  • 5-FU ectoderm
  • mesoderm mesoderm
  • endoderm a lineage that carries the highest dose of 5-FU.
  • NFH levels 60% at 0.01 ⁇ g/ml
  • FIG. 6A shows the gene expression profile of day 15 EBs following treatment with caffeine.
  • NFH expression decreased by 80% at a dose of 50 ⁇ g/ml
  • keratin by 85% at a dose of 50 ⁇ g/ml
  • ACTCl by 40% at 500 ⁇ g/ml
  • AFP by 80% at a dose of 50 ⁇ g/ml (FIG. 6B).
  • NANOG CD34
  • BMP4 BMP4
  • MSXl BMP5
  • FIG. 7A depicts the toxic effects of penicillin G on various tissue specific markers. There was no significant change observed in any of the markers of ectoderm, mesoderm, and endoderm, except a significant decrease in AFP and ALB levels. The AFP levels decreased to 40% at a dose as low as 100 ⁇ g/ml, whereas ALB levels decreased at a dose of 10 ⁇ g/ml. There was a complete loss of expression of AFP and ALB at the highest dose of penicillin G, suggesting the toxic effects of penicillin G are more prominent in the endoderm lineage than the other two lineages (FIG. 7B).
  • Table 2 shows a list of genes used for performing real time PCR to study the genotoxic effects of strong, weak and non embryotoxic compounds.
  • ectoderm nestin
  • KRTl 5 keratin 15
  • NEFH neurofilament, heavy polypeptide 200 KDa
  • mesoderm brachyury
  • GATA4 GATA binding protein 4
  • actin alpha, cardiac muscle
  • AFP alpha fetoprotein
  • NKX6-1 NK6 homeobox 1
  • OCT4 pluripotency
  • OCT4 homeobox transcription factor Nanog
  • ABCG2 homeobox transcription factor-binding cassette, subfamily G, member 2
  • one petri dish was used per concentration of the test chemical, as well as for the untreated control (assay medium) and the solvent control.
  • the lid was turned carefully to its regular position and put on top of a petri dish filled with 5 ml PBS.
  • the "hanging drops" were incubated for 3 days in a humidified atmosphere with 5% CO 2 at 37°C.
  • Changes in mesodermal marker representative of cardiogenesis was used to calculate ID 5O values of the tested compounds, and the densitometry of the band intensities performed in case of RT-PCR, or CT values in case of qPCR, were used to calculate the ID 50 values.
  • inhibitory concentrations were calculated and are represented in Table 3 (values indicated are mean of 3 experiments).
  • the data suggest that 5-FU shows the lowest IC 50 values compared to the other two compounds.
  • the data also suggest that hEBs are more susceptible to toxic effects compared to the HFF and hES cell toxicity.
  • ID 50 inhibitory concentration at which 50% differentiation of cells is inhibited
  • ID 50 -NFH 0.00289 ⁇ g/ml
  • ID 50 -AFP was 0.0524 ⁇ g/ml
  • ID 5O -ALB was 0.000814 ⁇ g/ml, suggesting that ALB levels are affected more prominently, followed by NFH levels and AFP levels.
  • the ID 50 values were calculated for the most significant changes in gene expression.
  • the ID 50 -NFH was 17.38 ⁇ g/ml
  • the ID 50 -KRT15 was 17.08 ⁇ g/ml
  • the ID 50 -AFP was 1.28 ⁇ g/ml
  • the ID 50 -ALB was 57.30 ⁇ g/ml, suggesting that caffeine showed maximum inhibition of AFP expression, followed by NFH, KRTl 5, and ALB.
  • AFP and ALB ID 50 were calculated to be 84.11 ⁇ g/ml and 28.11 ⁇ g/ml respectively, suggesting that both the endodermal markers showed toxicity to penicillin G.
  • the ICs 0 values for the compounds were compared with those obtained in the EST (Table 5) (Genschow, et ah, 2000, supra). Results showed that although the readings were quite similar for most of the compounds, the IC 50 values obtained with human ES cells with the strong, weak, and non-embryotoxic compounds was lower than the IC 50 values obtained with mouse ES cells. Further, the IC 50 values of HFF were lower than the IC 50 values seen in 3T3 (mouse fibroblast cell line), indicating HFF were more sensitive than 3T3. TABLE 4
  • the therapeutic range for busulfan should be less than 0.6 ⁇ g/ml, and the IC 50 was 0.38 ⁇ g/ml, well within the therapeutic range.
  • the therapeutic range of penicillin G is 300-500 ⁇ g/ml, and the ICs 0 hEB is 1300 ⁇ g/ml.
  • Such high values in the serum would likely never be achieved, signifying that the compound is non-embryo toxic. So, in summary, the results using hEBs proved to be similar to that seen in the clinical context.

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Abstract

La présente invention concerne des procédés utiles pour le criblage de composés et/ou de compositions, par exemple des médicaments candidats potentiels. Les résultats des dosages de criblage sont corrélés aux effets des composés sur le niveau moléculaire et/ou cellulaire de l'organisme humain. L'invention concerne également des dosages de criblage mettant en œuvre des cellules souches embryonnaires humaines RELICELL®hES d'origine indienne. Les procédés selon la présente invention sont en parfaite corrélation avec des études de toxicité précliniques sur des animaux dans un contexte d'essais cliniques.
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